SITE-SPECIFIC NUCLEASE SINGLE-CELL ASSAY TARGETING GENE REGULATORY ELEMENTS TO SILENCE GENE EXPRESSION

Abstract

This invention relates to a single cell assay for determining the effect of chromosomal contact on the transcriptional activity of genes of interest in a cell and to methods of silencing gene expression in a cell by way of perturbing gene regulatory elements which are engaged in chromosomal contact.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Divisional Application of U.S. patent application Ser. No. 14/916,338, filed Mar. 3, 2016, which is a .sctn. 371 National Stage Application of PCT/IB2014/064259, filed 4 Sep. 2014 which claims priority to ZA 2013/06649, filed 4 Sep. 2013. Each of these applications is incorporated by reference in its entirety

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE (.txt)

[0002] Pursuant to the EFS-Web legal framework and 37 CFR .sctn..sctn. 1.821-825 (see MPEP .sctn. 2442.03(a)), a Sequence Listing in the form of an ASCII-compliant text file (entitled "3000012-002001_Sequence_Listing_ST25.txt" created on 15 Nov. 2018, and 79,384 bytes in size) is submitted concurrently with the instant application, and the entire contents of the Sequence Listing are incorporated herein by reference.

BACKGROUND

Field of the Invention

Description of Related Art

[0003] This invention relates to a single cell assay for determining the effect of chromosomal contact on the transcriptional activity of genes of interest in a cell and to methods of silencing gene expression in a cell by way of perturbing gene regulatory elements which are engaged in chromosomal contact.

[0004] Gene regulation begins with signal transduction cascades resulting in transcription in the nucleus. Gene expression is inherently stochastic with transcription occurring in bursts as gene switch from inactive to active states. Attempts have been made to decipher gene expression and relate it to biological noise and stochasticity, while simultaneously accounting for cell-to-cell variation.

[0005] The highly conserved endogenous eukaryotic RNA interference (RNAi) pathway is the standard approach used to silence gene expression. This approach involves the transfection of exogenous small interfering RNAs (siRNA) and analysis of alterations in gene expression at a population level. Therefore, siRNA-approaches fail to reveal cell-to-cell variability. Off-target effects represent another major challenge in the use of siRNA as gene knockdown tools.

[0006] The eukaryotic nucleus is an immensely crowded, yet paradoxically, highly organized environment. Its main constituent, DNA, is folded many times over reducing its one-dimensional length by over one million fold in space. Due to this tight compaction, regions of looped chromatin are permitted to interact or "kiss."

[0007] By identifying sites where DNA interacts, the population based chromosome conformation capture (3C) and derivative techniques (4C, 5C, HiC, ChIA-PET) allow the characterization of the global interactome (Dekker et al., 2002, Fullwood et al., 2009). Analyses of Hi-C data reveal that chromatin is divided into smaller chromosomal interacting domains, referred to as topologically associating domains (TADs), which range in size from several hundred Kb to .about.3 Mb (Lieberman-Aiden et al., 2009). By constraining the DNA topology, TAD structure may enhance the probability of specific loop-mediated interactions occurring thus playing a direct role in transcriptional activity.

[0008] The subdivision of the genome into subchromosomal domains, or TADs, represents a highly conserved feature of chromosomal organization (Dixon et al., 2012). This suggests that each cell across a population would possess a generally similar arrangement of its chromosomes. Paradoxically, traditional in situ studies reveal a large degree of heterogeneity across a population of genotypically identical cells (Li et al., 2012). Since C-techniques report an ensemble of many nuclei, the dynamic nature of TAD structure at the single cell level and any consequent effects on transcription are obscured in the data. Clearly, interrogating chromosomal interactions at the level of a single cell is imperative to the interpretation of global interactome studies.

[0009] A characteristic feature of TADs is the enrichment of intra-domain chromatin contacts. FISH results confirm a spatial distinction between domains and contact arrangements within domains, as loci within a single domain are closer in nuclear space than those in different domains despite having similar genomic distances from one another. TADS therefore may provide a layer of structural regulation governing the principle of long-range chromatin contact.

[0010] Within TADS, and/or at the interface between TADS, chromosomal contact is strongly correlated with the transcriptional activity of interacting DNA elements (Li et al., 2012). Recently, we developed a novel single cell microscopy-based assay, to directly address the role of loop-mediated contact on the transcription of interacting genes. Using this assay, we demonstrated that chromosomal contact plays a central role in supporting transcription of co-regulated genes.

[0011] The invention described here relates to this single cell assay to silence gene expression through the discrete targeting (by cutting) of gene regulatory elements engaged in chromosomal contact. Major regulatory elements include; (I) enhancers, (II) sites within chromatin loops that engage in intra- or interchromosomal contact in multigene complexes and (III) regulatory sites within chromatin loops that determine loop structure.

[0012] Transcription of co-regulated genes occurs concurrently with long-range chromosomal contacts to form multigene complexes. Such contacts and transcription are lost in knockout studies of transcription factors and structural chromatin proteins. However, these approaches do not reveal the necessity of chromosomal contacts for cotranscription in multigene complexes. To interrogate in a discrete manner the role of loop-mediated contact on co-transcription, we devised a novel strategy using TALENs to cleave and disrupt gene loops in a well-characterized multigene complex. Monitoring this disruption using RNA FISH and immunofluorescence microscopy revealed that perturbing the site of contact has a direct effect on transcription of other interacting genes. Unexpectedly this effect on co-transcription was hierarchical, with dominant and subordinate members of the multigene complex engaged in both intra- and inter-chromosomal contact. This observation reveals the unprecedented level of influence of these chromosomal contacts on the transcription of co-regulated genes in a multigene complex. Transcription is replete with proximal and distal chromatin looping interactions whose formation represents the basic organizing principle of nuclear architecture and gene activity (Tan-Wong et al., 2012). Loop-mediated chromosomal contacts are usually identified on a genome-wide scale using population-based `chromosome conformation capture` (3C) technologies (Dekker et al., 2002; Fullwood et al., 2009; Lieberman-Aiden et al., 2009; Li et al., 2012). Analyses of 3C-based data reveal a large heterogeneity in global chromatin interactions (Fullwood et al., 2009; Noordermeer et al., 2011; Li et al., 2012). Therefore, interacting DNA elements identified by 3C-based technologies are verified at the single cell level using fluorescent in situ hybridization (FISH) assays (Papantonis et al., 2012). These highly sensitive assays can target either DNA or nascent mRNA, and have revealed the co-localization between FISH foci in a fraction of the population (Papantonis et al., 2010). This suggests that a subset of cells within the population may be enriched for specific chromosomal interactions. Chromosomes are large and constrained in their ability to roam the entire nuclear volume. Thus, it is reasonable to surmise that the topological arrangements after each cellular division shuffle chromosomal proximities such that their 3D arrangements are altered in 1D space. This may lead to every cell in the population possessing unique spatial arrangements of its chromosomes.

[0013] Enhancer-promoter interactions utilize chromatin looping to trigger dynamic changes in transcription initiation (Deng et al., 2012). An example of this is the well-established model between the locus control region (LCR) and the promoter of the .beta.-globin gene. In a tissue-specific manner, the LCR has been shown to physically contact the promoter of the .beta.-globin gene, and initiate transcription (Deng et al., 2012). These LCR-mediated chromosomal interactions have been shown to result in variability in .beta.-globin genes transcript levels, or variegated gene expression, across the population (Noordermeer et al., 2011). In an otherwise identical population of cells, presumably through chromosomal interactions, such "jackpot" cells display higher levels of .beta.-globin transcription (Noordermeer et al., 2011). Accordingly, the specific set of chromosomal interactions (and consequent gene expression that may depend on LCR-mediated interactions) will vary between cells across the population. This heterogeneity reveals the absolute requirement of single cell analysis in global interactome and gene loop studies.

[0014] Looping also brings distal genes into close proximity, enabling chromosomal contact in "multigene complexes" at a single focus of multiple RNA polymerases (Papantonis et al., 2012; Li et al., 2012). Numerous studies have demonstrated that the formation of loop-mediated contact coincides with alterations in gene expression (Fullwood et al., 2009). Indeed, chromosomal contacts in multigene complexes appear to be the main modality of transcription in metazoan cells, as they are associated with over 95% of transcriptional activity (Li et al., 2012). In a comparable manner to enhancer-promoter interactions, specific chromatin interactions in multigene complexes are detected in a subset of cells within the population (Papantonis et al., 2010). Genome-wide chromatin interaction analysis with paired end tags (ChIA-PET), uncovered a multigene complex including the GREB1 locus and 3 other genes (Li et al., 2012). Of the 4 interacting genes, only GREB1 transcription is activated by the estrogen receptor-.alpha. (ER.alpha.) (Li et al., 2012). Intriguingly, despite the fact that this multigene complex may not assemble in every cell in the population, siRNAs targeting ER.alpha. disrupted all 4 interacting genes (Li et al., 2012). Therefore, even though these chromosomal contacts may only occur in a fraction of the population they clearly play a significant role in gene regulation. Moreover, this data supports a model of synergistic transcription, where chromosomal contact influences the transcription of the interacting genes. This would connote that the topological framework for transcriptional regulation is physical contact via chromosomal looping in multigene complexes.

[0015] Current siRNA and 3C-based experimental approaches cannot be applied to multigene complexes where all interacting genes are activated by the same transcription factor. Tumor necrosis factor alpha (TNF.alpha.) has been shown to induce the formation of such multigene complexes, where all interacting genes are activated by NF-.kappa.B (Papantonis et al., 2012). Ten minutes after TNF.alpha. stimulation, the promoters of genes located on the same chromosome (SAMD4A and TNFAIP) and on a different chromosome (SLC6A5) associate to form a NF-.kappa.B-dependent multigene complex (Papantonis et al., 2010). RNA FISH assays targeting the approximate sites of interaction identified by 3C, suggest an association between the formation of this NF-.kappa.B-regulated multigene complex and the co-transcription of interacting genes (Papantonis et al., 2010). However, both 3C and FISH approaches fail to reveal the necessity of chromosomal contacts for co-transcription of these interacting genes. Therefore, to accurately interrogate a model of synergistic regulation, a discrete perturbation of a single site within a gene loop, whilst monitoring the transcriptional status of other members of the multigene complex, is required. Importantly, owing to variegated gene expression (Noordermeer et al., 2011), this can only be achieved with a single cell approach.

[0016] Here we devise a single cell strategy using TALE nucleases (TALENs) to discretely perturb sites within gene loops that are established to engage in chromosomal contact in the well-characterized NF-.kappa.B-regulated multigene complex (Papantonis et al., 2010). This enabled us to address the longstanding question of the requirement of loop-mediated contact for transcriptional co-regulation in a multigene complex. Using RNA FISH and immunofluorescence (IF), we imaged the site of the disrupted loop simultaneously with the transcriptional activity of other interacting genes in the NF-.kappa.B-regulated multigene complex. This unique single cell perspective revealed that perturbing loop-mediated contact between the NF-.kappa.B-regulated genes, altered the transcriptional status of interacting genes. In addition, this effect on co-transcription was hierarchical, with dominant and subordinate members of the multigene complex engaged in intra-chromosomal contact at distances over 48 mbp, as well as inter-chromosomal interactions. Furthermore, restoration of a disrupted gene loop re-established both chromosomal contacts and transcription of interacting genes in a sequence independent manner. The unexpected hierarchical organization within the TNF.alpha.-induced multigene complex reveals the unprecedented level of influence of these chromosomal contacts on the transcription of co-regulated genes in a multigene complex.

SUMMARY OF THE INVENTION

[0017] The present invention relates to a method for silencing gene expression and an assay for determining the effects of chromosomal contact on gene silencing.

[0018] In a first aspect of the invention there is provided for a method for silencing gene expression at a single cell level. The method includes a step of perturbing or interfering with at least one chromosomal contact in the cell. The point of chromosomal contact may include any region of chromatin and/or DNA. The method further includes a step of detecting the site of perturbation of the chromosomal contact; and further includes a step of detecting the effect of the perturbation of chromosomal contact on the transcriptional activity of a gene of interest. It will be appreciated, that a gene of interest may include a single gene and or a multigene complex.

[0019] In one embodiment, the chromosomal contact is perturbed by inducing a site-specific double stranded break in the chromatin or DNA.

[0020] In another embodiment, the transcriptional activity of the gene of interest may be abrogated or prevented by recruitment of proteins involved in a repair process to the site of the double stranded break, which when bound to the chromatin or DNA at the site of the double stranded break obstruct chromosomal contacts. Alternatively, the transcriptional activity of the gene of interest may be abrogated or prevented by means of enhancement of mobility of the region of chromatin or DNA containing the double stranded break, which results in a reduction in the capacity of a gene loop to engage in chromosomal contact. In a further alternative, the transcriptional activity of the gene of interest may be abrogated or prevented through the loss of structural integrity of a gene loop, which results in the abrogation of chromosomal contact.

[0021] In yet another embodiment of the invention, the chromosomal contact may be an intergenic contact, an intragenic contact or both an intergenic and intragenic contact.

[0022] In another embodiment of the invention, the region of chromatin or DNA that is perturbed or interfered with may comprise a gene or regulatory element selected from the group consisting of an enhancer and/or promoter, a site within a chromatin or DNA loop that engages in intra-chromosomal or inter-chromosomal contact, or a regulatory site within a chromatin or DNA loop which determines the loop structure. It will be appreciated that the chromosomal contact may be between chromatin and/or DNA located either inter-chromosomally, intra-chromosomally or both inter-chromosomally and inter-chromosomally.

[0023] In yet another embodiment of the invention the double stranded break may be induced by a site-specific nuclease. The site-specific nuclease may be selected from the group consisting of a meganuclease, a zinc finger nuclease, a TALE nuclease, a BUD1 nuclease, and a CrispR nuclease. It will be appreciated that the site-specific nuclease may be delivered to the cell by transfection of the cell with a vector encoding the site-specific nuclease and wherein the site-specific nuclease is endogenously expressed in the cell. Alternatively, the site-specific nuclease may be exogenously expressed and the exogeounsly expressed site-specific nuclease may consequently delivered to the cell.

[0024] In yet another embodiment of the invention, the double stranded break is detected by the immunofluorescent staining of a protein involved in a cellular repair process in the cell, or by detecting the location of a recombinant protein expressing a fluorescent label which is involved in a cellular repair process in the cell.

[0025] In a further embodiment of the invention, the effect of the double stranded break on the transcriptional activity of the gene of interest is detected using a method selected from the group consisting of RNA fluorescent in situ hybridisation, live RNA fluorescent in situ hybridisation, immunogold labelling, molecular beacons and MS2 tagging.

[0026] In yet another embodiment of the invention the cell is a eukaryotic cell or prokaryotic cell.

[0027] A second aspect of the invention provides for a single cell assay for determining the effect of chromosomal contact on the transcriptional activity of at least one gene of interest in a cell. In one embodiment, a site-specific nuclease is used to induce or cause a double stranded break in a region of chromatin or DNA which is engaged in chromosomal contact in the cell. Consequently, an immunofluorescent probe or recombinant protein expressing a fluorescent label is used to detect the site of the double stranded break and one or more fluorescent oligonucleotide probes which are capable of hybridising to a target mRNA sequence produced by the transcription of at least one gene of interest are used to detect the presence or absence of transcription of the gene of interest by monitoring the fluorescence and the relative intensity of fluorescence of the fluorescent oligonucleotide probe hybridised to the target mRNA sequence. The fluorescence and relative intensity of fluorescence will be indicative of the effect of the chromosomal contact on the transcriptional activity of the gene of interest.

[0028] In one embodiment, of this aspect of the invention the transcriptional activity of the gene of interest may be abrogated or prevented by recruitment of proteins involved in a repair process to the site of the double stranded break, which when bound to the chromatin or DNA at the site of the double stranded break obstruct chromosomal contacts. Alternatively, the transcriptional activity of the gene of interest may be abrogated or prevented by means of enhancement of mobility of the region of chromatin or DNA containing the double stranded break, which results in a reduction in the capacity of a gene loop to engage in chromosomal contact. In a further alternative, the transcriptional activity of the gene of interest may be abrogated or prevented through the loss of structural integrity of a gene loop, which results in the abrogation of chromosomal contact.

[0029] In another embodiment, the chromosomal contact may be an intergenic contact, an intragenic contact or both an intergenic and intragenic contact.

[0030] In a further embodiment, the double stranded break perturbs or interfers with the chromatin or DNA engaged in chromosomal contact and consequently perturbs or interfers with the transcriptional activity of the gene of interest.

[0031] In another embodiment of the invention, the region of chromatin or DNA that is perturbed or interfered with may comprise a gene or regulatory element selected from the group consisting of an enhancer and/or promoter, a site within a chromatin or DNA loop that engages in intra-chromosomal or inter-chromosomal contact, or a regulatory site within a chromatin or DNA loop which determines the loop structure.

[0032] In yet another embodiment of the invention, the double stranded break may be induced by a site-specific nuclease. The site-specific nuclease may be selected from the group consisting of a meganuclease, a zinc finger nuclease, a TALE nuclease, a BUD1 nuclease, and a CrispR/Cas 9 nuclease. It will be appreciated by those skilled in the art that the site-specific nuclease may be delivered to the cell by transfection of the cell with a vector encoding the site-specific nuclease and wherein the site-specific nuclease is endogenously expressed in the cell. Alternatively, the site-specific nuclease may be exogenously expressed and the exogeounsly expressed site-specific nuclease may consequently delivered to the cell.

[0033] In yet another embodiment of the invention the immunofluorescent probe may be an antibody which binds to at least one of the proteins involved in the cellular repair process of a double strand break in the cell.

[0034] In another embodiment, the location of the hybridized fluorescent oligonucleotide probes in the cell is observable by a technique selected from the group consisting of diffraction limited imaging techniques, sub-diffraction limit image resolution and other imaging techniques, such as imaging in three dimensions, wherein detection of the chromosomal location of gene expression for the target mRNA sequences and the diffraction limited or sub-diffractive location of the hybridised immunofluorescent probe is indicative of the location of the double stranded break.

[0035] In yet another embodiment, the cell is a eukaryotic cell or prokaryotic cell.

BRIEF DESCRIPTION OF THE FIGURES

[0036] Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

[0037] FIG. 1: Enhancer-promoter interactions. Enhancer-promoter interactions utilize looping to trigger dynamic changes in transcription. An example of this is the well-established model between the locus control region (LCR) and the promobter of the .beta.-globin gene. These LCR-mediated chromosomal interactions have been shown to result in variability in .beta.-globin genes transcript levels, or variegated gene expression, across the population (Noordmeer et al., (2011)). Therefore, preventing enhancer-promoter interactions will have a significant impact on gene expression. To date, no assay exists to discretely perturb enhancer elements to silence gene expression.

[0038] FIG. 2: Intra- or inter-chromosomal contact in multigene complexes. Looping brings distal genes into close proximity, enabling chromosomal contact in multigene complexes. In a comparable manner to enhancer-promoter interactions, specific chromatin interactions in multigene complexes are detected in a subjet of cells within the population (Papantonis et al. (2010)). Loop-mediated contact between genes in a multigene complex, influences their transcriptional status of interacting genes. Therefore, preventing loop-mediated contact will have a significant impact on the gene expression of interacting genes in a multigene complex. To date, no assay exists to discretely perturb interacting genes in a multigene complex to silence expression.

[0039] FIG. 3: Regulatory sites that determine loop structure. Many distinct regulatory elements have been identified that organize chromatin into loops, thereby altering gene expression. Knockout studies of these structural chromatin proteins reveal their role in regulating transcription. However, the interpretation of these studies is complicated by the global alterations in gene expression that inevitably occurs. Preventing chromating remodelling and structural proteins from bining to their target sites will have significant impact on gene expression of related genes. To date, no assay exists to discretely perturb sites where chromatin structural proteins bind, and monitor associated gene expression at a single cell level.

[0040] FIG. 4: The transcriptional response of co-regulated genes in a multigene complex is asymmetric. The promoters of genes located on the same chromosome (SAMD4A and TNFAIP) and on a different chromosome (SLC6A5) associate via RNA Polymerase II (orange) to form a NF-.kappa.B-dependent multigene complex. BMP4 and RCOR1 are non-responsive to TNF.alpha., do not interact with SAMD4A (blue), TNFAIP2 (green) or SLC6A5 (red), and therefore serve as controls in this study.

[0041] FIG. 5: The transcriptional response of co-regulated genes in a multigene complex is asymmetric. Chromosomal contact in a multigene complex is associated with cotranscription. Spectrally distinct RNA FISH intronic probes targeting the region of these genes involved in chromosomal contact reveal co-localization of nascent intronic RNA, as visualized by overlapping foci. Two-tailed Fisher exact test; ***P=4.5E-4, n, number of alleles.

[0042] FIG. 6: RNA Pol II-mediated SAMD4A, TNFAIP2 and SLC6A5 co-transcription may occur within TADs. Overlapping SAMD4A, TNFAIP2 and/or SLC6A5 RNA FISH foci consistently co-localize with the active, poised form of RNA Pol II (phosphorylated at Serine 5). Bar, 10 .mu.m.

[0043] FIG. 7: RNA Pol II-mediated SAMD4A, TNFAIP2 and SLC6A5 co-transcription may occur within TADs. HUVECs always display two spatially distinct DNA FISH foci, both before and after TNF.alpha. treatment. Cells were counterstained with DAPI. Bar, 5 .mu.m.

[0044] FIG. 8: RNA Pol II-mediated SAMD4A, TNFAIP2 and SLC6A5 co-transcription may occur within TADs. Analysis of overlapping DNA FISH foci revealed co-localized foci in both the unstimulated and the TNF.alpha.-treated HUVEC population. Cells were counterstained with DAPI. Bar, 5 .mu.m.

[0045] FIG. 9: The transcriptional response of co-regulated genes in a multigene complex is asymmetric. The response to TNF.alpha. is asymmetric in diploid HUVECs. Allelic expression of each gene in the population of cells is shown. TNFAIP2 transcription occurs predominantly when SAMD4A is also transcribed (.about.86%) and SLC6A5 transcription occurs mainly when SAMD4A (.about.92%), or both SAMD4A and TNFAIP2 are transcribed (.about.62%). N, number of cells (N=166), n, number of alleles (n=332).

[0046] FIG. 10: The transcriptional response of co-regulated genes in a multigene complex is asymmetric. Data in (C) was replotted to show the combined allelic transcriptional status of SAMD4A, TNFAIP2 and SLC6A5 in the total population (T/P). Colocalization frequencies relative to cells co-expressing SAMD4A, TNFAIP2 and/or SLC6A5 is also shown. n, number of alleles, Bar, 5 .mu.m. See also FIGS. 6, 7 and 8.

[0047] FIG. 11: Visualization of TALEN-mediated disruption of a gene loop. The SAMD4A TALEN targets the region in intron 1 involved in chromosomal contact 10 mins post TNF.alpha. stimulation. TALE repeat domains are colored to indicate the identity of repeat variable diresidues (RVD); each RVD is related to the cognate targeted DNA base by the following code; NI=A, HD=C, NN=G/A, NG=T.

[0048] FIG. 12: Detection of double strand breaks in HUVECs. Indirect immunofluorescence was performed using an antibody specific to Ser139 phosphorylation of histone variant, H2A.X. DSBs labeled by H2A.X were detected with a donkey-anti rabbit antibody conjugated to Atto488. Distinct nuclear staining was evident in HUVECs treated with 100 .mu.M Etoposide, a DSB-inducer, for 1 hr. Cells were counterstained with DAPI. Bar, 5 .mu.m.

[0049] FIG. 13: Visualization of TALEN-mediated disruption of a gene loop. Successful detection of TALEN transfection at a single cell level. Discrete sites of H2A.X pSer139 were evident in .about.60% of HUVECs transfected with the SAMD4A TALEN. A higher portion of successfully transfected cells displayed single allelic DSBs than dual allelic DSBs.

[0050] FIG. 14: A time course of SAMD4A TALEN nuclease activity. DSBs, labeled by an H2A.X pSer139 primary antibody, were detected with a donkey-anti rabbit secondary antibody conjugated to Atto488. Nuclease activity (a measure of transfection efficiency) was first evident after 6 hr as evidenced by distinct sites of H2A.X pSer139 in .about.40% of HUVECs transfected with the SAMD4A pcDNA TALENs. At this time point, the majority of cell displayed no H2A.X foci and low levels of apoptotic cells, as evidenced by cells displaying multiple breaks (more than 2). Post 24 hr transfection, .about.60% of cells displayed DSBs, with slightly higher levels of apoptotic cells (.about.5%). After 48 hr post transfection, .about.70% of cells displayed DSBS and the level of apoptotic cells increased to .about.12%. At all time points, a higher portion of transfected cells displayed single allelic DSBs than dual allelic DSBs (at a ratio of .about.3:1).

[0051] FIG. 15: Visualization of TALEN-mediated disruption of a gene loop. H2A.X immunofluorescence accurately labels the sites of the disrupted SAMD4A gene loop. SAMD4A DNA FISH foci consistently co-localize with SAMD4A TALEN-induced DSBs.

[0052] FIG. 16: Visualization of TALEN-mediated disruption of a gene loop. Gel showing the surveyor nuclease result from the SAMD4A TALEN pair. NT, un-transfected control; GFP, GFP transfected cells; L, SAMD4A left TALEN only; R, SAMD4A right TALEN only; L+R, cells transfected with pcDNA left and right TALENs. Red arrow, 246 bp, blue arrow, 114 bp. Cells were counterstained with DAPI. Bar, 5 .mu.m. See also FIGS. 12, 14, 21, 32 and 41.

[0053] FIG. 17: TALEN-mediated disruption of the SAMD4A gene loop abrogates SAMD4A RNA and protein expression. SAMD4A TALEN-induced DSB does not alter transcription up to the DSB. Nascent intronic SAMD4A RNA (detected by probe set i) transcribed 5' of the DSB was evident in 42% of HUVECs displaying DSB, as evidenced by H2A.X staining. n, number of alleles (n=194), NS, no significant difference.

[0054] FIG. 18: TALEN-mediated disruption of the SAMD4A gene loop abrogates SAMD4A RNA and protein expression. The SAMD4A TALEN abrogates transcription downstream of the DSB. Nascent intronic SAMD4A RNA (detected by probe set ii) transcribed 3' of the DSB was never observed. n, number of alleles (n=84), Two-tailed Fisher exact test; ***P<0.001.

[0055] FIG. 19: TALEN-mediated disruption of the SAMD4A gene loop abrogates SAMD4A RNA and protein expression. Disrupting the SAMD4A gene loop is sufficient to abrogate protein expression. TNF.alpha. induces SAMD4A protein expression 16 hr post TNF.alpha. stimulation. Cells harboring dual allelic DSBs, as detected by H2A.X, displayed a significant reduction in protein expression, whilst cells harboring single allelic DSBs still express SAMD4A protein. R.F.U., relative fluorescent units, mean.+-.SD, *P<0.01, Two-tailed unpaired Students t test, cells were counterstained with DAPI, Bar, 5 .mu.m.

[0056] FIG. 20: TALEN-mediated disruption of a single gene loop and the associated chromosomal contacts in a multigene complex alters the transcriptional status of other genes occupying the same complex. A DSB induced in the non-TNF.alpha. responsive gene, BMP4, did not alter transcription of SAMD4A, TNFAIP2 or SLC6A5 relative to the normal TNF.alpha. response (FIG. 9).

[0057] FIG. 21: Detection of TALEN efficiency. Gel showing the surveyor nuclease result from the BMP4 TALEN pair. NT, control from un-transfected cells; GFP, cells transfected with GFP; L, BMP4 left TALEN only; R, BMP4 right TALEN only; L+R, cells transfected with the BMP4 pcDNA left and right TALENs. MW, O'GeneRuler 1 kp Plus DNA ladder.

[0058] FIG. 22: TALEN-mediated disruption of a single gene loop and the associated chromosomal contacts in a multigene complex alters the transcriptional status of other genes occupying the same complex. The disruption of the SAMD4A gene loop abrogates TNFAIP2 and SLC6A5 transcription and co-localization. SAMD4A loop disruption detected by H2A.X was simultaneously monitored with transcription of TNFAIP2 and SLC6A5 by RNA FISH.

[0059] FIG. 23: Comparison between the phenotype in cells displaying a single allelic DSB to cells displaying dual allelic DSBs. In cells where a single allele of SAMD4A was targeted, virtually all transcription of TNFAIP2 and SLC6A5 was lost at the corresponding allele, with multigene transcription confined to the intact allele. Where both SAMD4A alleles were targeted, most transcription from TNFAIP2 and SLC6A5 was lost at both alleles. There was a significant reduction in TNFAIP2/SLC6A5 colocalization frequencies in both cells displaying single and dual allelic DSBs. Two-tailed Fisher exact test; *P<0.05, ***P<0.001, n, number of DSBs. Cells were counterstained with DAPI. Bar, 5 .mu.m.

[0060] FIG. 24: TALEN-induced dual allelic DSBs abrogate protein expression of other members of the NF-kB multigene complex. Disrupting the SAMD4A gene loop is sufficient to abrogate protein expression of TNFAIP2 and SLC6A5. Dual indirect immunofluorescence was performed using a donkey-anti rabbit antibody conjugated to Atto488 to detect H2A.X and donkey-anti-goat secondary antibody conjugated to Atto565 to detect TNFAIP2 or SLC6A5. Cells displaying dual SAMD4A-induced DSBs, as detected by H2A.X, displayed a severe reduction in protein expression of TNFAIP2 and SLC6A5.

[0061] FIG. 25: The SAMD4A TALEN does affect the transcriptional status of RCOR1. RCOR1, a gene with transcriptional activity comparable to GAPDH, located approximately 400 kb 5' of TNFAIP2 was unaffected by the SAMD4A TALEN. Two foci of RCOR1 are evident in each cell in the field of view. Cells were counterstained with DAPI. Bar, 10 .mu.m

[0062] FIG. 26: TALEN-mediated disruption of a single gene loop and the associated chromosomal contacts in a multigene complex alters the transcriptional status of other genes occupying the same complex. Disruption of the TNFAIP2 gene loop does not affect SAMD4A gene expression, but alters SLC6A5 transcription and SAMD4A/SLC6A5 colocalization.

[0063] FIG. 27: TALEN-mediated disruption of the TNFAIP2 gene loop abrogates TNFAIP2 gene expression. The TNFAIP2 TALEN targets the approximate region in intron 2 involved in chromosomal contact at 10 mins post TNF.alpha. stimulation. Left and right TALENs were designed to contain 18 full monomer repeats and target a 20 bp target region, where the first and last space specify the T at the N terminus and the 0.5 repeat respectively.

[0064] FIG. 28: TALEN-mediated disruption of the TNFAIP2 gene loop abrogates TNFAIP2 gene expression. Post 24 hr transfection, .about.60% of cells displayed TNFAIP2 TALEN-induced DSBs and low levels of apoptotic cells, as evidenced by cells displaying multiple breaks (more than 2). A higher portion of transfected cells displayed single allelic DSBs as opposed to dual allelic DSBs.

[0065] FIG. 29: TALEN-mediated disruption of the TNFAIP2 gene loop abrogates TNFAIP2 gene expression. The TNFAIP2 TALEN abrogates transcription downstream of the DSB. Dual-transfected HUVECs were stimulated with TNF.alpha. for 10 mins to allow for the first .about.1.5 kbp of the TNFAIP2 to be transcribed. Nascent intronic TNFAIP2 RNA (Atto565) transcribed downstream of the DSB (H2A.X--Atto488) was never observed. Two-tailed Fisher exact test; ***P<0.001. Cells were counterstained with DAPI. Bar, 5 .mu.m.

[0066] FIG. 30: TALEN-mediated disruption of the TNFAIP2 gene loop abrogates TNFAIP2 gene expression. TNF.alpha. induces TNFAIP2 protein expression 16 hr post stimulation. Cells were counterstained with DAPI. Bar, 5 .mu.m.

[0067] FIG. 31: TALEN-mediated disruption of the TNFAIP2 gene loop abrogates TNFAIP2 gene expression. Disrupting the TNFAIP2 gene loop is sufficient to abrogate protein expression. Dual indirect immunofluorescence of TNFAIP2 and H2A.X (DSBs) was performed using a donkey-anti rabbit antibody conjugated to Atto488 and donkey-anti-goat conjugated to Atto565 respectively. DSBs, as detected by H2A.X, displayed a severe reduction in protein expression. R.F.U., relative fluorescent units, mean.+-.SD, **P<0.01, Two-tailed unpaired Students t test, cells were counterstained with DAPI, Bar, 5 .mu.m.

[0068] FIG. 32: Detection of TALEN efficiency. Gel showing the surveyor nuclease result from the TNFAIP2 TALEN pair. NT, control from un-transfected cells; GFP, cells transfected with GFP; L, TNFAIP2 left TALEN only; R, TNFAIP2 right TALEN only; L+R, cells transfected with the pcDNA left and right TNFAIP2 TALENs.

[0069] FIG. 33: Comparison between the phenotype in cells displaying a single allelic DSB to cells displaying dual allelic DSBs. In cells displaying single allelic TNFAIP2 TALEN-induced DSBs, SAMD4A transcription remained unaffected, whilst SLC6A5 transcription was limited to the intact TNFAIP2 allele. SLC6A5 transcription was rarely observed in cells harboring dual allelic DSBs, whereas SAMD4A transcription remained unaffected. There was a reduction in SAMD4A/SLC6A5 co-localization frequencies in both cells displaying single and dual allelic DSBs. Two-tailed Fisher exact test; *P<0.05, cells were counterstained with DAPI. Bar, 5 .mu.m.

[0070] FIG. 34: TALEN-induced dual allelic DSBs abrogate protein expression of other members of the NF-kB multigene complex. Disrupting the TNFAIP2 gene loop is sufficient to abrogate SLC6A5, but not SAMD4A protein expression. Dual indirect immunofluorescence of SAMD4A or SLC6A5 and DSBs was performed using a donkey-anti rabbit antibody conjugated to Atto488 and donkey-anti-goat conjugated to Atto565 respectively. Cells displaying dual TNFAIP2-induced DSBs, as detected by H2A.X, displayed a reduction in protein expression of SLC6A5, whereas SAMD4A protein expression did not appear to be affected.

[0071] FIG. 35: TALEN-mediated disruption of a single gene loop and the associated chromosomal contacts in a multigene complex alters the transcriptional status of other genes occupying the same complex. Disruption of the SLC6A5 gene loop does not alter SAMD4A/TNFAIP2 transcription or co-localization. Two-tailed Fisher exact test; *P<0.05, **P<0.01, ***P<0.001, n, number of DSBs. Cells were counterstained with DAPI. Bar, 5 .mu.m. See also FIGS. 23, 24, 27, 28, 29, 30, 31, 33, 34, 36, 37, 38, 39, 40, 42 and.

[0072] FIG. 36: TALEN-mediated disruption of the SLC6A5 gene loop abrogates SLC6A5 gene expression. The SLC6A5 TALEN targets the approximate region in intron 1 involved in chromosomal contact at 10 min post TNF.alpha. stimulation. Left and right TALENs were designed to contain 18 full monomer repeats and target a 20 bp target region, where the first and last space specify the T at the N terminus and the 0.5 repeat respectively.

[0073] FIG. 37: TALEN-mediated disruption of the SLC6A5 gene loop abrogates SLC6A5 gene expression. Post 24 hr transfection, .about.60% of cells displayed SLC6A5 TALEN-induced DSBs and low levels of apoptotic cells, as evidenced by cells displaying multiple breaks (more than 2). Consistent with SAMD4A and TNFAIP2 TALENs, a higher portion of transfected cells displayed single allelic DSBs as opposed to dual allelic DSBs.

[0074] FIG. 38: TALEN-mediated disruption of the SLC6A5 gene loop abrogates SLC6A5 gene expression. The SLC6A5 TALEN abrogates transcription downstream of the DSB. Dual-transfected HUVECs were stimulated with TNF.alpha. for 10 min to allow for the first .about.1.5 kbp of SLC6A5 to be transcribed. Nascent intronic SLC6A5 (Atto647N) RNA transcribed downstream of the DSB (H2A.X--Atto488) was never observed. Two-tailed Fisher exact test; ***P<0.001. Cells were counterstained with DAPI. Bar, 5 .mu.m.

[0075] FIG. 39: TALEN-mediated disruption of the SLC6A5 gene loop abrogates SLC6A5 gene expression. TNF.alpha. induces SLC6A5 protein expression 16 hr post stimulation. Cells were counterstained with DAPI. Bar 5 .mu.m.

[0076] FIG. 40: TALEN-mediated disruption of the SLC6A5 gene loop abrogates SLC6A5 gene expression. Disrupting the SLC6A5 gene loop is sufficient to abrogate protein expression. Dual indirect immunofluorescence of SLC6A5 and H2A.X (DSBs) was performed using a donkey-anti rabbit antibody conjugated to Atto488 and donkey-anti-goat conjugated to Atto565 respectively. DSBs, as detected by H2A.X, displayed a significant reduction in protein expression. R.F.U., relative fluorescent units, mean.+-.SD, **P<0.01, Two-tailed unpaired Students t test, cells were counterstained with DAPI, Bar, 5 .mu.m.

[0077] FIG. 41: Detection of TALEN efficiency. Gel showing the surveyor nuclease result from the SLC6A5 TALEN pair. NT, control from un-transfected cells; GFP, cells transfected with GFP; L, SLC6A5 left TALEN only; R, SLC6A5 right TALEN only; L+R, cells transfected with the SLC6A5 pcDNA left and right TALENs.

[0078] FIG. 42: Comparison between the phenotype in cells displaying a single allelic DSB to cells displaying dual allelic DSBs. Transcription, as well as co-localization of SAMD4A and TNFAIP2 was not affected in cells harboring both single allelic or dual allelic SLC6A5 TALENmediated DSBs. n, number of DSBs. Cells were counterstained with DAPI. Bar, 5 .mu.m.

[0079] FIG. 43: TALEN-induced dual allelic DSBs abrogate protein expression of other members of the NF-kB multigene complex. Disrupting the SLC6A5 gene loop does not alter protein expression of SAMD4A or TNFAIP2. Dual indirect immunofluorescence of SAMD4A or TNFAIP2 and DSBs was performed using a donkey-anti rabbit antibody conjugated to Atto488 and donkey-anti-goat conjugated to Atto565 respectively. Cells displaying dual SLC6A5-induced DSBs, as detected by H2A.X, did not display any alteration in protein expression of TNFAIP2 and SLC6A5. R.F.U., relative fluorescent units, mean.+-.SD, Mann Whitney U test, *P<0.05, cells were counterstained with DAPI, Bar, 5 .mu.m.

[0080] FIG. 44: The SAMD4A gene loop was successfully repaired. Graphical representation of the repair strategy. The repair construct consists of an IRES and eGFP, flanked by splice sites.

[0081] FIG. 45: The SAMD4A gene loop was successfully repaired. TNF.alpha.-induced activation of SAMD4A induces eGFP expression. eGFP signal was observed in .about.1% of HUVECs dual transfected with the pDT vector and PCR product for 72 hr and stimulated with TNF.alpha. for 24 hr. Cells were counterstained with DAPI. n, number of cells. Bar, 5 .mu.m. See also FIGS. 46 and 47.

[0082] FIG. 46: The SAMD4A gene loop was successfully repaired with the IRES-GFP construct. HUVECs were transfected with a CMV-eGFP construct. eGFP positive or `green` cells coincided with a large number of RNA FISH foci.

[0083] FIG. 47: The SAMD4A gene loop was successfully repaired with the IRES-GFP construct. The SAMD4A promoter does not constitutively express the IRES-eGFP mRNA. eGFP signal was observed in .about.1% of HUVECs dual transfected with the pDT vector and PCR product for 72 hr and stimulated with TNF.alpha. for 24 hr. .about.90% of cells displaying eGFP mRNA transcripts were not visualized as a `green` cell. Cells were counterstained with DAPI. Bar 10 .mu.m.

[0084] FIG. 48: Repairing the disrupted SAMD4A gene loop restores transcription of genes in a multigene complex. HUVECs were stimulated for 20 min with TNF.alpha., recapitulating transcription of the first .about.1.5 kbp of SAMD4A as well as the IRES-eGFP. Distinct eGFP foci were evident in .about.10% of transfected cells, and these foci overlapped with SAMD4A mRNA. n, number of cells (n=118), Bar, 5 .mu.m.

[0085] FIG. 49: Repairing the disrupted SAMD4A gene loop restores transcription of genes in a multigene complex. There was no significant difference in the co-localization frequencies between eGFP/SAMD4A, TNFAIP2 and SLC6A5 and mock-transfected cells. Cells were counterstained with DAPI. N, number of cells (N=448), n, number of eGFP foci (n=45), NS, no significant difference, Bar, 5 .mu.m.

[0086] FIG. 50: Hypothetical model of hierarchical transcription between coregulated genes in a multigene complex. TADs constrain genes to compartments in the nucleus that are admissible to long-range chromosomal interactions. Upon induction by TNF.alpha., NF-kB responsive genes engage in chromosomal interactions. As two of these genes reside on the same chromosome but almost 50 Mbp apart, and another resides on a different chromosome, these interactions most likely occur within a TAD. Single cell analysis 10 mins post TNF.alpha. stimulation, reveals that the transcriptional response of interacting genes in a multigene complex is asymmetric (FIG. 9). Allelic analysis revealed the following 4 categories were most prevalent; (a) no transcription of any gene, (b) SAMD4A expression only, (c) SAMD4A, SLC6A5 and TNFAIP2 combined expression and (d) combined expression of SAMD4A and TNFAIP2. Despite the low frequency of these interactions (FIG. 10), disrupting the sites within the gene loops that engage in chromosomal contact revealed a hierarchical organization between this TNF.alpha.-induced multigene complex.

[0087] FIG. 51: Disrupting the IL8 enhancer abrogates transcription of the CXC proinflammatory genes. (A) The IL-8 enhancer is brought in close proximity to the IL8 by chromosomal looping. (B) Inducing a DSB in the IL8 promoter abrogates IL8 expression as well as the expression of a non coding RNA that is transcribed in the enhancer. Two-tailed Fisher exact test; *P<0.05, **P<0.01, ***P<0.001, n, number of DSBs. Cells were counterstained with DAPI. Bar, 5 .mu.m.

[0088] FIG. 52: TALEN-mediated disruption of a single gene loop and the associated chromosomal contacts in a multigene complex alters the transcriptional status of other genes occupying the same complex. (A) A DSB induced in the non-TNF.alpha. responsive gene, BMP4, did not alter transcription of SAMD4A, TNFAIP2 or SLC6A5 relative to the normal TNF.alpha. response. (B) The disruption of the SAMD4A gene loop abrogates TNFAIP2 and SLC6A5 transcription and co-localization. SAMD4A loop disruption detected by H2A.X was simultaneously monitored with transcription of TNFAIP2 and SLC6A5 by RNA FISH. (C) Disruption of the TNFAIP2 gene loop does not affect SAMD4A gene expression, but alters SLC6A5 transcription and SAMD4A/SLC6A5 co-localization. (D) Disruption of the SLC6A5 gene loop does not alter SAMD4A/TNFAIP2 transcription or co-localization. Two-tailed Fisher exact test; *P<0.05, **P<0.01, ***P<0.001, n, number of DSBs. Cells were counterstained with DAPI. Bar, 5 .mu.m.

[0089] FIG. 53: Disrupting the CTCF site 3' of the TNFAIP2 gene abrogates transcription of TNFAIP2. (A) A CRISPR was designed to induce a DSB in the consensus CTCF site located 3' of the TNFAIP2 gene. (B) The disruption of the 3' CTCF site was detected by H2A.X was simultaneously monitored with transcription of TNFAIP2 by RNA FISH. Two-tailed Fisher exact test; *P<0.05, **P<0.01, ***P<0.001, n, number of DSBs. Cells were counterstained with DAPI. Bar, 5 .mu.m.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

[0090] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

[0091] The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention.

[0092] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. As used throughout this specification and in the claims which follow, the singular forms "a", "an" and "the" include the plural form, unless the context clearly indicates otherwise.

[0093] The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms "comprising", "containing", "having" and "including" and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0094] The terms "nucleic acid" or "nucleic acid molecule" encompass both ribonucleotides (RNA) and deoxyribonucleotides (DNA), genomic DNA, and synthetic DNA. The nucleic acid may be double-stranded or single-stranded. Where the nucleic acid is single-stranded, the nucleic acid may be the sense strand or the antisense strand. A nucleic acid molecule may be any chain of two or more covalently bonded nucleotides, including naturally occurring or non-naturally occurring nucleotides, or nucleotide analogs or derivatives. By "RNA" is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides. The term "DNA" refers to a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides.

[0095] The term "chromatin" refers to the nucleoprotein structure comprising the cellular genome. Cellular chromatin comprises nucleic acid, primarily DNA, and protein, including histones and non-histone chromosomal proteins. The majority of eukaryotic cellular chromatin exists in the form of nucleosomes, wherein a nucleosome core comprises approximately 150 base pairs of DNA associated with an octamer comprising two each of histones H2A, H2B, H3 and H4; and linker DNA which extends between nucleosome cores. For the purposes of the present disclosure, the term "chromatin" is meant to encompass all types of cellular nucleoprotein, both prokaryotic and eukaryotic.

[0096] The term "gene of interest," refers to a nucleic acid sequence comprising a nucleotide sequence which includes a transcription unit, and which can be transcribed and translated into a protein. Using the methods and/or assay of the present invention the expression of a gene of interest may be interrupted or silenced as a result of perturbation of a chromosomal contact in the cell.

[0097] Chromosomal contact may be perturbed by means of by interfering with a region of chromatin or DNA in a cell, resulting in the abrogation of the transcriptional activity of a gene of interest. Transcriptional activity may be abrogated, stopped, prevented or decreased as a result of recruitment of proteins involved in DNA repair to a site of a double stranded break in the chromatin or DNA in a cell. As a result of the recruitment of these proteins chromosomal contact may be prevented. Alternatively, chromosomal contact may be perturbed by a protein which interferes with the transcriptional machinery of a cell as a result of being "parked" at a site of chromosomal contact and thus occluding chromosomal contact or blocking the assembly of transcription machinery at the site where the protein is parked, a non-limiting example of such a protein would include a deactivated Cas9 protein, which when bound to the chromatin or DNA obstructs chromosomal contact. Typically chromosomal contact is disrupted or prevent by the introduction of a double stranded break in the DNA.

[0098] Chromosomal contact may also be disrupted as a result of the enhancement of mobility of a region of chromatin or DNA which is in the vicinity of a double stranded break. Increased mobility of the region of chromatin or DNA results in a reduction in the capacity of a gene loop to engage in chromosomal contact, as a result the transcriptional activity of the gene of interest will be negatively affected.

[0099] Chromosomal contact may further be affected by the loss of structural integrity of a gene loop as a result of a double stranded DNA break, which results in the prevention of chromosomal contact, and consequently the disruption of transcriptional activity.

[0100] The terms "protein," "peptide" or "polypeptide" refers to any chain of two or more amino acids, including naturally occurring or non-naturally occurring amino acids or amino acid analogues, irrespective of post-translational modification (e.g., glycosylation or phosphorylation).

[0101] The term "recombinant" means that something has been recombined. When used with reference to a nucleic acid construct the term refers to a molecule that comprises nucleic acid sequences that are joined together or produced by means of molecular biological techniques. The term "recombinant" when used with reference to a protein or polypeptide refers to a protein or polypeptide molecule which is expressed from a recombinant nucleic acid construct created by means of molecular biological techniques. Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Accordingly, a recombinant nucleic acid construct indicates that the nucleic acid molecule has been manipulated using genetic engineering. Recombinant nucleic acid constructs may be introduced into a host cell by transformation. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species.

[0102] The present invention relates to a single cell assay to silence gene expression through the discrete targeting (by cutting) of gene regulatory elements engaged in chromosomal contact. It will be appreciated that the cell used in the assay may be selected from a eukaryotic or prokaryotic cell. It will be further appreciated that a eukaryotic cell may include, without limitation, a cell selected from a diploid or polyploid cell line, a diploid or polyploid oncogenic cell, a primary cell, stem cell, pluripotent stem cell or tissue sample or any other eukaryotic cell.

[0103] It will be appreciated by those of skill in the art that the fluorescent moiety used in the methods of the invention may include fluorophore selected from the group consisting of the Alexafluor family of dyes, FAM, TET or CAL FluorGold 540, HEX or JOE, VICB, CAL Fluor Orange 560A; Cy3C or NEDB, Quasar 570A, Oyster 556D; TMR or CAL Fluor Red 590A; ROX or LC red 610E, CAL FLuor Red 610A; Texas red or LC red 610E, CAL Fluor Red 610A; LC red 640E or CAL Fluor Red 635A; Cy5C or LC red 670E, Quasar 670A, Oyster 645D; LC red 705E or Cy5.5C or 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), fluorescein, anthranilamide, coumarin, and terbium chelates.

[0104] Major regulatory elements include; (I) enhancers, (II) sites within chromatin loops that engage in intra- or interchromosomal contact in multigene complexes and (III) regulatory sites within chromatin loops that determine loop structure. Each of the different types of regulatory elements is expanded below.

[0105] Enhancer-Promoter Interactions (FIG. 1)

[0106] Enhancers are DNA elements that regulate the activity of specific gene promoters. Enhancers may be separated from the genes they regulate by large genomic distances. Enhancers are brought in close proximity to target genes by chromatin looping. Genome-wide chromatin interactome studies reveal that enhancer-promoter interactions are pervasive, and may occur in cis even over large genomic distances, or in trans across different chromosomes.

[0107] Enhancer-promoter interactions are the primary means in which cell-specific gene expression is achieved. Enhancers contain binding sites for transcription factors, chromatin remodelers and transcriptional co-activators. Enhancers with large numbers of regulatory protein binding sites (transcription factors, chromatin remodelers, co-activators), are termed "super-enhancers".

[0108] Super-enhancers have been shown to be more sensitive to small changes in factor concentration than those with small numbers of binding sites. Super-enhancer regulated genes may be identified by identifying genes located with the same TAD. Super-enhancer regulated genes have been shown to have important roles in embryonic stem cell identity. Super-enhancer regulated genes have been shown to be more highly expressed than genes regulated by typical enhancers.

[0109] A remarkable recent observation is the transcription of a novel class of non-coding RNAs, including enhancer RNA (eRNA), activating RNAs and long non coding RNAs (IncRNA), arising from enhancer loci throughout the genome, including the chemokine locus. eRNAs are enriched for H3K4me1 and H3K27Ac chromatin marks, whilst IncRNAs possess, H3K4me3 and H3K36me3 chromatin marks. Through their interaction with promoters, cohesin and the mediator protein complex, eRNAs have been proposed to regulate chromatin looping.

[0110] Recent studies have used a combination of predictive polymer modelling followed by imaging and deletion of structural DNA regulatory elements such as CTCF/cohesin in cells, to ascertain variation in the internal structure of TADs between single cells. These studies have revealed the fluctuating structural environment within a TAD and the tremendous heterogeneity in TAD configurations between single cells that could be linked to transcriptional activity. Though DNA regulatory elements are identical in all cells, eRNA activity is highly tissue specific. eRNAs are speculated to play key roles in organizing TAD structure, though this remains functionally uninterrogated.

[0111] A well-characterized example of enhancer-promoter interactions is the well-established model between the locus control region (LCR) and the promoter of the .beta.-globin gene. These LCR-mediated chromosomal interactions have been shown to result in variability in .beta.-globin genes transcript levels, or variegated gene expression, across the population (Noordermeer et al., 2011). In cells unable to form this enhancer-promoter loop, forceful tethering of Ldb1, a transcription factor thought to mediate the LCR-.beta.-globin interaction, to the .beta.-globin promoter showed that formation of the LCR-.beta.-globin loop underlies transcriptional activation (Deng et al., 2012). Therefore, preventing enhancer-promoter interactions will have a significant impact on gene expression. To date, no assay exists to discretely perturb enhancer elements to silence gene expression.

[0112] Regulatory Sites within Chromatin Loops that Determine Loop Structure (FIG. 2)

[0113] CTCF, a sequence-specific DNA-binding protein, binds to consensus sites (CS) that frequently flank genes. At CS sites, the multiprotein cohesin `ring-like` complex (including the Smc1-Smc3 heterodimer, Rad21 and Scc3/SA1/SA2) is loaded onto chromatin by Nipbl. The mediator complex (a multiprotein complex composed of over 30 proteins) may also be recruited to CTCF and/or cohesin occupied chromatin to stabilize loop topology, and regulate transcription initiation and elongation.

[0114] Studies to identify the architectural proteins shaping the three dimensional genome have revealed CCTC-binding factor (CTCF), Mediator and cohesin to have widespread and specific roles across the genome. Consistent with the above-mentioned role in enhancer-promoter interactions, Mediator and cohesin were found to specifically bridge short-range, cell-type-distinct interactions. On the other hand, long-range interactions were found to be bridged by CTCF and cohesin. Boundaries of TADs are enriched in binding sites for CTCF and cohesin complex, implicating their importance in maintaining domain integrity and loop-mediated transcription. Experiments in cells containing a deletion that spans the boundary between the Xist and Tsix TADs in the X-chromosome inactivation center, directly demonstrated that the loss of boundary had led to the partial fusion of the adjacent TADs and the formation of new and ectopic contacts, causing long-range transcriptional misregulation (Dixon et al., 2012). Furthermore, depletion of CTCF and cohesin has revealed that these factors contribute differentially to domain organization and transcriptional regulation. Particularly, disruption of cohesin reduces local chromatin interactions although TADs remain intact, whereas depletion of CTCF leads to a reduction in local intradomain interactions but also to an increase in interdomain interactions. In each case, different classes of genes are misregulated, indicating that each factor has a distinct capacity in chromatin organization and gene regulation.

[0115] Knockout studies of these structural chromatin proteins (mediator, cohesin, CTCF) reveal their role in regulating transcription. However, the interpretation of these studies is complicated by the global alterations in gene expression that inevitably occurs. Preventing chromatin remodeling and structural proteins from binding to their target sites will have significant impact on gene expression of related genes. To date, no assay exists to discretely perturb sites where chromatin structural proteins bind, and monitor associated gene expression at a single cell level.

[0116] Intra- or Interchromosomal Contact in Multigene Complexes (FIG. 3)

[0117] Within a TAD, or at the interface between TADs, chromosomal looping may also bring co-regulated genes into close proximity to permit chromosomal contact. These interactions are proposed to occur at discrete foci of active, hyper-phosphorylated RNA Pol II that have been referred to as multigene complexes or transcription factories.

[0118] Sites within a chromatin or DNA loop which engage in intra-chromosomal or inter-chromosomal contact may include both coding and noncoding regions, including, but not limited to, introns, exons and 3' un-translated regions.

[0119] Numerous studies have demonstrated that loop-mediated contact between coregulated genes coincides with alterations in the transcription of interacting genes (Papantonis et al., 2010, Li et al., 2012). Over 95% of genes engaged in promoter-mediated chromosomal contact in multigene complexes are associated with transcriptional activity. Therefore, chromosomal contact between coregulated genes in multigene complexes appears to be a critical component of transcriptional regulation.

[0120] The present inventors developed a novel single cell microscopy-based assay, to directly address the role of loop-mediated contact on the transcription of interacting genes. Using this assay, they demonstrated that chromosomal contact plays a central role in supporting transcription of co-regulated genes. Therefore, preventing loop-mediated contact will have a significant impact on the gene expression of interacting genes in a multigene complex. This is the first assay described to date, that enables the discrete perturbation of interacting genes in a multigene complex to silence expression.

[0121] The present invention provides a means to enhance the study of cellular processes through the discrete perturbation of chromatin regulatory elements that are engaged in chromosomal contact.

[0122] The invention provides a means for the discrete perturbation of chromatin regulatory elements resulting in the abrogation of transcription that can be applied in translational medicine. For example through the precise delivery of the nuclease to the correct cell type and location. Or through the manipulation ex vivo of patient cells with the nuclease that are then autologously transplanted into the patient with the nuclease modification having been conducted in the patient cells.

[0123] The perturbation is induced by site-specific nucleases (including meganucleases, zinc fingers (ZF), transcription activator-like effectors (TALE), the Type II CRISPR system (clustered regularly interspaced short palindromic repeats, BUD nucleases) which are designed to induce a double strand break (DSB) within the regulatory site (Li et al., 2011).

[0124] Typically, these site-specific nucleases are used in `cut and paste` experiments, whereby the cell's own repair responses, nonhomologous end-joining (NHEJ) or homologous recombination (HR), are exploited to repair the DSB. This is the first description of the use of these site-specific nucleases for the purpose of disrupting chromosomal contact.

[0125] Through the use of a catalytically inactive Cas9 coupled to a repressor domain, the CRISPRi system has been used as an efficient means to silence gene expression at a population level. However, the CRISPRi approach fails to reveal cell-to-cell variability. Further, as this approach only silences at the level of transcription, it is unable to perturb chromosomal interactions between regulatory elements.

[0126] Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells. Site-specific nucleases are delivered into live cells by transfection (including microporation, electroporation, lipid transfection, calcium phosphate transfection) and endogenously expressed.

[0127] Cells for use for the assay encompass any type of eukaryotic cell, including but not limited to primary and induced pluripotent stem cell lines.

[0128] The DSB, or site of disruption, is detected at a single cell level by immunofluorescent staining of a factor of the DSB repair process.

[0129] The disruption caused by the DSB may serve to do one of the following: (I) A large number of other proteins involved in the repair process are recruited to the DSB site. Therefore, one possibility could be that chromosomal contacts between genes or gene elements may still occur, but are "bridged" via the repair complex. This may occlude the ability of gene loops to "deliver" Pol II to other interacting genes. (II) DSB induction has been shown to enhance the mobility of damaged chromatin. Therefore, due to increased movement, the probability that the disrupted gene loop will be able to interact with other loci is significantly reduced. (III) Alternatively, it is unknown how the DSB, and associated repair factors, influence gene topology. Therefore, the DSB may prevent loop-mediated contact by destabilizing or collapsing the gene loop.

[0130] In parallel to the detection of the DSB by immunofluorescent staining of a factor of the DSB repair process, transcriptional activity of: (i) genes of interest, or (ii) interacting genes in the multigene complex, are detected using intronic single molecule RNA fluorescent in situ hybridization (smFISH). As introns are typically excised and degraded co-transcriptionally, intronic FISH foci represent the transcriptional start site (TSS).

[0131] The following examples are offered by way of illustration and not by way of limitation.

Example 1

[0132] Cell Culture

[0133] Early passage HUVECs from pooled donors (Lonza) were grown to .about.80% confluence in Endothelial Basal Medium-2 (EGM-2) with supplements (Lonza), serum-starved (18 hr) in EGM-2+0.5% FBS, and treated with TNF.alpha. (10 ng/ml; Sigma) for up to 30 mins. Prior to transfection cells were grown in antibiotic free EGM-2.

[0134] TALEN Design

[0135] Software developed by the Bogdanove laboratory was used to identify TALEN candidate binding sites (Doyle et al., 2012). Left and right TALENs were designed to contain 18 full monomer repeats, which target a 20 bp sequence, where the first and last bases are specified by the thymine at the N terminus, and the 0.5 repeat, respectively. To facilitate FokI dimerization, the left and right TALEN target sites were chosen with a spacer of 16-19 bp. For the pDT TALEN vector, the SAMD4A left and right arms were cloned into the pBI_CMV1 bidirectional promoter vector (Clontech). The left TALEN was cloned into MCS1 of pBI_CMV1 (MluI and HindIII) and the right TALEN was cloned into MCS2 of pBI_CMV1 (EcoRI and BglII).

[0136] TALEN Binding Sites and Spacer Regions

[0137] The SAMD4A TALEN recognition sequences (for both pcDNA and pDT vectors) are: left TALEN 5'-TCC ACG TTT ATA AAT AGC TG-3' (SEQ ID NO:1) and right TALEN 5'-CAC TGG GGT GTG GAA GCA TA-3' (SEQ ID NO:2), with a 16 bp spacer. The TNFAIP2 TALEN recognition sequences are: left TALEN 5'-TTC GCG GCC CAC CTG GCC GC-3' (SEQ ID NO:3) and right TALEN 5'-CTG TGC GAG CGC GAC ACC TA-3' (SEQ ID NO:4), with a 16 bp spacer. The SLC6A5 TALEN recognition sequences are: left TALEN 5'-TTG TCC CTT TAA AAC TTG AA-3' (SEQ ID NO:5) and right TALEN 5'-TTA TCA AAC TTG TAT TAT CA-3' (SEQ ID NO:6), with a 17 bp spacer. The BMP4 TALEN recognition sequences are: left TALEN 5'-TGC AGC GCC ACA GTC CCC GG-3' (SEQ ID NO:7) and right TALEN 5'-CAA CCG TTC AGA GGT CCC CAG-3' (SEQ ID NO:8), with a 19 bp spacer.

[0138] TALEN Synthesis

[0139] TALENs were generated using the protocol by Sanjana et al. 2012. Briefly, specific primers were used to amplify monomers from each of four plasmids to add the appropriate adapters (each plasmid corresponding to one RVD; NI=A, HD=C, NG=T and NN=A/G). Monomers generated by PCR were then purified by gel extraction and DNA concentration was normalized to create a monomer library. In the first Golden Gate reaction step, monomers were simultaneously digested with the type IIS restriction enzyme BsmBI and ligated to generate circularized hexamers. Non-hexamers were removed by exonuclease treatment. Hexamers were then amplified by PCR, gel purified and the DNA concentration between samples was normalized using the Qubit High Sensitivity DNA quantification kit (Life Technologies). In the second Golden Gate step, hexamers were simultaneously digested with BsaI, and ligated into the appropriate TALEN cloning backbone (corresponding to one of the four different bases targeted by the 0.5 repeat) to generate the final TALEN expression construct. Colony PCR was used on E. coli transformants to identify successful TALEN clones. HUVECs were then microporated with the respective TALENs using the Neon.RTM. Transfection System (Life Technologies) according to manufacturers instructions. Nuclease activity was assessed by the surveyor assay and the consistent co-localization of DNA FISH with double strand breaks, as assessed by H2A.X immunofluorescence (Keogh et al., 2005).

[0140] Surveyor Assay

[0141] Genomic DNA of transfected cells was extracted using QuickExtract DNA extraction solution (Epicenter). The genomic region encompassing the nuclease target sites in human SAMD4A, TNFAIP2, SLC6A5 or BMP4 were PCR amplified and amplicons cleaned up with QIAquick PCR Purification Kit (Qiagen). For SAMD4A the primers were 5'-TGA GGG AGA TTC CAT TGA GC-3' (SEQ ID NO:9) and 5'-GGA AAA AGT GCT GCT CCA AC-3' (SEQ ID NO:10). For TNFAIP2 the primers were 5'-TGC AGG ACA GAC TCA GGA CA-3' (SEQ ID NO:11) and 5'-ATT TGG GTT GAG CAT TCC AC-3' (SEQ ID NO:12). For SLC6A5 the primers were 5'-TGA TTT AAC CCC CTC CTT CC-3' (SEQ ID NO:13) and 5'-CTT TAG GAG CCA CAG CCA AC-3' (SEQ ID NO:14). For BMP4 the primers were 5'-CTA GTA CCT CCG CAC GTG GT-3' (SEQ ID NO:15) and 5'-TCC AGC ACC ACT ATT GGA AA-3' (SEQ ID NO:16). The DNA fragments were then subjected to digestion with mismatch-sensitive T7 endonuclease I (T7E1; New England BioLabs). For the T7E1 assay, DNA was denatured at 95.degree. C. for 5 min, slowly cooled down to room temperature to allow for formation of heteroduplex DNA, treated with 5 U of T7E1 for 1 hr min at 37.degree. C., and then analyzed by 1.2% agarose gel electrophoresis.

[0142] RNA FISH Probes

[0143] RNA FISH was performed according to the protocol by Raj et al. 2008 using 48 20-mer probes (Biosearch) targeting the following genes: SAMD4A (probes set i=.about.1.5 kbp into intron 1, probe set ii=.about.34 kbp into intron 1, TNFAIP2 (intron2), SLC6A5 (intron1), RCOR1 (intron1) and 32 20-mer probes targeting eGFP (Table 1). Each 20-mer bares a 3'-amino-modifier C6-dT. The amino group was subsequently conjugated to the following NHS-ester dyes: ATTO-488, ATTO-565, ATTO-647N (ATTO-TEC) or Alexa Fluor 647 (Invitrogen). Briefly, oligonucleotide probes were ethanol precipitated and resuspended in 0.1 M sodium tetraborate (Sigma). Approximately 0.3 mg of the NHS-ester dye (ATTO-TEC) was dissolved in dimethyl sulphoxide (Sigma). The dye solution was added to the probe solution and incubated overnight in the dark at 37.degree. C. Following conjugation reaction, the probes were ethanol precipitated overnight, and resuspended in 0.1 M Triethyl ammonium (TEA, Sigma). Conjugated probes were separated and purified to enrich for dye-conjugated probes by reverse phase HPLC on a C18 column.

TABLE-US-00001 TABLE 1 Sequences of RNA FISH probes SAMD4A 1.5 kbp into intron 1 SAMD4A 34 kbp into intron 1 Sequence Sequence Identity Identity Probe Sequence (5'-3') Number Probe Sequence (5'-3') Number ATTCATGCTACCGTAGCTAC SEQ ID NO: 25 CCCCACCCAATATGAATGAA SEQ ID NO: 73 GGAAACTGCATGAGAGAAAA SEQ ID NO: 26 CTGTTGAGACTCATTCTTGT SEQ ID NO: 74 GACCTGACTTACTTATTTCC SEQ ID NO: 27 GCTTTAAAATGTCGCTGGGT SEQ ID NO: 75 CCAGAAAATCCAGACTCTAC SEQ ID NO: 28 GTGATGGCCCTTTGATTTAC SEQ ID NO: 76 GGAGGAAGGAAAAAACACAC SEQ ID NO: 29 TAATCCTGCCATTCCTAAGC SEQ ID NO: 77 AGCCACGTTGCCCAAAAGAA SEQ ID NO: 30 CCACTGTTTAACCGCAACAA SEQ ID NO: 78 CATGTGTTGCTGAAATCCAG SEQ ID NO: 31 AATTCCAACATTGGAGCCTC SEQ ID NO: 79 ATCCAAGCTTGGCTTCTGAA SEQ ID NO: 32 CACACAGACACAAGATTCAA SEQ ID NO: 80 TTCATGTACTCCTCACACAG SEQ ID NO: 33 GGTTTCTGCCTAAAACAGCA SEQ ID NO: 81 CCAGAATATCTGTGGGGAAA SEQ ID NO: 34 ATTCTCTCCAAGTCATCAGC SEQ ID NO: 82 TTCTGAAGACGAAGCTCTAA SEQ ID NO: 35 TAAGAGTGTCTGAGGGGTTA SEQ ID NO: 83 TTACTAGTCTCTAGCGTCAC SEQ ID NO: 36 GTCTAGCCAGACTGTAGTTT SEQ ID NO: 84 GCAGTAAGCTTAACCGCATT SEQ ID NO: 37 CTCACCCCAAAGTCTTTAAG SEQ ID NO: 85 CAAGATCCGTATCAATATGG SEQ ID NO: 38 TCTAAGGACTTAGCACCATC SEQ ID NO: 86 CCTTTCTCCAAGACCCTTTT SEQ ID NO: 39 GCCTTCGGAGTTTTCTTCTT SEQ ID NO: 87 AAGTAACCCACTTCATGCCT SEQ ID NO: 40 TTTCTCTCCAACATGGTTGC SEQ ID NO: 88 GCAGGGTAATATGAAACGAT SEQ ID NO: 41 GTTTCTACATTGCTTCCCTC SEQ ID NO: 89 CATACTAGTTGAGGTGTCTG SEQ ID NO: 42 TTTCTGTCCTGCTTTCTCCT SEQ ID NO: 90 GCTGGACCTTTCGACTATAT SEQ ID NO: 43 AAAGAAGTCTCTGGTACCAG SEQ ID NO: 91 CCACGCTAGCAAATAGGAAA SEQ ID NO: 44 GTCTAGCCCAAGTGAGAGAA SEQ ID NO: 92 CCTACCCTCCAGGATATAAT SEQ ID NO: 45 TAAACCAGAAGCCTGTCTCT SEQ ID NO: 93 TCACAACCATCAGACTTTCC SEQ ID NO: 46 ATCTAATCCATGGCCAGCCA SEQ ID NO: 94 GCAGCAGCATGAACTAAAGA SEQ ID NO: 47 AAAGGGCATTGATTCCACAG SEQ ID NO: 95 TAAACACTGGGGACTCTGTT SEQ ID NO: 48 ACTTGGAAAGATGTCCCAAG SEQ ID NO: 96 ACCCTGCATTCTTTTCTCTG SEQ ID NO: 49 CCCAGTTAATCCTAAGGAAA SEQ ID NO: 97 AACATGGAACAGCTGGAAGA SEQ ID NO: 50 GCTTCTTCTTTGTACAGTGA SEQ ID NO: 98 ACCTTGTCATCAAATGGCAG SEQ ID NO: 51 CAGTCCTCTAGCAATGGAAA SEQ ID NO: 99 ACTCACTTTAGTGTCTCCCA SEQ ID NO: 52 GTGTGTGTGTGTGTGTGTAT SEQ ID NO: 100 TCGCTTCTTGCTGCTCTGAA SEQ ID NO: 53 TTTCGTGTGTGTGTGTGTGT SEQ ID NO: 101 ATACTAGGGAGGAGGAATGA SEQ ID NO: 54 AAAGCCACTGTCTGTGCTAT SEQ ID NO: 102 TGTTTTCACCATCGTGCACA SEQ ID NO: 55 AAAGGAAGTATCTTCATCCC SEQ ID NO: 103 ATATGGAAGCATCCCATTCT SEQ ID NO: 56 CAGAAGAGACTAGAACAGAC SEQ ID NO: 104 CACTTCCACCCTATGATTCT SEQ ID NO: 57 ACTAGTTTCTGTTCCACGCA SEQ ID NO: 105 AACTGTGAAGATTTCCAGCG SEQ ID NO: 58 CTTCATCTCCTTAAGCTAGC SEQ ID NO: 106 AGTCCTGTCTGGTCAGGAAA SEQ ID NO: 59 CATGTCATTCCCACAAGAAC SEQ ID NO: 107 TTGGCCATGCAGGATCTTTC SEQ ID NO: 60 TGAGAAGGTTTCTGTCCATG SEQ ID NO: 108 ATGGAATCTCCCTCAATGTG SEQ ID NO: 61 AAAGCTTCCCAATTCTAGCC SEQ ID NO: 109 AAACGTGGAGCTCAACCAAA SEQ ID NO: 62 CCGAAGGTGAATGTCTTAAG SEQ ID NO: 110 AATTATGCTTCCACACCCCA SEQ ID NO: 63 ATCTCCTAATGCTATCCCTC SEQ ID NO: 111 AGAGGGTGGATCATCAGTTA SEQ ID NO: 64 CTGCACCCATTAACTCATCA SEQ ID NO: 112 CCGCTGAAATTAAGGAAGGA SEQ ID NO: 65 TATACATGTGCCGTGTTGGT SEQ ID NO: 113 AAAACCGAGCAGGTAAACCA SEQ ID NO: 66 CAACGTGCAGGTTTGTTACA SEQ ID NO: 114 CCTTGTTTGTCAAACCTGAG SEQ ID NO: 67 GAGAGAATGTAAGAAGGCGA SEQ ID NO: 115 GCGTAAATCCTCTGCAATCT SEQ ID NO: 68 TTTAGCCTGACAGGCATGAA SEQ ID NO: 116 CCAGCACTGAAATCAAGCAT SEQ ID NO: 69 GTGCACTTGAAGTAACAAGG SEQ ID NO: 117 AGATGAGTTTGAACAGTCCC SEQ ID NO: 70 TGATGTCCTTGATACTGGTC SEQ ID NO: 118 GTGCTGCTCCAACATTTGAA SEQ ID NO: 71 GCTGCATTTCCAAGAAGATC SEQ ID NO: 119 CTCCATTACTCAAAGGGAAA SEQ ID NO: 72 ATTCAGTAGGTCTGCAGTGT SEQ ID NO: 120 TNFAIP2 intron 2 SLC6A5 intron 1 Sequence Sequence Identity Identity Probe Sequence (5'-3') Number Probe Sequence (5'-3') Number TACCTGCCCTATCACCCCTC SEQ ID NO: 121 CCCTTTCCTCTTGAAAGAAC SEQ ID NO: 169 ACCTTCCTCTCACCTTCTCC SEQ ID NO: 122 AACGAATCTGCTTTCCCTGT SEQ ID NO: 170 AGAGGACATGCTCAGGAGCA SEQ ID NO: 123 GCACAGACATCTCCAAAAGA SEQ ID NO: 171 TAGCTCTGCTCAGCTCCAGA SEQ ID NO: 124 AGAATTGTTCTCCTCGTCGC SEQ ID NO: 172 ATCACTTGGAACTCTCCTGC SEQ ID NO: 125 AGTACAAATACCTAGGGCTG SEQ ID NO: 173 TAACAAGACACGTGGCCCTA SEQ ID NO: 126 ACAGCCTAGGAAACCTCTTT SEQ ID NO: 174 ACAGGGTAACCCTACACAAT SEQ ID NO: 127 TCTTTAGGAGCCACAGCCAA SEQ ID NO: 175 TGTACTCAGCAGGGGTACCA SEQ ID NO: 128 TTTTCAGCACCGAGGACAGT SEQ ID NO: 176 ATCTATGGAGTCAAGGGACG SEQ ID NO: 129 AGAGCAACTCGCCTCTGTAC SEQ ID NO: 177 ATGCAGACCGTGCGAATGAC SEQ ID NO: 130 TTTGTCTCAGTAGCCTCTAG SEQ ID NO: 178 ACACTGCAGTAAGCCTCTCT SEQ ID NO: 131 AAATCTAGGTACCTCGGCTT SEQ ID NO: 179 ACAATAACAGCCCCTGGACT SEQ ID NO: 132 GCAGAATAGAACTCCTCGAT SEQ ID NO: 180 TTCACACTAGGATCTGGGGC SEQ ID NO: 133 TCTAACCCCCTTTCACAAAC SEQ ID NO: 181 CTCCAGGCTTCCTTCCTAAA SEQ ID NO: 134 TCCTATTATCTTCGCCTCCA SEQ ID NO: 182 ACACTGCCCACACTGTCTAT SEQ ID NO: 135 GCTTCCTAAACATTAGCACC SEQ ID NO: 183 TTCAACACGAACTGAGCCCA SEQ ID NO: 136 TCCCCTCGTGTTATCTTTCA SEQ ID NO: 184 ATCCTTCTGCCAGAGGCTGA SEQ ID NO: 137 CGGTGGTTTTTCTATCCACT SEQ ID NO: 185 CTCTCCACTGGACCCTTTCT SEQ ID NO: 138 CGCTTTTTCCCAATTCACTT SEQ ID NO: 186 TATGCCCTGCTGACTCCTCT SEQ ID NO: 139 GAGTACCAAAACTCATGACT SEQ ID NO: 187 TTATCTCTCCCGTGGCCCCT SEQ ID NO: 140 CTTCACAACTCTAAGCTTGC SEQ ID NO: 188 AGCAAGCGGGAACTGGCTCT SEQ ID NO: 141 CGTAACTATTCACAGGAGTC SEQ ID NO: 189 CATTTCCTGGTGAGTCAGGA SEQ ID NO: 142 CTTCTTCTTCCTCTTCTTCT SEQ ID NO: 190 TCACTGGGACAGAAAGACTG SEQ ID NO: 143 TCTTCTTCTTCTTCTTCTTC SEQ ID NO: 191 TTGCCTGTTGCTAACCCCAG SEQ ID NO: 144 CTTCTTCTTCTTCTTCTTCT SEQ ID NO: 192 TCCATGCCCAGCTTGGCTTT SEQ ID NO: 145 GGTCTTCTTCTTCTTCTTCT SEQ ID NO: 193 ACACAGGCTTCAACGATGCC SEQ ID NO: 146 CCCAGACATGGAAAAGAATA SEQ ID NO: 194 CTCCGTTCCCCACATTTCTG SEQ ID NO: 147 CAACTCGTATTCACTTCCAG SEQ ID NO: 195 CAATCCAGATGCAGCTGTGT SEQ ID NO: 148 GACACAGAGTAGTAACTAGA SEQ ID NO: 196 AAATGACCTTCTCTCTGCCC SEQ ID NO: 149 CCACAAAAAAACCTACAACG SEQ ID NO: 197 CTTTGTCTGCCTAGAGGTTT SEQ ID NO: 150 CAGGTAGCAAAGACAAGGTT SEQ ID NO: 198 AGCCCTGGCACTTCCTAAGG SEQ ID NO: 151 TGAGGCCAAATTGGTTGAGT SEQ ID NO: 199 ATCTTGGCTCACTGTCCTGG SEQ ID NO: 152 ACAAAGCTGATGCCCTGGCA SEQ ID NO: 200 CCCCAAACTGCTCCACAGAC SEQ ID NO: 153 TTTTCCCCCATAGACAAGCT SEQ ID NO: 201 TAGGCCAGAATGGGCAGGAA SEQ ID NO: 154 CATTCAACAATGCACACTGC SEQ ID NO: 202 AGGTCTTGGGCATCTCACCA SEQ ID NO: 155 TTTTCCGCCCCATTTCCTTT SEQ ID NO: 203 ACTCACAGCAGCCCTGGAAT SEQ ID NO: 156 CCATCACCACTGTAGAAAGA SEQ ID NO: 204 TGTCACCCAGATAAAACCCT SEQ ID NO: 157 TTGGATTCCACATCACAGCT SEQ ID NO: 205 TTCTTCCTGCTCCAAGACTA SEQ ID NO: 158 AAATAGCCCTGGAGTCATGA SEQ ID NO: 206 GAGCCACCTTTAAGATCTGA SEQ ID NO: 159 TGTAAGGGTAAAGGGGGAAT SEQ ID NO: 207 AATTCCTTCTTCACCCAGCA SEQ ID NO: 160 GGATGAACTAAGAAAAGAGG SEQ ID NO: 208 TGCTCCTTCCCTCACCTCCA SEQ ID NO: 161 CTAATATTCCCTCCCTAGTG SEQ ID NO: 209 TCACTGCAAAAGCCTCCTCC SEQ ID NO: 162 CCCGTTTCATCAATAACCAG SEQ ID NO: 210 AAATCAGCAGCCTCATGCCA SEQ ID NO: 163 GGCTGCAATCAGTATTTCTC SEQ ID NO: 211 TAGGTCCTGCTCCAAAATAG SEQ ID NO: 164 GGGAAGCAAATTGGAATCTG SEQ ID NO: 212 ATCATGCCTCCTCTGACTCT SEQ ID NO: 165 TCAGGTCCAGACACAATATC SEQ ID NO: 213 TTGGAACAGAAACTCTGAAG SEQ ID NO: 166 TAGATATGGAGACGTTCGCA SEQ ID NO: 214 TCCTCCATCTACTTAGTTTG SEQ ID NO: 167 CAAAACAAAGTCACAACAGC SEQ ID NO: 215 GGTCCCCATTTCAGATGATG SEQ ID NO: 168 CAAACACAATGTCAAGTTCG SEQ ID NO: 216 RCOR1 intron 1 eGFP intron 1 Sequence Sequence Identity Identity Probe Sequence (5'-3') Number Probe Sequence (5'-3') Number CAAAAAGAAAGTTGTCGGCG SEQ ID NO: 217 TCCTCGCCCTTGCTCACCAT SEQ ID NO: 265 GTTTGCAGAACACTCGTGTG SEQ ID NO: 218 ATGGGCACCACCCCGGTGAA SEQ ID NO: 266 GCTCACCTGGACGCAGGGAC SEQ ID NO: 219 TTACGTCGCCGTCCAGCTCG SEQ ID NO: 267 GCAGGACACTAACTCTCCGG SEQ ID NO: 220 GACACGCTGAACTTGTGGCC SEQ ID NO: 268 GGACCTCCTAAGTCCGGGGC SEQ ID NO: 221 GGCATCGCCCTCGCCCTCGC SEQ ID NO: 269 GTTCCGAGAAAGGGGTCTCT SEQ ID NO: 222 TCAGGGTCAGCTTGCCGTAG SEQ ID NO: 270 CAGTCAGGAGCCAGCAAGAG SEQ ID NO: 223 TTGCCGGTGGTGCAGATGAA SEQ ID NO: 271 GTTCTTCTACTCGCCTTCTG SEQ ID NO: 224 GGTGGGCCAGGGCACGGGCA SEQ ID NO: 272 AGGTCCTAAAATCCAACTGC SEQ ID NO: 225 CGTAGGTGAAGGTGGTCACG SEQ ID NO: 273 GGCTAAAGCTTCTGATGCTT SEQ ID NO: 226 TAGCGGCTGAAGCACTGCAC SEQ ID NO: 274 CTAGGGTGTAGGGCTTTCTT SEQ ID NO: 227 GTGCTGCTTCATGTGGTCGG SEQ ID NO: 275 CCAGGGAACTAAGTGTTCCA SEQ ID NO: 228 GCATGGCGGACTTGAAGAAG SEQ ID NO: 276 TTGCTTCTACACAGAATAGC SEQ ID NO: 229 CGCTCCTGGACGTAGCCTTC SEQ ID NO: 277 TGACGAATTCCTTTGTCTGA SEQ ID NO: 230 GTCGTCCTTGAAGAAGATGG SEQ ID NO: 278 TGGCTAGAATCTCATGCATT SEQ ID NO: 231 CGGCGCGGGTCTTGTAGTTG SEQ ID NO: 279 AAAGACAGTCTCATACAGAC SEQ ID NO: 232 GTGTCGCCCTCGAACTTCAC SEQ ID NO: 280 CTCCAGCTTTACAAGATGCT SEQ ID NO: 233 TTCAGCTCGATGCGGTTCAC SEQ ID NO: 281 TCCAACCATGGAGGATTGTT SEQ ID NO: 234 CGTCCTCCTTGAAGTCGATG SEQ ID NO: 282 TGGTATAACCCACATACAGA SEQ ID NO: 235 AGCTTGTGCCCCAGGATGTT SEQ ID NO: 283

TTTCCAAGGAAAAAACATGT SEQ ID NO: 236 GTGGCTGTTGTAGTTGTACT SEQ ID NO: 284 ATTGCAATTTTCTGCAGTCT SEQ ID NO: 237 TGTCGGCCATGATATAGACG SEQ ID NO: 285 CGCCTTTTTCAAGGACTAGA SEQ ID NO: 238 ACCTTGATGCCGTTCTTCTG SEQ ID NO: 286 TTAATAACAGCTTGAGAGCA SEQ ID NO: 239 TGTTGTGGCGGATCTTGAAG SEQ ID NO: 287 CTTCAAAGTGCAGTTTAGTA SEQ ID NO: 240 AGCTGCACGCTGCCGTCCTC SEQ ID NO: 288 GTCCAAGAGCTCACCTAGGA SEQ ID NO: 241 TGTTCTGCTGGTAGTGGTCG SEQ ID NO: 289 AGGAAGTCATTTATTTCCAG SEQ ID NO: 242 CACGGGGCCGTCGCCGATGG SEQ ID NO: 290 TCAAAATTTAGAAGGCCAAT SEQ ID NO: 243 CTCAGGTAGTGGTTGTCGGG SEQ ID NO: 291 AAGAAACACAAACGCGAGTA SEQ ID NO: 244 CTTTGCTCAGGGCGGACTGG SEQ ID NO: 292 AGAGGAAACTATCAAAAACA SEQ ID NO: 245 TGATCGCGCTTCTCGTTGGG SEQ ID NO: 293 CCTGTCACTCATAAACGAGA SEQ ID NO: 246 CACGAACTCCAGCAGGACCA SEQ ID NO: 294 ATAGCCTGGCCACCTAGTTC SEQ ID NO: 247 CGAGAGTGATCCCGGCGGCG SEQ ID NO: 295 AGGAGCCAAGCATACAAGAG SEQ ID NO: 248 CACTTGTACAGCTCGTCCAT SEQ ID NO: 296 CTGGTGGCCAACCAAGGAAC SEQ ID NO: 249 GTAACCAAAAAACAGTATGC SEQ ID NO: 250 CAAAATGAAGACAGGGTCAA SEQ ID NO: 251 AAGCAAAGATAGACTTGACC SEQ ID NO: 252 ACAAGCTTTATGTTCTGTGT SEQ ID NO: 253 CTCAAAGATTTGTGGAAGTC SEQ ID NO: 254 TGTTGTTGACAAATATGCCC SEQ ID NO: 255 TCGATGAATGTCAGAAATCT SEQ ID NO: 256 AATGCAGAGCTGATGTCAAT SEQ ID NO: 257 GTATAACTCCAAAGAAGTCA SEQ ID NO: 258 AACAAAAAGCCCAAAGACAG SEQ ID NO: 259 GCTGGTCACTAGCAAGTTTA SEQ ID NO : 260 GACTATGCAGCAAAACAGTT SEQ ID NO: 261 ATACTCAACCAATTGGCAGC SEQ ID NO: 262 GCTTAGGTTGGCAAAACAAG SEQ ID NO: 263 GTAAAGTCTCAATTTTCTGC SEQ ID NO: 264

[0144] Immuno-RNA RNA FISH

[0145] For each experiment, early passage HUVECs on coverslips were grown to .about.80% confluence, treated with TNF.alpha., fixed in 3.7% formaldehyde for 10 min at room temperature, then washed three times in PBS. Cells were permeabilized in ice-cold 90% methanol for ten minutes, then washed twice with PBS and incubated in blocking buffer (1% BSA/PBS) for 30 minutes at room temperature on an orbital shaker. Cells were then incubated in 1.degree. antibody solution (diluted in 1% BSA/PBS) for 1 hr. Double strand breaks were detected with rabbit polyclonal anti-phospho-histone H2A/X (Ser139) (Sigma). Goat polyclonal anti-SAMD4A C-15 (SantaCruz), mouse monoclonal anti-TNFAIP2 F-6 (SantaCruz) and goat polyclonal anti-SLC6A5/GLYT2 N-20 (SantaCruz) were used to detect SAMD4A, TNFAIP2 and SLC6A5 proteins respectively. Coverslips were then washed 5 times with wash buffer (0.05% Tween-20/PBS), following incubation with secondary antibodies conjugated to either Atto-488 or Atto-565 for 1 hr. Coverslips were then washed 5 times with wash buffer (0.05% Tween-20/PBS), and post-fixed with 3.7% formaldehyde/PBS for 10 minutes at room temperature, followed by further permeabilization in 70% ethanol overnight. For RNA FISH detection, coverslips were washed twice in PBS and incubated in wash buffer (10% formamide, 2.times.SCC-1.times.SCC is 0.15 M NaCl plus 0.015 M sodium citrate) for 5 min. Cells were then hybridized overnight in a humidified chamber at 37.degree. C. in 50 .mu.l of Hyb buffer (10% dextran sulfate, 1 .mu.g/.mu.l E. coli tRNA, 2 mM Vanadyl ribonucleoside complex, 0.02% RNAse-free BSA, 10% formamide) combined with 50 ng of single molecule FISH probes. Coverslips were then washed 3.times. (30 min each on the orbital shaker) in wash buffer (10% formamide, 2.times.SCC). Cells were then incubated in equilibration buffer (0.4% glucose, 2.times.SCC) for 5 min and counter stained with 1 .mu.g/ml DAPI (4',6-diamidino-2-phenylindole; Life Technologies). Coverslips were mounted in glox buffer (3.7 .mu.g/.mu.l glucose oxidase, 1 U catalase) and imaged.

[0146] Image Acquisition and Processing

[0147] Cells were imaged on a custom built Nikon Ti Eclipse widefield TIRF microscope using a 100.times.N.A. 1.49 Nikon Apochromat TIRF oil immersion objective. Imaging was done using mercury lamp illumination through the appropriate filter sets at low camera gain in each of the fluorescent channels using an Andor iXion897 EMCCD camera cooled to -80.degree. C. The microscope was controlled using pmanager open source microscope management software (NIH and UCSF, USA). A 20 ms exposure time was used for DAPI. Exposure times ranged from 200 to 500 ms for other dyes. Each field of view was captured as a series of images acquired on multiple focal planes through the samples, across a range of 2-10 .mu.m in the axial plane. A 0.2 .mu.m piezo step-size was used for these z-stacks. Chromatic aberration was verified before image capture by alignment of Focal Check Fluorescent Microspheres (Molecular Probes). Signal intensities were measured using Fiji. The contrast of pictures shown was adjusted to fit a 16 bit grey scale. To facilitate the comparison between different fields of view on the same coverslip, IDV values were normalized relative to the intensity of fluorescent beads.

[0148] DNA FISH Probes

[0149] DNA FISH probes were constructed using either one of two methods. In the first method, they were constructed from BAC clones RP11-299D5 (SAMD4A), RP11-1102D6 (TNFAIP2) and RP11-207A15 (SLC6A5) (Empire Genomics). Using the FISH Tag DNA Multicolor Kit (Molecular Probes), these clones were nick-translated to incorporate aminoallyl-dUTP, followed by dye-labeling of the amine-modified DNA. Alexa Fluor 488, 555 or 647 dye was incorporated. Alternatively, a PCR-based protocol was followed to construct "high definition" FISH probes (Bienko et al., 2012). Each locus was amplified from genomic DNA using twenty pairs of PCR primers obtained from the van Oudenaarden FISH probe database. Amplified DNA was pooled, purified and labeled with a fluorescent dye (FISHBright 495, 550 or 647) using the FISHBright Nucleic Acid Labeling Kit (Kreatech).

TABLE-US-00002 TABLE 2 Sequences of PCR primers used to construct ''high definition'' FISH probes SAMD4A HD DNA FISH probe primers Sequence Primer Name Primer Sequence (5'-3') Identity Number SAMD4A5'1F CTCCAAAAACCTGACTTCGC SEQ ID NO: 297 SAMD4A5'1R TGTCCTAATTGGCCTGATCC SEQ ID NO: 298 SAMD4A5'2F CCACCCAACATGCTCTCTTT SEQ ID NO: 299 SAMD4A5'2R CCATCATTTACCAGCCCTGT SEQ ID NO: 300 SAMD4A5'3F GCTCATTGCCCTGCAGATAG SEQ ID NO: 301 SAMD4A5'3R TGATCCTCCAGTAACCGCAT SEQ ID NO: 302 SAMD4A5'4F AGACCTGCATTTCCTTCCCT SEQ ID NO: 303 SAMD4A5'4R ACTGTGGCTCTGTTGCCTTT SEQ ID NO: 304 SAMD4A5'5F CCCTCAGACCCACTTCACAT SEQ ID NO: 305 SAMD4A5'5R CCCTTCTGCACGTCTCTTCT SEQ ID NO: 306 SAMD4A5'6F CAGTGTCTATTCAGAAACCACAGAA SEQ ID NO: 307 SAMD4A5'6R CGTGAGTTCAGGCCTACTGTC SEQ ID NO: 308 SAMD4A5'7F CATGCAGAGAAGATCACCATT SEQ ID NO: 309 SAMD4A5'7R GGGCCAAGTCCTCAAGATAA SEQ ID NO: 310 SAMD4A5'8F TGGCAATTATCCATTGTCATTT SEQ ID NO: 311 SAMD4A5'8R TGTTTAAGAGGATGCCTGGG SEQ ID NO: 312 SAMD4A5'9F TTAGTAATCTTTGGAACATCTGAACA SEQ ID NO: 313 SAMD4A5'9R GGCACTGAATACACGATGGG SEQ ID NO: 314 SAMD4A5'10F TTATTGCCTAACCCAGTGCC SEQ ID NO: 315 SAMD4A5'10R TCCTATGTCAACCTGGACCC SEQ ID NO: 316 SAMD4A5'11F TCAAGCCTTTCTGCCTCTACA SEQ ID NO: 317 SAMD4A5'11R GACACGTGATAGCACCTAACCA SEQ ID NO: 318 SAMD4A5'12F ACATATGCCGGAGTTGGAAA SEQ ID NO: 319 SAMD4A5'12R TCTGGGCTATCTCCGTGATT SEQ ID NO: 320 SAMD4A5'13F GCACTCTCCAGATCCAGGTG SEQ ID NO: 321 SAMD4A5'13R CTGGCTGAGTCCCACTCTTC SEQ ID NO: 322 SAMD4A5'14F AACCACATTCCCAAGGACAA SEQ ID NO: 323 SAMD4A5'14R GGTCGGGTAGAAGGAACCTC SEQ ID NO: 324 SAMD4A5'15F GTCCCCACTTAGGAATCCGT SEQ ID NO: 325 SAMD4A5'15R GCTGCTGCCTCCAGTGTC SEQ ID NO: 326 SAMD4A5'16F ACTCGAAGCACCGCACTC SEQ ID NO: 327 SAMD4A5'16R GAGGGGACCCCCAGTGAC SEQ ID NO: 328 SAMD4A5'17F AGCAGGCTTCTCGCTGTTAC SEQ ID NO: 329 SAMD4A5'17R GGAGAGAAAGAGCTGCAGTGA SEQ ID NO: 330 SAMD4A5'18F ACCACCGGAACGTAACTGAA SEQ ID NO: 331 SAMD4A5'18R CGAAACATCATGGTTAGGGG SEQ ID NO: 332 SAMD4A5'19F GGATTTTGCCCACTGTGAAC SEQ ID NO: 333 SAMD4A5'19R CGGGCATCTATCATGTGTTG SEQ ID NO: 334 SAMD4A5'20F TTCCTATTTGCTAGCGTGGG SEQ ID NO: 335 SAMD4A5'20R ATGGAACAGCTGGAAGAGGA SEQ ID NO: 336 TNFAIP2 HD DNA FISH probe primers Sequence Primer Name Primer Sequence (5'-3) Identity Number TNFAIP23'1F GGGCAAAGCAATCAATAACC SEQ ID NO: 337 TNFAIP23'1R TTCTCCCCTACCTGCCCTAT SEQ ID NO: 338 TNFAIP23'2F TACCCCTGCTGAGTACAGCC SEQ ID NO: 339 TNFAIP23'2R ACCTCCAGGCTTCCTTCCTA SEQ ID NO: 340 TNFAIP23'3F GAGGAGTCAGCAGGGCATAG SEQ ID NO: 341 TNFAIP23'3R AGCTTGGCTTTAGTTGCCTG SEQ ID NO: 342 TNFAIP23'4F AGGGGCAGAGAGAAGGTCAT SEQ ID NO: 343 TNFAIP23'4R AGGTCTTGGGCATCTCACC SEQ ID NO: 344 TNFAIP23'5F TTTGGAGCAGGACCTAATGG SEQ ID NO: 345 TNFAIP23'5R ACCACTCAAGCTAGAGCCCA SEQ ID NO: 346 TNFAIP23'6F CTGCCTGCAGTGACATCATC SEQ ID NO: 347 TNFAIP23'6R CTGCCCATGTCCTGTCTGT SEQ ID NO: 348 TNFAIP23'7F GCCAATGTGAGGGAGTTGAT SEQ ID NO: 349 TNFAIP23'7R CATGGGTATGCACACAGGAC SEQ ID NO: 350 TNFAIP23'8F CTGGACTTGGGCTCACAGAT SEQ ID NO: 351 TNFAIP23'8R GCTGAGTGGGAAACAACTCC SEQ ID NO: 352 TNFAIP23'9F TCTGACCTCCACCAGGATTC SEQ ID NO: 353 TNFAIP23'9R TAGGAGTGAGCGTGTGGTTG SEQ ID NO: 354 TNFAIP23'10F AGATGGGCTGGTACCCTCTT SEQ ID NO: 355 TNFAIP23'10R GTAGCTGGCGAAACCAGAAG SEQ ID NO: 356 TNFAIP23'11F TTCTGGAGAGAGGCAAGCAG SEQ ID NO: 357 TNFAIP23'11R TCCTCACACATGTTGCTGGT SEQ ID NO: 358 TNFAIP23'12F CAAGCACACAGGCAGATGTT SEQ ID NO: 359 TNFAIP23'12R CAATGGCTCCCCATTCTCTA SEQ ID NO: 360 TNFAIP23'13F GGATGTCCATGGAGCAGAAT SEQ ID NO: 361 TNFAIP23'13R CTTTGCTTCTGCCACTCCTC SEQ ID NO: 362 TNFAIP23'14F ATTCAGACACAGCCCAGTCC SEQ ID NO: 363 TNFAIP23'14R CCCCACTTTACACCCTGCTA SEQ ID NO: 364 TNFAIP23'15F GGGTGACCTTGGATAAGGGT SEQ ID NO: 365 TNFAIP23'15R GAGACAGGAGAGGCAGGATG SEQ ID NO: 366 TNFAIP23'16F ATCATCCAACTCAGCAAGGG SEQ ID NO: 367 TNFAIP23'16R GGGCTTAGAGAGGCACCAG SEQ ID NO: 368 TNFAIP23'17F ATTGAGGTGGCCACTTATGC SEQ ID NO: 369 TNFAIP23'17R TCGTAGTGCTGTGGTGAAGG SEQ ID NO: 370 TNFAIP23'18F TGGCCCACACTCTTAGCTTT SEQ ID NO: 371 TNFAIP23'18R AACATGTCAGAGGACCCAGC SEQ ID NO: 372 TNFAIP23'19F AAGGGAAGCCAGGTCTCAGT SEQ ID NO: 373 TNFAIP23'19R CTTCCCACCCCTTAGGTCTC SEQ ID NO: 374 TNFAIP23'20F AGTAGGGGTGTGGGTGACAG SEQ ID NO: 375 TNFAIP23'20R GCCACAGGAAAAGCTAACCA SEQ ID NO: 376 SLC6A5 HD DNA FISH probe primers Sequence Primer Name Primer Sequence (5'-3') Identity Number SLC3'1F GTTGGCTGTGGCTCCTAAAG SEQ ID NO: 377 SLC3'1R CGCACTCCTCTCGTCTAACC SEQ ID NO: 378 SLC3'2F CCTTGTCTTTGCTACCTGGC SEQ ID NO: 379 SLC3'2R TCCCCATCACCACTGTAGAA SEQ ID NO: 380 SLC3'3F CCATTCCCCCTTTACCCTTA SEQ ID NO: 381 SLC3'3R GCAACTCAGGTCCAGACACA SEQ ID NO: 382 SLC3'4F ATAAACTGCCAGCCAACAGC SEQ ID NO: 383 SLC3'4R GAGTTTGCAAGACCCCACTC SEQ ID NO: 384 SLC3'5F CGCTGCACTGTAAGATCCCT SEQ ID NO: 385 SLC3'5R AATGCAACCTGCTTACCTGG SEQ ID NO: 386 SLC3'6F ACGTATGCTGATGGGGAAAC SEQ ID NO: 387 SLC3'6R CAACTCTTTCGTGGATCTGGA SEQ ID NO: 388 SLC3'7F AGGTGGGTTTACCTGAGGCT SEQ ID NO: 389 SLC3'7R GTGGGTTTTTCACGCCTCTA SEQ ID NO: 390 SLC3'8F ATCTCCCACTTGCCTTTCCT SEQ ID NO: 391 SLC3'8R CCATTCCTAATCCCACCCTT SEQ ID NO: 392 SLC3'9F TGTCCTGCACACCCTGTAGA SEQ ID NO: 393 SLC3'9R GGTCCTGGAATGCCTCACTA SEQ ID NO: 394 SLC3'10F GCATCTTCGTGTGAGCTTGA SEQ ID NO: 395 SLC3'10R TGGGAAGAGACAAGCCATCT SEQ ID NO: 396 SLC3'11F GAAAGTAAGGCAGGGGCTCT SEQ ID NO: 397 SLC3'11R CCCAACCCCTTACCAAGAAT SEQ ID NO: 398 SLC3'12F TACAGGTTCAAGGTTTGGGG SEQ ID NO: 399 SLC3'12R ATGCATCAGCAAGCAGGAC SEQ ID NO: 400 SLC3'13F GCCTGCTTGTGGACCTACTC SEQ ID NO: 401 SLC3'13R AGAGCGGAAAAGCCATACCT SEQ ID NO: 402 SLC3'14F ACAGCCTCCTTAGGCTCCAG SEQ ID NO: 403 SLC3'14R AACACTTGACCGCTAGCACC SEQ ID NO: 404 SLC3'15F GTTGAGGCTGTGCTGTCAAA SEQ ID NO: 405 SLC3'15R AGCCGATGCAAGCCTAACTA SEQ ID NO: 406 SLC3'16F CACTGCTTCACCCCATAGGT SEQ ID NO: 407 SLC3'16R ATGGCAAATGCTCTGAGGTC SEQ ID NO: 408 SLC3'17F ACACGAATAGAGGCACCCAG SEQ ID NO: 409 SLC3'17R AACCCTGAGATCTGCCCC SEQ ID NO: 410 SLC3'18F TTTTCAGGAGCAGAGGAGGA SEQ ID NO: 411 SLC3'18R AGCGGAGGGAAAACTGATCT SEQ ID NO: 412 SLC3'19F CCTCAAAGAATGCTGAAGGG SEQ ID NO: 413

SLC3'19R CCTTCTGAGTCCTCCCACAG SEQ ID NO: 414 SLC3'20F TGACCTTGAAGGATGGAAGG SEQ ID NO: 415 SLC3'20R CCCAGGACCCAAAATCTTCT SEQ ID NO: 416

[0150] Immuno-DNA FISH

[0151] Immunofluorescence was performed as above and cells were post-fixed with 3.7% formaldehyde/PBS for 20 min at room temperature. DNA FISH hybridization was performed as follows. Cells were washed twice in PBS for 5 min each and permeabilized in 0.5% Triton X-100 for 10 minutes. Cells were treated with 10 U of RNAseA in 2.times.SCC for 1 hr at 37.degree. C. Cells were then washed twice with 2.times.SSC, and dehydrated in 70%, 85% and 100% ethanol for 2 min each. After air-drying, cells were denatured in 70% formamide, 2.times.SCC for 3 min at 73.degree. C., and dehydrated in ice-cold 70%, 85% and 100% ethanol for 2 min each. Cells were then hybridized overnight in a humidified chamber at 37.degree. C. in 10 .mu.l of Hyb buffer (10% dextran sulfate, 50% formamide, 4.times.SSC) combined with 30 ng of DNA FISH probes that were freshly denatured for 5 min at 72.degree. C. and cooled on ice. Coverslips were then washed 3.times. (5 min each on the orbital shaker) in each of three wash buffers-1) 50% formamide, 2.times. SCC, 2) 1.times.SSC and 3) 4.times.SSC, 0.01% Tween 20. Cells were counter stained with DAPI, mounted in glox buffer (3.7 .mu.g/.mu.l glucose oxidase, 1 U catalase) and imaged.

[0152] Repair Construct

[0153] The repair construct pCR-SAMD4A-IRES-GFP includes two segments, SAMD4A 5' Intron 1 and SAMD4A 3' Intron 1 of 564 bp and 563 bp respectively which are homologous to regions either side of the SAMD4A intron 1 TALEN cleavage site. Segment SAMD4A 5' Intron 1 includes 3' splice acceptor site and 52 bp of 3' intronic sequence derived from a modified chimeric intron used in the mammalian expression vector pCI-neo (Promega, Wis., USA). Similarly, segment SAMD4A 3' Intron 1 includes a 5' splice donor site and 52 bp of 5' intronic sequence derived from a modified chimeric intron used in the mammalian expression vector pCI-neo (Promega, Wis., USA). The resulting artificial exon, which includes an IRES-GFP cassette, allowed for independent in situ GFP expression off the SAMD4A promoter following repair (homologous recombination) and splicing. The primers used to amplify the 564 bp 5' SAMD4A fragment were as follows: SAMD4A 5' Intron F: 5'-TGC TGC TGC AGG AGG GTG-3' (SEQ ID NO:17)

TABLE-US-00003 SAMD4A 5' Intron R: (SEQ ID NO: 18) 5'-GAT CCC TAG CAC CTG TGG AGA GAA AGG CAA ACT GGA TGT CAG TAA GAC CAA TAG GTG CCT ATC ATG GCC CTC CAG CTA TTT ATA AAC GTG-3'

(Bold=splice acceptor site; Underlined=intron extension from a modified chimeric intron derived from pCI-neo [Promega]). The primers used to amplify the 563 bp 3' SAMD4A fragment were as follows:

TABLE-US-00004 SAMD4A 3' Intron F: (SEQ ID NO: 19) 5'-GAT CCC TAG CAG GTA AGT ATC AAG GTT ACA AGA CAG GTT TAA GGA GAC CAA TAG AAA CTG GGG CCT CCT TCA CTG GGG TGT G-3'

(Bold=splice donor site; Underlined=intron extension from a modified chimeric intron derived from pCI-neo [Promega]); SAMD4A 3' Intron R: 5'-CTT TTG TAT ATC TAC ATC ATT TAG CAG CAT G-3' (SEQ ID NO:20). Both amplicons were digested with NheI (NEB) and ligated. Full length ligated products were selected by PCR with primers SAMD4A 5' intron F and SAMD4A 3' intron R and cloned into the TA vector pCR.2.1 (Invitrogen) to produce pCR-SAMD4A-5/3. The (HCV) IRES-GFP sequence was amplified from pHIV7-IRES-GFP (a gift from John Rossi) and cloned into the NheI site of pCR-SAMD4A-5/3 to produce pCR-SAMD4A-IRES-GFP. The primers used for amplification include: IRES F: 5'-GAT CGC TAG CCC CCC TAA CGT TAC TGG CCG-3' (SEQ ID NO:21) and GFP R: 5'-GAT CGC TAG CGG ATC CTC ACT TGT ACA GCT CGT CCA TGC C-3' (SEQ ID NO:22). To generate double stranded PCR product harboring the 20 and 18 bp homologous arms, the following primers were used: 5'-CCA CAC CCC AGT GAA GGA G-3' (SEQ ID NO:23) and 5'-TAA ATA GCT GGA GGG CCA TG-3' (SEQ ID NO:24). The PCR product was purified by QIAquick PCR Purification Kit (Qiagen) prior to transfection.

[0154] The Transcriptional Response of Co-Regulated Genes in a Multigene Complex is Asymmetric.

[0155] TNF.alpha. has been shown to rapidly and synchronously shape the transcriptional response in early passage human umbilical vein endothelial cells (HUVECs) by systematically inducing the formation of a large variety of different multigene complexes (Papantonis et al., 2012). Interacting genes in TNF.alpha.-induced multigene complexes are activated by NF-.kappa.B (Papantonis et al., 2010). The SAMD4A, TNFAIP2 and SLC6A5 genes associate in a NF-.kappa.Bdependent multigene complex (FIG. 4), which has been extensively characterized by 3C, 4C (3C capture-on-chip), tiling microarray and FISH, and thus is an ideal model to interrogate the role chromosomal contacts play in cotranscription at a single cell level (Papantonis et al., 2010; Papantonis et al., 2012). HUVECs were arrested in the GO phase of the cell cycle by serum deprivation. Importantly, the transcription of SAMD4A, TNFAIP2 and SLC6A5 is rapidly induced 10 mins post TNF.alpha. stimulation (FIG. 5; Papantonis et al., 2010). Concurrent to their expression at 10 mins, prior 3C data including our own (data not shown) (Papantonis et al., 2010), indicate that chromosomal contacts between these genes occur at sites .about.1.5 kbp downstream of the TSS (FIG. 5). To interrogate whether the formation of these contacts are associated with cotranscription, we designed RNA FISH probes to target the intronic sites where these contacts occur 10 mins post TNF.alpha. treatment (FIG. 5). As these introns are typically spliced and degraded co-transcriptionally, these probes label the transcriptional start site (TSS). Each set of gene-specific RNA FISH intronic probes was conjugated to spectrally distinct fluorophores. By simultaneously performing RNA FISH on the 3 NF-.kappa.B regulated genes, we were able to investigate the frequency of these interactions across a population of cells. Consistent with previous studies (Papantonis et al., 2010, Papantonis et al, 2012), analysis of overlapping RNA FISH foci revealed co-localized foci in only a fraction of all alleles in the total population (.about.5%) (FIG. 5). Chromosomal interactions between transcribed genes have been shown to occur at discrete foci of active RNA Pol II, as well as at nuclear speckles. Overlapping SAMD4A, TNFAIP2 and/or SLC6A5 RNA FISH foci consistently co-localize with the active, poised form of RNA Pol II (phosphorylated at Ser5) (FIG. 6). Collectively, these data suggest that the co-transcription of these genes at RNA pol II foci may only occur in a small fraction of the HUVEC population. Chromosomes in primary cells occupy distinct territories in the nucleus. As HUVECs are primary cells, they always display two spatially distinct DNA FISH foci, both before and after TNF.alpha. treatment (FIG. 7). Within territories, chromosomes are segregated further into topologically associating domains (TADs). As a consequence of this compartmentalization, interacting genes in multigene complexes may be confined within much smaller genomic neighborhoods. By simultaneously performing DNA FISH on these 3 co-regulated genes, we were able to investigate whether the DNA of these 3 genes were in close vicinity prior to activation by TNF.alpha.. Interestingly, analysis of overlapping DNA FISH foci revealed proximal foci in both the unstimulated and the TNF.alpha.-treated HUVEC population (FIG. 8). This data suggest that in a fraction of the HUVEC population, the DNA of the SAMD4A, TNFAIP2 and SLC6A5 genes may be constrained within a TAD, prior to TNF.alpha. induction.

[0156] Through an enrichment of chromosomal interactions in TADs, and higher levels of transcription, "jackpot" cells may contribute to variable, or stochastic effects, in gene expression (Noordermeer et al., 2011). As these NF-.kappa.B regulated genes respond stochastically to TNF.alpha. (Papantonis et al., 2010), we assessed the monoallelic and biallelic expression of the three genes. Nine of the 33 possible phenotypes were observed in the majority of the population (.about.84%) (FIG. 9). With the exception of cells displaying no foci (.about.29%), all cells in this category displayed either a single or dual SAMD4A foci. With respect to the individual expression of each of the 3 genes, not all cells respond similarly to TNF.alpha., with approximately half of the alleles expressing SAMD4A, a lower proportion expressing TNFAIP2 and approximately one fifth expressing SLC6A5 (FIG. 9). As chromosomal contact occurs between these 3 genes (FIG. 5; Papantonis et al., 2010), the asymmetric transcriptional response of these genes to TNF.alpha. suggests a hierarchical regulation in the assembly of this multigene complex. Supporting this hypothesis, TNFAIP2 transcription occurs predominantly when SAMD4A is also transcribed (.about.86%) and SLC6A5 transcription occurs mainly when SAMD4A (.about.92%), or both SAMD4A and TNFAIP2 are transcribed (.about.62%) (FIG. 9).

[0157] As SAMD4A, TNFAIP2 and SLC6A5 are not always co-expressed (FIG. 9), we also represented the co-localization frequencies relative to cells coexpressing SAMD4A, TNFAIP2 and/or SLC6A5 (FIG. 10). Allelic coexpression analyses between the 3 genes revealed that co-expression of SAMD4A and TNFAIP2, as well as triple expression of the 3 genes was most prevalent across the population (.about.12% and .about.14% respectively) (FIG. 10). SLC6A5 was rarely expressed (.about.4%) in the absence of TNFAIP2 expression at the corresponding allele (FIG. 10). Moreover, co-transcription of TNFAIP2 and SLC6A5 in the absence of SAMD4A transcription was extremely rare (.about.1%) (FIG. 10). This provides further evidence for a relationship between the transcriptional status of SAMD4A and the transcriptional activation of TNFAIP2 and SLC6A5. With respect to coexpressed alleles, SAMD4A and TNFAIP2 were co-localized at .about.26% of coexpressed alleles (3% in the total population (T/P)), whilst SAMD4A, TNFAIP2 and SLC6A5 were co-localized at .about.40% of all co-expressed alleles (5% in the T/P) (FIG. 10). Collectively, these data suggest a hierarchical mode of regulation between these genes, in which chromosomal contact favors cotranscriptional activation.

[0158] Visualization of TALEN-Mediated Gene Loop Disruption

[0159] Hierarchical regulation dependent on chromosomal contact was revealed in the GREB1 multigene complex (Li et al., 2012). qPCR analysis revealed that siRNAs targeting ER.alpha. disrupted not only GREB1 transcription, but were sufficient to cause more than a 2 to 4-fold reduction in the transcription of interacting members. siRNA approaches cannot be applied to the TNF.alpha.-induced multigene complex, as these 3 genes are transcriptionally activated by NF-.kappa.B (Papantonis et al., 2010). Toward this end, we developed a single cell assay allowing the discrete disruption of individual gene loops at the sites of chromosomal contacts (FIG. 5). In parallel, to decrypt the role of a single individual gene loop on co-transcription in the same multigene complex, we visualized transcriptional activity of other genes in the multigene complex using highly sensitive RNA FISH (FIG. 5). We constructed our microscopy-based assay upon TALENs, the orthogonal and robust, well established genome editing tool derived from TAL effectors of Xanthomonas sp. (Christian et al., 2010, Li et al., 2011). Within the TALE structure, a central repeat domain mediates highly specific DNA recognition (Boch et al., 2009; Christian et al., 2010). Fusion of this domain to FokI endonuclease enables TALENs to induce site-specific double strand breaks (DSB) (Christian et al., 2010, Li et al., 2011). As the TALENs used in this study induce a DSB at the approximate site of chromosomal contact that occurs 10 mins post TNF.alpha. treatment (FIG. 11), we reasoned that should loop-mediated contact be required for co-transcription, the DSB would serve to rupture chromosomal contact between these genes. To ascertain whether the TALEN had successfully disrupted a gene loop, we stained for induction of a DSB using histone variant H2A.X (FIGS. 12 and 13). Rapidly phosphorylated at Ser139 within minutes following DNA rupture, this modification persists throughout the entire DNA repair process. To initially test our TALEN system and its ability to disrupt a single gene loop and concurrent assembly of the multigene complex, we introduced into HUVECs, by high efficiency microporation, our TALEN vectors that were able to disrupt the gene loop of the longest gene in the multigene complex, SAMD4A. We used H2A.X staining of DSBs to carefully establish a time course of TALEN activity (also a measure of transfection efficiency), noting that nuclease activity was first evident after .about.6 hrs, and was sustained until .about.48 hrs (FIG. 14). We chose to assay for nuclease activity after 24 hrs, as there were high levels of DSBs and low levels of cytotoxicity at this time point. Discrete sites of H2A.X phosphorylation were evident in .about.60% of cells 24 hrs post transfection, with a higher proportion of these cells displaying single allelic DSBs, with fewer cells displaying dual allelic DSBs (FIG. 13). The high specificity of the SAMD4A TALEN was demonstrated by the consistent co-localization of SAMD4A DNA with the DSB, visualized by DNA FISH (FIG. 15). TALEN cleavage efficiency was further supported by the results of "surveyor assays" (FIGS. 16, 21, 32 and 42). Thus, we were able to assay uniquely for the disruption of the SAMD4A gene loop in its native environment at a single cell level.

[0160] TALEN-Mediated Disruption of a Gene Loop Abrogates RNA and Protein Expression

[0161] Upon stimulation by TNF.alpha., RNA pol II engages the SAMD4A promoter, triggering a wave of transcription that propagates down the gene. RNA FISH tiling array analysis revealed that the transcriptional cycle takes approximately 85 min (Papantonis et al., 2010). Accordingly, RNA transcribed .about.1.5 kbp downstream of the TSS appears within 10 mins (probe set i, FIG. 17), and .about.34 kbp into intron 1 after 30 mins post TNF.alpha. stimulation (probe set ii, FIG. 18). Importantly, the DSB induced by the SAMD4A TALEN occurs at a site in between these two regions where different sets of intronic RNA FISH probes bind. HUVECs were dual transfected for 24 hrs with the SAMD4A TALENs and exposed to TNF.alpha. for 10 mins. This recapitulated SAMD4A transcriptional activity, allowing for the first .about.1.5 kb of SAMD4A to be transcribed. By using RNA FISH to monitor intronic RNA transcription 10 mins post TNF.alpha. stimulation, SAMD4A transcription was frequently detected (.about.42%), either overlapping or in close proximity to the DSB (FIG. 17). Thus, these data indicate that despite disrupting the SAMD4A gene loop, the DSB did not appear to influence the ability of RNA Pol II to access the SAMD4A promoter, permitting transcriptional initiation and elongation up to the DSB. The half-life of SAMD4A intronic RNA transcripts transcribed at 10 mins is between 3 and 6 mins. Consequently, RNA transcripts transcribed downstream of the TSS at 10 mins, have degraded by the 30 mins time point. In a separate experiment, dual-transfected HUVECs were stimulated with TNF.alpha. for 30 mins to allow for the first .about.34 kbp of SAMD4A to be transcribed (FIG. 18). Notably, the TALEN-induced perturbation was able to abrogate transcription of SAMD4A in this region, as no transcription of the intronic region of SAMD4A was ever evident beyond the DSB (FIG. 18)

[0162] It has been published that DNA lesions alter RNA Polymerase II occupancy distal to DSBs, resulting in transcriptional inhibition (Shanbhag et al., 2010). This effect is mediated by ATM kinase activity, which results in the abrogation of Pol II-dependent elongation beyond the DSB. Importantly, ATM-mediated local inhibition of Pol II occurs in cis to DSBs (Shanbhag et al., 2010). Hence, in this study, the local inhibition of Pol II that occurs distal to the DSB, should not affect the intact allele in cells displaying single allelic DSBs.) IF in untransfected cells revealed that TNF.alpha. induction resulted not only in the robust transcription of members of the multigene complex, but also in an increase in protein expression (FIG. 19). Consistent with the ability of the TALEN to abrogate transcription, there was a significant reduction in SAMD4A protein expression in cells harboring dual allelic DSBs as detected by IF, whilst cells harboring single allelic DSBs were still able to express SAMD4A protein presumably from the intact allele (FIG. 19). Overall this robust assay offered an unprecedented perspective into the dynamics of TALEN activity, transcriptional status of the targeted gene and related protein levels at single cell resolution. Importantly, the observed disruption in SAMD4A RNA (FIG. 18) and protein expression (FIG. 19) strengthened our assay, as it validates that the DSB occurs at the intended site of TALEN activity where H2A.X staining occurred.

[0163] TALEN-Mediated Disruption of a Gene Loop Abrogates Expression of Interacting Members

[0164] Satisfied that our TALEN assay was able to discretely disrupt SAMD4A at the site that engages in chromosomal contact, we repeated the prior experiment with an important modification: we used RNA FISH to visualize transcription of two other interacting genes that engage in intra- and inter-chromosomal with the region of SAMD4A that had been disrupted. By simultaneously monitoring SAMD4A gene loop disruption, as well as transcription of additional members of this multigene complex, we were able to interrogate discretely the effect of the disrupted SAMD4A gene loop on the transcriptional status of two other members of the complex.

[0165] Firstly, to exclude the possibility that the DSB was capable of inducing cell cycle arrest, thereby altering global transcription, we designed a TALEN to rupture BMP4, a non-TNF.alpha. responsive gene, located .about.600 kb 5' of SAMD4A on chromosome 14 (FIG. 20). We observed no change in the transcription of any of the three members of the multigene complex relative to the normal TNF.alpha.-induced transcriptional response (FIG. 9). Therefore, inducing a DSB 5' of SAMD4A has no effect on transcription of members of the multigene complex.

[0166] We then monitored the effects of perturbation of the chromatin loop of SAMD4A at its first intron. We observed a significant reduction of transcription of TNFAIP2 and SLC6A5 at the SAMD4A TALEN-induced DSB (FIG. 22). In cells where a single allele of SAMD4A was targeted, virtually all transcription of TNFAIP2 and SLC6A5, as assessed by RNA FISH, was lost at the corresponding allele or DSB, with multigene transcription confined to the intact allele (FIG. 23). Importantly, as the SAMD4A protein is still observed in cells harboring a single allelic DSB (FIG. 18), this observation excludes the possibility that the SAMD4A protein is required for the transcription of TNFAIP2 and SLC6A5.

[0167] Initially, a possible explanation for the transcriptional response observed in FIGS. 20, 22, 26 and 35 could be that the SAMD4A protein is required for the expression of TNFAIP2 and SLC6A5. However, microarray analysis demonstrates that it takes .about.85 mins for RNA Pol II to transcribe the SAMD4A gene. The observed reduction in TNFAIP2 and SLC6A5 expression by the SAMD4A TALEN-induced DSB is observed 10 mins post TNF.alpha. stimulation (FIGS. 22 and 26). No transcription, or chromosomal contact, is observed between these co-regulated genes in unstimulated HUVECs (Papantonis et al., 2010). Therefore, these effects cannot be attributed to the entire SAMD4A transcript, or protein, as neither is present at the 10 mins time point. HUVECs are primary cells with defined chromosomal territories, and therefore always display two spatially distinct DNA FISH foci (FIG. 7). The comparison between cells harboring single allelic DSBs and dual allelic DSBs (FIG. 19) offers a unique perspective on the influence of protein expression in the assembly of the multigene complex. In cells displaying a single allelic break (disrupted SAMD4A transcription at one allele) the following is observed; reduced transcription of two other genes that interact with SAMD4A near to the TALEN-induced DSB (FIG. 23), and translation of SAMD4A protein, presumably from the intact allele (FIG. 19). The reduction of TNFAIP2 and SLC6A5 transcription was also observed in cells harboring dual allelic DBSs for SAMD4A (FIG. 23). Cells displaying dual allelic SAMD4A TALEN induced DSBs also have reduced protein expression (FIG. 19). Collectively, these data clearly show that the protein product of the SAMD4A gene is not required for the expression of TNFAIP2 and SLC6A5. Currently, no other biochemical technique offers the same insight.) Supporting the observed reduction in transcription at single allelic DSBs, in cells where both SAMD4A alleles were targeted, virtually all transcription from TNFAIP2 and SLC6A5 was lost at both alleles (FIGS. 23, 33 and 42). Furthermore, protein levels of TNFAIP2 and SLC6A5 were also severely reduced, as assessed by IF, in cells where both SAMD4A alleles were successfully targeted (FIGS. 24, 34 and 43). Hence, in a manner analogous to GREB1 (Li et al., 2012), SAMD4A appeared to be influencing the transcription of other co-regulated and interacting genes within the same multigene complex.

[0168] We sought to determine whether the loss of transcription of TNFAIP2 was due to the DSB abrogating the transcription of genes located between SAMD4A and TNFAIP2, on chromosome 14. RCOR1 is a gene located 400 kb 5' of TNFAIP2 and displays transcriptional activity comparable to GAPDH (Papantonis et al., 2012). We repeated the TALEN-mediated disruption of SAMD4A gene loop whilst monitoring transcription of RCOR1 (FIG. 25). Transcription remained unaffected at the RCOR1 locus (FIG. 25). This observation concurred with the hypothesis that loop-mediated chromosomal contact of SAMD4A specifically impacts the transcription of other members of the multigene complex but has no effect on genes outside of the complex. This extended to genes on the same chromosome that were interspersed between two genes in the multigene complex.

[0169] We sought to determine whether the presence of other gene loops in the multigene complex were equally required for co-transcription, since all genes in the multigene complex were bound by the NF-.kappa.B transcription factor (Papantonis et al., 2010). We hypothesized that if gene loops were equally required for co-transcription, then the disruption of any other gene loop would have a similar effect to that observed for SAMD4A. Alternatively, an asymmetrical relationship between gene loops infers that the disruption of one gene loop may have no bearing on the transcriptional status of other genes in the complex. To differentiate between these two hypotheses we designed TALENs targeted to TNFAIP2 (FIGS. 27, 28, 29, 30 and 31) and SLC6A5 (FIGS. 36, 37, 38, 39 and 40). The sites for these TALENs were identified by 3C (Papantonis et al., 2010) indicating where the chromosomal contacts occurred between these genes. Using an identical approach to our initial assay, we delivered a TALEN to TNFAIP2 whilst monitoring transcription of SAMD4A and SLC6A5. The TNFAIP2 TALEN targets the site of chromosomal contact (.about.1.5 kbp downstream of the TSS), whilst our RNA FISH probes interrogated a region downstream of this site (FIG. 27). Remarkably, we observed that the disruption of the TNFAIP2 loop had no significant effect on SAMD4A transcription, yet transcription of both TNFAIP2 (FIG. 29) and SLC6A5 was reduced (FIG. 26). Transcriptional loss was observed at either a single or both targeted alleles (FIG. 33). When we repeated the experiment with a TALEN targeting the SLC6A5 gene loop on chromosome 11 (FIG. 36), the hierarchical effect was more pronounced with transcription unaffected in both SAMD4A and TNFAIP2 (FIG. 35 and FIG. 42). However at all successfully targeted single and dual alleles of SLC6A5, transcription was lost (FIG. 38). This result lends credence to the hypothesis that the disrupted site of chromosomal contact precludes the ability of the gene loop to access critical transcriptional machinery due to a requirement for loop-mediated chromosomal contacts. This unique single cell perspective reveals a highly unexpected hierarchical organization within the TNF.alpha.-induced multigene complex, providing the first direct evidence that chromosomal contacts play a central role in supporting transcription and determining the hierarchy.

[0170] Disrupted SAMD4A Gene Loops can be Successfully Repaired

[0171] If the integrity of the gene loop topology and chromosomal contacts were essential for co-transcription, then we hypothesized that restoring the chromosomal contacts would restore transcription of interacting genes in the multigene complex. DSBs are generally repaired in most cells by two highly conserved mechanisms: rapid but error-prone non-homologous end joining (NHEJ), or the slower, but highly precise, homology-directed repair (HDR). We generated a repair construct designed to span the SAMD4A TALEN target site, to exploit HDR to restore SAMD4A gene loop integrity by inserting an exonic eGFP sequence into intron 1 of SAMD4A (FIG. 44). We included an internal ribosome entry sequence (IRES), as well as splice sites flanking the IRES-eGFP, to facilitate the co-transcriptional splicing and independent translation of the repair construct (FIG. 44). Once repaired, through the activation of SAMD4A, TNF.alpha. stimulation would then be able to induce SAMD4A/IRES-eGFP transcription. We used the identification of eGFP positive cells to establish a time course for TNF.alpha. treatment. Due to HDR being rare, and as the IRES-eGFP is under the control of the endogenous SAMD4A promoter, we sought to enhance the efficiency of the repair. We constructed a novel bidirectional TALEN system able to combine both targeting arms of the TALEN into a single bi-directional promoter plasmid (pDT; FIG. 45), to ensure that equimolar quantities of both left and right TALENs were synthesized in the same cell throughout the DSB-induction process. HUVECs dual transfected with the pDT and the repair plasmids demonstrated poor repair efficiency in the transfected cells as evidenced by the lack of eGFP signal (data not shown). To modify the repair construct, we amplified the IRES-eGFP sequence with associated flanking splice sites and included short 5' 20 bp and 3' 18 bp homologous extensions on either side of the SAMD4A DSB to induce efficient HDR (FIG. 45). Notably, eGFP protein signal was observed in .about.1% of HUVECs dual transfected with the pDT plasmid and PCR product for 72 hr and stimulated with TNF.alpha. for 24 hr (FIG. 45). However, eGFP mRNA transcripts, which can only arise following successful HDR, were observed in .about.10% of cells in the population (FIGS. 46 and 47). Notably, eGFP positive or `green` cells coincided with a large number of RNA FISH foci (FIGS. 46 and 47). As the SAMD4A promoter does not constitutively express the IRES-eGFP mRNA, it is reasonable that very few cells in the population would transcribe sufficient IRES-eGFP mRNA to enable the detection of a `green` cell. Therefore, detection of IRES-eGFP mRNA was the more sensitive approach to identifying successful HDR and was used to interrogate the effects of a repaired gene loop.

[0172] Restoration of the SAMD4A Gene Loop Restores Transcription of Interacting Members in a Sequence-Independent Manner

[0173] Satisfied that the repair experiment was fully functional, we sought to investigate whether the re-establishment of contact was sufficient to restore transcription of interacting genes. We stimulated HUVECs for 20 mins with TNF.alpha., recapitulating transcription of the first .about.1.5 kbp of SAMD4A and IRESeGFP. We detected transcription of eGFP using RNA FISH probes binding to its RNA (FIGS. 46 and 47), and related the position of eGFP transcription to members of the multigene complex. Distinct eGFP foci were evident in .about.10% of transfected cells and these foci overlapped with SAMD4A intronic mRNA (FIG. 48). In the instances where we had successful HDR mediated repair, we observed no change in the transcription of any of TNFAIP2 and SLC6A5 relative to the normal TNF.alpha.-induced transcriptional response (FIG. 49). Moreover, there was no significant difference in the co-localization frequencies between eGFP/SAMD4A, TNFAIP2 and SLC6A5 in the repaired and mock-transfected cells (FIG. 49). This result indicates that reestablishment of an intact SAMD4A loop, in a sequence-independent manner, restores chromosomal contacts as well as transcription of TNFAIP2 and SLC6A5 in this multigene complex.

Example 2

[0174] Disrupting the IL8 Enhancer Abrogates IL8 Expression

[0175] Recently, Hi-C studies revealed that the pro-inflammatory chemokines CC Chr.17 (CCL2, CCL7, CCL11) and CXC Chr.4 (IL8, CXCL1, CXCL3, CXCL2) are organized into TADs and engage in chromosomal contact.

[0176] Using intronic smFISH, we were able to show that the CXC chemokines are only induced following TNF induction. Furthermore, the smFISH foci of co-expressed CXC genes always co-localize.

[0177] Deeper bioinformatic analysis of both Hi-C and ChIA-PET data in the CC and CXC TADs identified a large cluster of transcriptional enhancers. At the 5' end of the CXC TAD, we identified a putative `super-enhancer` region, spanning .about.80 kb and forming extensive chromosomal contacts with the proinflammatory genes (unpublished data). Typically, such regions are densely occupied by chromatin regulators over tens of kb. Accordingly, this region is highly enriched for eRNA chromatin marks, H3K4me1 and H3K27Ac. Using the recently published `eRNA encyclopedia`, we identified several eRNAs transcribed from this region in a tissue- and cell-specific manner. Using smFISH, we were able to detect some of these eRNAs across different primary cell types and observed different expression patterns (unpublished data).

[0178] Through their interaction with the mediator complex, eRNAs have been proposed to be the architects of chromatin organization and therefore putatively TADs. We found that depleting Mediator 12 (Med12) abrogates the expression of all the CXC cytokines. We designed TALENs to induce a DSB at various sites within the superenhancer region that was established by ChIA-PET to engage in contact. In one instance the DSB was induced at the site of an eRNA. The DSB, or site of disruption, was detected by immunofluorescent staining of H2A.X pSer139, a factor of the DSB repair process. In parallel to the detection of the DSB, transcriptional activity of interacting genes in the multigene complex was determined using intronic RNA FISH. We observed that perturbing the superenhancer in the CXC TAD silenced all of the pro-inflammatory CXC genes (IL8, CXCL2, CXCL1 and CXCL3).

Example 3

[0179] Disrupting Intra- or Interchromosomal Contact in Multigene Complexes Abrogates the Transcription of Interacting Genes (See FIG. 52)

[0180] TNF.alpha., a major proinflammatory cytokine, is a stimulus that induces the coordinated assembly of coregulated genes in NF-.kappa.B-dependent multigene complexes (Papantonis et al., 2012). SAMD4A, a .about.221 Kb gene on chromosome 14, is rapidly switched on by TNF.alpha. in primary human umbilical vein endothelial cells (HUVEC). 4C analysis reveals that prior to stimulation with TNF.alpha., SAMD4A seldom interacts with other genes.

[0181] After activation by TNF.alpha., SAMD4A interacts with multiple coregulated genes to form a multigene complex. TNFAIP2, a gene located on the same chromosome but .about.50 Mb downstream, and SLC6A5 on chromosome 11, are two well-characterized interacting partners of SAMD4A. siRNA approaches cannot be utilized to interrogate loop-mediated dynamics in the SAMD4A/TNFAIP2/SLC6A5 multigene complex, as all three genes are activated by the same transcription factor. This exposes the necessity to develop functional assays able to discretely alter gene loop topology, without the global ablation of transcriptional regulators.

[0182] We used TALENs to discretely perturb sites within gene loops that are established to engage in chromosomal contact in the well-characterized NF-.kappa.B-regulated multigene complex (Papantonis et al., 2010).

[0183] Using the TALEN single cell assay, we observed that perturbing the SAMD4A gene loop did not alter transcription 5' of the DSB. However, consistent with other studies, we observed silencing of SAMD4A transcription 3' of the DSB. Intriguingly, despite occupying distal genomic locations, the transcription of TNFAIP2 and SLC6A5 were also significantly reduced (FIG. 52(I)). Further, the effect on co-transcription was hierarchical, with the disruption of TNFAIP2 altering SLC6A5 expression, but having no influence on SAMD4A (FIG. 52(II)), whereas perturbing SLC6A5 did not influence SAMD4A or TNFAIP2 transcription (FIG. 52(III)). This suggests a hierarchical assembly between these three genes, whereby TNFAIP2 `collects` RNA Pot II from SAMD4A, which are then able to recruit SLC6A5 to the multigene complex.

Example 4

[0184] Perturbing CTCF Sites Flanking TNFAIP2 Abrogates Transcription

[0185] CTCF, binds to consensus sites that frequently flank genes. TNFAIP2, a .about.11 Kb gene on chromosome 14, is flanked by CTCF consensus binding sites. We used CRISPRs to discretely perturb sites within CTCF consensus binding site (FIG. 53). We observed that perturbing the 3' CTCF binding site with a DSB in the silenced the expression of TNFAIP2.

DISCUSSION

[0186] Here we show that disrupting sites within gene loops that engage in chromosomal contact significantly impacts the transcription of interacting genes in a multigene complex. We revealed this level of gene regulation by implementing a novel single cell strategy that allows discrete perturbation within chromatin loops. Initial evidence for this regulation was our single cell based observation of the hierarchical transcriptional response of these 3 genes to TNF.alpha. induction (FIG. 9). Despite the clear asymmetric transcriptional response, co-localization of RNA FISH foci was only observed in a subset of the population (FIG. 5). The functional relationship between chromosomal contact and co-transcription of interacting members of a multigene complex remains opaque. However, if loop-mediated contact is indeed a prerequisite for co-transcription, the RNA FISH co-localization data suggests that only certain cells in the population may possess the correct spatial arrangements of their chromosomes to permit such regulation (FIG. 5). Such variegated gene expression is suggested to occur in TADs, highly conserved compartments within mammalian chromosome territories (Dixon et al., 2012). We speculate that TADs may ensure robust SAMD4A, TNFAIP2 and SLC6A5 gene regulation by constraining these genes to areas permissible to long-range interactions (FIG. 50). Indeed, analysis of DNA in unstimulated HUVECs revealed that despite occupying distal genomic locations, in a fraction of the HUVEC population, the SAMD4A, TNFAIP2 and SLC6A5 DNA are in close proximity prior to TNF.alpha. induction (FIG. 8). The enrichment of chromosomal interactions in these `jackpot` cells, may contribute to hierarchical SAMD4A, TNFAIP2 and SLC6A5 gene expression. However, current biochemical and imaging technologies lack the spatiotemporal resolution to interrogate whether loop-mediated co-transcription between these genes can only occur in these `jackpot` cells. Alternatively, given more time, whether these chromosomal interactions may occur in most cells across the population.

[0187] Chromosomal translocations are natural perturbations in chromosome structure, which alter the spatial positioning of DNA within chromosome territories. Interestingly, these discrete perturbations in chromosome structure do not only influence genes located near to the breakpoint, but are sufficient to modify gene expression in cis and trans. Therefore, through the repositioning of chromosomes and relocation of DNA into different TADs, translocations may alter transcription by disrupting intra- and interchromosomal interactions. We were able to reveal that perturbing the SAMD4A gene loop has a direct effect on the transcriptional status of TNFAIP2 and SLC6A5 (FIG. 22). In addition, corresponding to the transcriptional response (FIG. 9), the effect on co-transcription was hierarchical, with the perturbation of TNFAIP2 altering SLC6A5 expression, but having no influence on SAMD4A (FIG. 26). Furthermore, perturbing SLC6A5 did not impact either SAMD4A or TNFAIP2 transcription (FIG. 35). This observation raises the question: if all the factors necessary for transcription are present, why does transcription of these interacting genes not occur? A possible explanation is that the recruitment of the multitude of repair proteins to the DSB acts as an obstruction to the normal chromatin contacts between these genes loops. Alternatively, contacts between interacting genes may still be present but are `bridged` by the repair machinery. Although this "bridging" maintains the contact between gene loops, it is still inadequate for transcriptional activity to occur. It is important to note that the DNA of these 3 loci appear to be in close proximity in unstimulated HUVECs, despite the absence of 3C contact (Papantonis et al., 2010). Thus, it would be very difficult to definitively show that the disruption of a gene loop abrogates contact between other interacting genes, at least as measured by diffraction limited co-localized DNA FISH foci. Another possible explanation could be that the DNA repair machinery is occluding the entry of RNA Pol II to the other interacting genes. However, the observed unidirectional loss of transcription between these three genes (FIGS. 20, 22, 26 and 35) excludes this possibility. Thus, taken together, the most likely conclusion is that the DSB serves to disrupt chromosomal contact between interacting genes. The repair experiment reveals that restoration of an intact SAMD4A gene loop, in a sequence-independent manner, is sufficient to restore co-transcription, as well as co-localization, of TNFAIP2 and SLC6A5 (FIG. 49). Therefore, we speculate that by disrupting chromosomal interactions, the topological framework (comprised of gene loops and RNA Pol II) is unable to assemble, thus, disrupting transcription. Collectively, these data provide strong evidence that intact chromatin is a requirement for loop-mediated co-transcription.

[0188] One way enhancer-promoter interactions are proposed to enhance transcription, is by bringing protein complexes to the promoter (Deng et al., 2012). In an analogous manner, through interchromosomal interactions, NF-KB has been shown to be delivered to the promoter of inducible genes. Similarly, we propose SAMD4A to be the dominant member of the NF-.kappa.B multigene complex that "organizes" transcription through loop-mediated contact. Recent published data in live cells reveals that RNA Pol II is mobile and clustering precedes transcriptional elongation and is linked to transcriptional initiation (Cisse et al., 2013). Therefore, we speculate that the SAMD4A gene loop provides a topological platform that serves as a scaffold on which a focus of many RNA Pol II molecules can cluster and engage in transcription of subordinate members of the multigene complex. When TNFAIP2 or SLC6A5 cannot engage in chromosomal contact, their ability to access the focus of Pol II is limited. The strict hierarchical relationship between the interacting members further suggests a hand off or "collector" process, where dynamic chromosomal contacts with the dominant gene loop are formed (FIG. 50). This could occur by a mechanism where TNFAIP2 via chromosomal contact "collects" its Pol II from an intact SAMD4A gene loop and in turn SLC6A5 from an intact TNFAIP2 loop. Therefore, these data argue neither in favor of nor against putative "transcription factories," as they are currently defined, but suggest that such factories may be dynamically assembled rather than immobile structures. A recent study revealed that sequences within the promoter might drive the co-localization between these NFkB-regulated genes (Papantonis et al., 2013). Therefore, an alternative model might involve sequence specific elements within the promoters that facilitate the accumulation of different thresholds of general transcription factors for the 3 genes. Therefore, only upon the creation of a nuclear subcompartment of sufficient activity by SAMD4A can TNFAIP2 and then SLC6A5 be activated.

[0189] Our observation shifts the general paradigm of how transcriptional regulation in three dimensions occurs. Although these chromosomal interactions are rare and stochastic (Noordermeer et al., 2011), our single cell view strongly suggests that chromosomal contact between genes engaged in multigene complexes have a significant impact on co-transcription of interacting genes. Such long-range interactions of co-transcribed genes could serve to organize transcription in nuclear space, using hierarchical relationships between gene loops. Without gene loops of dominant members, subordinate members of the multigene complex cannot engage in long-range chromosomal contacts, nor can they participate in transcription. Finally, as looping enables stochastic chromosomal contact between genes in multigene complexes, this study provides supporting evidence to prior work showing that gene looping is a fundamental requirement of transcriptional activity (Tan-Wong et al., 2012). Importantly, such chromatin looping within multigene complexes (Li et al., 2012) may be governed by similar hierarchical regulation. Perturbation of members of multigene complexes, through knockouts, gene deletions, chromosomal translocations or silencing of transcription factors, all of which disrupt loop-mediated contact, may inadvertently result in unintended consequences to transcription of other members of a given multigene complex.

REFERENCES

[0190] Boch, J., et al. (2009) Science 326, 1509-1512.

[0191] Christian, M., et al. (2010) Genetics 186, 757-761.

[0192] Cisse, I. I., et al. (2013) Science 341, 664-7.

[0193] Dekker, J., et al. (2002) Science 295, 1306-1311.

[0194] Deng, W., et al. (2012) Cell 149, 1233-1244.

[0195] Dixon, J. R., et al. (2012) Nature 485, 376-380.

[0196] Fanucchi et al. (2013) Cell 155, 606-20.

[0197] Fullwood, M. J., et al. (2009) Nature 462, 58-64.

[0198] Li, T., et al. (2011) Nucl. Acids Res. 39, 359-372.

[0199] Li, G., et al. (2012) Cell 148, 84-98.

[0200] Lieberman-Aiden, E., et al. (2009) Science 326, 289-293.

[0201] Noordermeer, D., et al. (2011) Nat Cell Biol 13, 944-951.

[0202] Papantonis, A., et al. (2010) PLoS Biol 8: e1000419.

[0203] Tan-Wong, S. M., et al. (2012) Science 338, 671-675.

[0204] Bienko, M., et al. (2013) Nat Methods. 10, 122-124.

[0205] Keogh, M. C., et al. (2005) Nature 439, 497-501.

[0206] Shanbhag, N. M., et al. (2010) Cell 141, 970-981.

Sequence CWU 1

1

416120DNAArtificial SequenceOligonucleotide 1tccacgttta taaatagctg 20220DNAArtificial SequenceOligonucleotide 2cactggggtg tggaagcata 20320DNAArtificial SequenceOligonucleotide 3ttcgcggccc acctggccgc 20420DNAArtificial SequenceOligonucleotide 4ctgtgcgagc gcgacaccta 20520DNAArtificial SequenceOligonucleotide 5ttgtcccttt aaaacttgaa 20620DNAArtificial SequenceOligonucleotide 6ttatcaaact tgtattatca 20720DNAArtificial SequenceOligonucleotide 7tgcagcgcca cagtccccgg 20821DNAArtificial SequenceOligonucleotide 8caaccgttca gaggtcccca g 21920DNAArtificial SequenceOligonucleotide 9tgagggagat tccattgagc 201020DNAArtificial SequenceOligonucleotide 10ggaaaaagtg ctgctccaac 201120DNAArtificial SequenceOligonucleotide 11tgcaggacag actcaggaca 201220DNAArtificial SequenceOligonucleotide 12atttgggttg agcattccac 201320DNAArtificial SequenceOligonucleotide 13tgatttaacc ccctccttcc 201420DNAArtificial SequenceOligonucleotide 14ctttaggagc cacagccaac 201520DNAArtificial SequenceOligonucleotide 15ctagtacctc cgcacgtggt 201620DNAArtificial SequenceOligonucleotide 16tccagcacca ctattggaaa 201718DNAArtificial SequenceOligonucleotide 17tgctgctgca ggagggtg 181890DNAArtificial SequenceOligonucleotide 18gatcgctagc acctgtggag agaaaggcaa agtggatgtc agtaagacca ataggtgcct 60atcatggccc tccagctatt tataaacgtg 901982DNAArtificial SequenceOligonucleotide 19gatcgctagc aggtaagtat caaggttaca agacaggttt aaggagacca atagaaactg 60gggcctcctt cactggggtg tg 822031DNAArtificial SequenceOligonucleotide 20cttttgtata tctacatcat ttagcagcat g 312130DNAArtificial SequenceOligonucleotide 21gatcgctagc ccccctaacg ttactggccg 302240DNAArtificial SequenceOligonucleotide 22gatcgctagc ggatcctcac ttgtacagct cgtccatgcc 402319DNAArtificial SequenceOligonucleotide 23ccacacccca gtgaaggag 192420DNAArtificial SequenceOligonucleotide 24taaatagctg gagggccatg 202520DNAArtificial SequenceOligonucleotide 25attcatgcta ccgtagctac 202620DNAArtificial SequenceOligonucleotide 26ggaaactgca tgagagaaaa 202720DNAArtificial SequenceOligonucleotide 27gacctgactt acttatttcc 202820DNAArtificial SequenceOligonucleotide 28ccagaaaatc cagactctac 202920DNAArtificial SequenceOligonucleotide 29ggaggaagga aaaaacacac 203020DNAArtificial SequenceOligonucleotide 30agccacgttg cccaaaagaa 203120DNAArtificial SequenceOligonucleotide 31catgtgttgc tgaaatccag 203220DNAArtificial SequenceOligonucleotide 32atccaagctt ggcttctgaa 203320DNAArtificial SequenceOligonucleotide 33ttcatgtact cctcacacag 203420DNAArtificial SequenceOligonucleotide 34ccagaatatc tgtggggaaa 203520DNAArtificial SequenceOligonucleotide 35ttctgaagac gaagctctaa 203620DNAArtificial SequenceOligonucleotide 36ttactagtct ctagcgtcac 203720DNAArtificial SequenceOligonucleotide 37gcagtaagct taaccgcatt 203820DNAArtificial SequenceOligonucleotide 38caagatccgt atcaatatgg 203920DNAArtificial SequenceOligonucleotide 39cctttctcca agaccctttt 204020DNAArtificial SequenceOligonucleotide 40aagtaaccca cttcatgcct 204120DNAArtificial SequenceOligonucleotide 41gcagggtaat atgaaacgat 204220DNAArtificial SequenceOligonucleotide 42catactagtt gaggtgtctg 204320DNAArtificial SequenceOligonucleotide 43gctggacctt tcgactatat 204420DNAArtificial SequenceOligonucleotide 44ccacgctagc aaataggaaa 204520DNAArtificial SequenceOligonucleotide 45cctaccctcc aggatataat 204620DNAArtificial SequenceOligonucleotide 46tcacaaccat cagactttcc 204720DNAArtificial SequenceOligonucleotide 47gcagcagcat gaactaaaga 204820DNAArtificial SequenceOligonucleotide 48taaacactgg ggactctgtt 204920DNAArtificial SequenceOligonucleotide 49accctgcatt cttttctctg 205020DNAArtificial SequenceOligonucleotide 50aacatggaac agctggaaga 205120DNAArtificial SequenceOligonucleotide 51accttgtcat caaatggcag 205220DNAArtificial SequenceOligonucleotide 52actcacttta gtgtctccca 205320DNAArtificial SequenceOligonucleotide 53tcgcttcttg ctgctctgaa 205420DNAArtificial SequenceOligonucleotide 54atactaggga ggaggaatga 205520DNAArtificial SequenceOligonucleotide 55tgttttcacc atcgtgcaca 205620DNAArtificial SequenceOligonucleotide 56atatggaagc atcccattct 205720DNAArtificial SequenceOligonucleotide 57cacttccacc ctatgattct 205820DNAArtificial SequenceOligonucleotide 58aactgtgaag atttccagcg 205920DNAArtificial SequenceOligonucleotide 59agtcctgtct ggtcaggaaa 206020DNAArtificial SequenceOligonucleotide 60ttggccatgc aggatctttc 206120DNAArtificial SequenceOligonucleotide 61atggaatctc cctcaatgtg 206220DNAArtificial SequenceOligonucleotide 62aaacgtggag ctcaaccaaa 206320DNAArtificial SequenceOligonucleotide 63aattatgctt ccacacccca 206420DNAArtificial SequenceOligonucleotide 64agagggtgga tcatcagtta 206520DNAArtificial SequenceOligonucleotide 65ccgctgaaat taaggaagga 206620DNAArtificial SequenceOligonucleotide 66aaaaccgagc aggtaaacca 206720DNAArtificial SequenceOligonucleotide 67ccttgtttgt caaacctgag 206820DNAArtificial SequenceOligonucleotide 68gcgtaaatcc tctgcaatct 206920DNAArtificial SequenceOligonucleotide 69ccagcactga aatcaagcat 207020DNAArtificial SequenceOligonucleotide 70agatgagttt gaacagtccc 207120DNAArtificial SequenceOligonucleotide 71gtgctgctcc aacatttgaa 207220DNAArtificial SequenceOligonucleotide 72ctccattact caaagggaaa 207320DNAArtificial SequenceOligonucleotide 73ccccacccaa tatgaatgaa 207420DNAArtificial SequenceOligonucleotide 74ctgttgagac tcattcttgt 207520DNAArtificial SequenceOligonucleotide 75gctttaaaat gtcgctgggt 207620DNAArtificial SequenceOligonucleotide 76gtgatggccc tttgatttac 207720DNAArtificial SequenceOligonucleotide 77taatcctgcc attcctaagc 207820DNAArtificial SequenceOligonucleotide 78ccactgttta accgcaacaa 207920DNAArtificial SequenceOligonucleotide 79aattccaaca ttggagcctc 208020DNAArtificial SequenceOligonucleotide 80cacacagaca caagattcaa 208120DNAArtificial SequenceOligonucleotide 81ggtttctgcc taaaacagca 208220DNAArtificial SequenceOligonucleotide 82attctctcca agtcatcagc 208320DNAArtificial SequenceOligonucleotide 83taagagtgtc tgaggggtta 208420DNAArtificial SequenceOligonucleotide 84gtctagccag actgtagttt 208520DNAArtificial SequenceOligonucleotide 85ctcaccccaa agtctttaag 208620DNAArtificial SequenceOligonucleotide 86tctaaggact tagcaccatc 208720DNAArtificial SequenceOligonucleotide 87gccttcggag ttttcttctt 208820DNAArtificial SequenceOligonucleotide 88tttctctcca acatggttgc 208920DNAArtificial SequenceOligonucleotide 89gtttctacat tgcttccctc 209020DNAArtificial SequenceOligonucleotide 90tttctgtcct gctttctcct 209120DNAArtificial SequenceOligonucleotide 91aaagaagtct ctggtaccag 209220DNAArtificial SequenceOligonucleotide 92gtctagccca agtgagagaa 209320DNAArtificial SequenceOligonucleotide 93taaaccagaa gcctgtctct 209420DNAArtificial SequenceOligonucleotide 94atctaatcca tggccagcca 209520DNAArtificial SequenceOligonucleotide 95aaagggcatt gattccacag 209620DNAArtificial SequenceOligonucleotide 96acttggaaag atgtcccaag 209720DNAArtificial SequenceOligonucleotide 97cccagttaat cctaaggaaa 209820DNAArtificial SequenceOligonucleotide 98gcttcttctt tgtacagtga 209920DNAArtificial SequenceOligonucleotide 99cagtcctcta gcaatggaaa 2010020DNAArtificial SequenceOligonucleotide 100gtgtgtgtgt gtgtgtgtat 2010120DNAArtificial SequenceOligonucleotide 101tttcgtgtgt gtgtgtgtgt 2010220DNAArtificial SequenceOligonucleotide 102aaagccactg tctgtgctat 2010320DNAArtificial SequenceOligonucleotide 103aaaggaagta tcttcatccc 2010420DNAArtificial SequenceOligonucleotide 104cagaagagac tagaacagac 2010520DNAArtificial SequenceOligonucleotide 105actagtttct gttccacgca 2010620DNAArtificial SequenceOligonucleotide 106cttcatctcc ttaagctagc 2010720DNAArtificial SequenceOligonucleotide 107catgtcattc ccacaagaac 2010820DNAArtificial SequenceOligonucleotide 108tgagaaggtt tctgtccatg 2010920DNAArtificial SequenceOligonucleotide 109aaagcttccc aattctagcc 2011020DNAArtificial SequenceOligonucleotide 110ccgaaggtga atgtcttaag 2011120DNAArtificial SequenceOligonucleotide 111atctcctaat gctatccctc 2011220DNAArtificial SequenceOligonucleotide 112ctgcacccat taactcatca 2011320DNAArtificial SequenceOligonucleotide 113tatacatgtg ccgtgttggt 2011420DNAArtificial SequenceOligonucleotide 114caacgtgcag gtttgttaca 2011520DNAArtificial SequenceOligonucleotide 115gagagaatgt aagaaggcga 2011620DNAArtificial SequenceOligonucleotide 116tttagcctga caggcatgaa 2011720DNAArtificial SequenceOligonucleotide 117gtgcacttga agtaacaagg 2011820DNAArtificial SequenceOligonucleotide 118tgatgtcctt gatactggtc 2011920DNAArtificial SequenceOligonucleotide 119gctgcatttc caagaagatc 2012020DNAArtificial SequenceOligonucleotide 120attcagtagg tctgcagtgt 2012120DNAArtificial SequenceOligonucleotide 121tacctgccct atcacccctc 2012220DNAArtificial SequenceOligonucleotide 122accttcctct caccttctcc 2012320DNAArtificial SequenceOligonucleotide 123agaggacatg ctcaggagca 2012420DNAArtificial SequenceOligonucleotide 124tagctctgct cagctccaga 2012520DNAArtificial SequenceOligonucleotide 125atcacttgga actctcctgc 2012620DNAArtificial SequenceOligonucleotide 126taacaagaca cgtggcccta 2012720DNAArtificial SequenceOligonucleotide 127acagggtaac cctacacaat 2012820DNAArtificial SequenceOligonucleotide 128tgtactcagc aggggtacca 2012920DNAArtificial SequenceOligonucleotide 129atctatggag tcaagggacg 2013020DNAArtificial SequenceOligonucleotide 130atgcagaccg tgcgaatgac 2013120DNAArtificial SequenceOligonucleotide 131acactgcagt aagcctctct 2013220DNAArtificial SequenceOligonucleotide 132acaataacag cccctggact 2013320DNAArtificial SequenceOligonucleotide 133ttcacactag gatctggggc 2013420DNAArtificial SequenceOligonucleotide 134ctccaggctt ccttcctaaa 2013520DNAArtificial SequenceOligonucleotide 135acactgccca cactgtctat 2013620DNAArtificial SequenceOligonucleotide 136ttcaacacga actgagccca 2013720DNAArtificial SequenceOligonucleotide 137atccttctgc cagaggctga 2013820DNAArtificial SequenceOligonucleotide 138ctctccactg gaccctttct 2013920DNAArtificial SequenceOligonucleotide 139tatgccctgc tgactcctct 2014020DNAArtificial SequenceOligonucleotide 140ttatctctcc cgtggcccct 2014120DNAArtificial SequenceOligonucleotide 141agcaagcggg aactggctct 2014220DNAArtificial SequenceOligonucleotide 142catttcctgg tgagtcagga 2014320DNAArtificial SequenceOligonucleotide 143tcactgggac agaaagactg 2014420DNAArtificial SequenceOligonucleotide 144ttgcctgttg ctaaccccag 2014520DNAArtificial SequenceOligonucleotide 145tccatgccca gcttggcttt 2014620DNAArtificial SequenceOligonucleotide 146acacaggctt caacgatgcc 2014720DNAArtificial SequenceOligonucleotide 147ctccgttccc cacatttctg 2014820DNAArtificial SequenceOligonucleotide 148caatccagat gcagctgtgt 2014920DNAArtificial SequenceOligonucleotide 149aaatgacctt ctctctgccc 2015020DNAArtificial SequenceOligonucleotide

150ctttgtctgc ctagaggttt 2015120DNAArtificial SequenceOligonucleotide 151agccctggca cttcctaagg 2015220DNAArtificial SequenceOligonucleotide 152atcttggctc actgtcctgg 2015320DNAArtificial SequenceOligonucleotide 153ccccaaactg ctccacagac 2015420DNAArtificial SequenceOligonucleotide 154taggccagaa tgggcaggaa 2015520DNAArtificial SequenceOligonucleotide 155aggtcttggg catctcacca 2015620DNAArtificial SequenceOligonucleotide 156actcacagca gccctggaat 2015720DNAArtificial SequenceOligonucleotide 157tgtcacccag ataaaaccct 2015820DNAArtificial SequenceOligonucleotide 158ttcttcctgc tccaagacta 2015920DNAArtificial SequenceOligonucleotide 159gagccacctt taagatctga 2016020DNAArtificial SequenceOligonucleotide 160aattccttct tcacccagca 2016120DNAArtificial SequenceOligonucleotide 161tgctccttcc ctcacctcca 2016220DNAArtificial SequenceOligonucleotide 162tcactgcaaa agcctcctcc 2016320DNAArtificial SequenceOligonucleotide 163aaatcagcag cctcatgcca 2016420DNAArtificial SequenceOligonucleotide 164taggtcctgc tccaaaatag 2016520DNAArtificial SequenceOligonucleotide 165atcatgcctc ctctgactct 2016620DNAArtificial SequenceOligonucleotide 166ttggaacaga aactctgaag 2016720DNAArtificial SequenceOligonucleotide 167tcctccatct acttagtttg 2016820DNAArtificial SequenceOligonucleotide 168ggtccccatt tcagatgatg 2016920DNAArtificial SequenceOligonucleotide 169ccctttcctc ttgaaagaac 2017020DNAArtificial SequenceOligonucleotide 170aacgaatctg ctttccctgt 2017120DNAArtificial SequenceOligonucleotide 171gcacagacat ctccaaaaga 2017220DNAArtificial SequenceOligonucleotide 172agaattgttc tcctcgtcgc 2017320DNAArtificial SequenceOligonucleotide 173agtacaaata cctagggctg 2017420DNAArtificial SequenceOligonucleotide 174acagcctagg aaacctcttt 2017520DNAArtificial SequenceOligonucleotide 175tctttaggag ccacagccaa 2017620DNAArtificial SequenceOligonucleotide 176ttttcagcac cgaggacagt 2017720DNAArtificial SequenceOligonucleotide 177agagcaactc gcctctgtac 2017820DNAArtificial SequenceOligonucleotide 178tttgtctcag tagcctctag 2017920DNAArtificial SequenceOligonucleotide 179aaatctaggt acctcggctt 2018020DNAArtificial SequenceOligonucleotide 180gcagaataga actcctcgat 2018120DNAArtificial SequenceOligonucleotide 181tctaaccccc tttcacaaac 2018220DNAArtificial SequenceOligonucleotide 182tcctattatc ttcgcctcca 2018320DNAArtificial SequenceOligonucleotide 183gcttcctaaa cattagcacc 2018420DNAArtificial SequenceOligonucleotide 184tcccctcgtg ttatctttca 2018520DNAArtificial SequenceOligonucleotide 185cggtggtttt tctatccact 2018620DNAArtificial SequenceOligonucleotide 186cgctttttcc caattcactt 2018720DNAArtificial SequenceOligonucleotide 187gagtaccaaa actcatgact 2018820DNAArtificial SequenceOligonucleotide 188cttcacaact ctaagcttgc 2018920DNAArtificial SequenceOligonucleotide 189cgtaactatt cacaggagtc 2019020DNAArtificial SequenceOligonucleotide 190cttcttcttc ctcttcttct 2019120DNAArtificial SequenceOligonucleotide 191tcttcttctt cttcttcttc 2019220DNAArtificial SequenceOligonucleotide 192cttcttcttc ttcttcttct 2019320DNAArtificial SequenceOligonucleotide 193ggtcttcttc ttcttcttct 2019420DNAArtificial SequenceOligonucleotide 194cccagacatg gaaaagaata 2019520DNAArtificial SequenceOligonucleotide 195caactcgtat tcacttccag 2019620DNAArtificial SequenceOligonucleotide 196gacacagagt agtaactaga 2019720DNAArtificial SequenceOligonucleotide 197ccacaaaaaa acctacaacg 2019820DNAArtificial SequenceOligonucleotide 198caggtagcaa agacaaggtt 2019920DNAArtificial SequenceOligonucleotide 199tgaggccaaa ttggttgagt 2020020DNAArtificial SequenceOligonucleotide 200acaaagctga tgccctggca 2020120DNAArtificial SequenceOligonucleotide 201ttttccccca tagacaagct 2020220DNAArtificial SequenceOligonucleotide 202cattcaacaa tgcacactgc 2020320DNAArtificial SequenceOligonucleotide 203ttttccgccc catttccttt 2020420DNAArtificial SequenceOligonucleotide 204ccatcaccac tgtagaaaga 2020520DNAArtificial SequenceOligonucleotide 205ttggattcca catcacagct 2020620DNAArtificial SequenceOligonucleotide 206aaatagccct ggagtcatga 2020720DNAArtificial SequenceOligonucleotide 207tgtaagggta aagggggaat 2020820DNAArtificial SequenceOligonucleotide 208ggatgaacta agaaaagagg 2020920DNAArtificial SequenceOligonucleotide 209ctaatattcc ctccctagtg 2021020DNAArtificial SequenceOligonucleotide 210cccgtttcat caataaccag 2021120DNAArtificial SequenceOligonucleotide 211ggctgcaatc agtatttctc 2021220DNAArtificial SequenceOligonucleotide 212gggaagcaaa ttggaatctg 2021320DNAArtificial SequenceOligonucleotide 213tcaggtccag acacaatatc 2021420DNAArtificial SequenceOligonucleotide 214tagatatgga gacgttcgca 2021520DNAArtificial SequenceOligonucleotide 215caaaacaaag tcacaacagc 2021620DNAArtificial SequenceOligonucleotide 216caaacacaat gtcaagttcg 2021720DNAArtificial SequenceOligonucleotide 217caaaaagaaa gttgtcggcg 2021820DNAArtificial SequenceOligonucleotide 218gtttgcagaa cactcgtgtg 2021920DNAArtificial SequenceOligonucleotide 219gctcacctgg acgcagggac 2022020DNAArtificial SequenceOligonucleotide 220gcaggacact aactctccgg 2022120DNAArtificial SequenceOligonucleotide 221ggacctccta agtccggggc 2022220DNAArtificial SequenceOligonucleotide 222gttccgagaa aggggtctct 2022320DNAArtificial SequenceOligonucleotide 223cagtcaggag ccagcaagag 2022420DNAArtificial SequenceOligonucleotide 224gttcttctac tcgccttctg 2022520DNAArtificial SequenceOligonucleotide 225aggtcctaaa atccaactgc 2022620DNAArtificial SequenceOligonucleotide 226ggctaaagct tctgatgctt 2022720DNAArtificial SequenceOligonucleotide 227ctagggtgta gggctttctt 2022820DNAArtificial SequenceOligonucleotide 228ccagggaact aagtgttcca 2022920DNAArtificial SequenceOligonucleotide 229ttgcttctac acagaatagc 2023020DNAArtificial SequenceOligonucleotide 230tgacgaattc ctttgtctga 2023120DNAArtificial SequenceOligonucleotide 231tggctagaat ctcatgcatt 2023220DNAArtificial SequenceOligonucleotide 232aaagacagtc tcatacagac 2023320DNAArtificial SequenceOligonucleotide 233ctccagcttt acaagatgct 2023420DNAArtificial SequenceOligonucleotide 234tccaaccatg gaggattgtt 2023520DNAArtificial SequenceOligonucleotide 235tggtataacc cacatacaga 2023620DNAArtificial SequenceOligonucleotide 236tttccaagga aaaaacatgt 2023720DNAArtificial SequenceOligonucleotide 237attgcaattt tctgcagtct 2023820DNAArtificial SequenceOligonucleotide 238cgcctttttc aaggactaga 2023920DNAArtificial SequenceOligonucleotide 239ttaataacag cttgagagca 2024020DNAArtificial SequenceOligonucleotide 240cttcaaagtg cagtttagta 2024120DNAArtificial SequenceOligonucleotide 241gtccaagagc tcacctagga 2024220DNAArtificial SequenceOligonucleotide 242aggaagtcat ttatttccag 2024320DNAArtificial SequenceOligonucleotide 243tcaaaattta gaaggccaat 2024420DNAArtificial SequenceOligonucleotide 244aagaaacaca aacgcgagta 2024520DNAArtificial SequenceOligonucleotide 245agaggaaact atcaaaaaca 2024620DNAArtificial SequenceOligonucleotide 246cctgtcactc ataaacgaga 2024720DNAArtificial SequenceOligonucleotide 247atagcctggc cacctagttc 2024820DNAArtificial SequenceOligonucleotide 248aggagccaag catacaagag 2024920DNAArtificial SequenceOligonucleotide 249ctggtggcca accaaggaac 2025020DNAArtificial SequenceOligonucleotide 250gtaaccaaaa aacagtatgc 2025120DNAArtificial SequenceOligonucleotide 251caaaatgaag acagggtcaa 2025220DNAArtificial SequenceOligonucleotide 252aagcaaagat agacttgacc 2025320DNAArtificial SequenceOligonucleotide 253acaagcttta tgttctgtgt 2025420DNAArtificial SequenceOligonucleotide 254ctcaaagatt tgtggaagtc 2025520DNAArtificial SequenceOligonucleotide 255tgttgttgac aaatatgccc 2025620DNAArtificial SequenceOligonucleotide 256tcgatgaatg tcagaaatct 2025720DNAArtificial SequenceOligonucleotide 257aatgcagagc tgatgtcaat 2025820DNAArtificial SequenceOligonucleotide 258gtataactcc aaagaagtca 2025920DNAArtificial SequenceOligonucleotide 259aacaaaaagc ccaaagacag 2026020DNAArtificial SequenceOligonucleotide 260gctggtcact agcaagttta 2026120DNAArtificial SequenceOligonucleotide 261gactatgcag caaaacagtt 2026220DNAArtificial SequenceOligonucleotide 262atactcaacc aattggcagc 2026320DNAArtificial SequenceOligonucleotide 263gcttaggttg gcaaaacaag 2026420DNAArtificial SequenceOligonucleotide 264gtaaagtctc aattttctgc 2026520DNAArtificial SequenceOligonucleotide 265tcctcgccct tgctcaccat 2026620DNAArtificial SequenceOligonucleotide 266atgggcacca ccccggtgaa 2026720DNAArtificial SequenceOligonucleotide 267ttacgtcgcc gtccagctcg 2026820DNAArtificial SequenceOligonucleotide 268gacacgctga acttgtggcc 2026920DNAArtificial SequenceOligonucleotide 269ggcatcgccc tcgccctcgc 2027020DNAArtificial SequenceOligonucleotide 270tcagggtcag cttgccgtag 2027120DNAArtificial SequenceOligonucleotide 271ttgccggtgg tgcagatgaa 2027220DNAArtificial SequenceOligonucleotide 272ggtgggccag ggcacgggca 2027320DNAArtificial SequenceOligonucleotide 273cgtaggtgaa ggtggtcacg 2027420DNAArtificial SequenceOligonucleotide 274tagcggctga agcactgcac 2027520DNAArtificial SequenceOligonucleotide 275gtgctgcttc atgtggtcgg 2027620DNAArtificial SequenceOligonucleotide 276gcatggcgga cttgaagaag 2027720DNAArtificial SequenceOligonucleotide 277cgctcctgga cgtagccttc 2027820DNAArtificial SequenceOligonucleotide 278gtcgtccttg aagaagatgg 2027920DNAArtificial SequenceOligonucleotide 279cggcgcgggt cttgtagttg 2028020DNAArtificial SequenceOligonucleotide 280gtgtcgccct cgaacttcac 2028120DNAArtificial SequenceOligonucleotide 281ttcagctcga tgcggttcac 2028220DNAArtificial SequenceOligonucleotide 282cgtcctcctt gaagtcgatg 2028320DNAArtificial SequenceOligonucleotide 283agcttgtgcc ccaggatgtt 2028420DNAArtificial SequenceOligonucleotide 284gtggctgttg tagttgtact 2028520DNAArtificial SequenceOligonucleotide 285tgtcggccat gatatagacg 2028620DNAArtificial SequenceOligonucleotide 286accttgatgc cgttcttctg 2028720DNAArtificial SequenceOligonucleotide 287tgttgtggcg gatcttgaag 2028820DNAArtificial SequenceOligonucleotide 288agctgcacgc tgccgtcctc 2028920DNAArtificial SequenceOligonucleotide 289tgttctgctg gtagtggtcg 2029020DNAArtificial SequenceOligonucleotide 290cacggggccg tcgccgatgg 2029120DNAArtificial SequenceOligonucleotide 291ctcaggtagt ggttgtcggg 2029220DNAArtificial SequenceOligonucleotide 292ctttgctcag ggcggactgg 2029320DNAArtificial SequenceOligonucleotide 293tgatcgcgct tctcgttggg 2029420DNAArtificial SequenceOligonucleotide 294cacgaactcc agcaggacca 2029520DNAArtificial SequenceOligonucleotide 295cgagagtgat cccggcggcg 2029620DNAArtificial SequenceOligonucleotide 296cacttgtaca gctcgtccat 2029720DNAArtificial SequenceOligonucleotide 297ctccaaaaac ctgacttcgc 2029820DNAArtificial SequenceOligonucleotide 298tgtcctaatt ggcctgatcc 2029920DNAArtificial SequenceOligonucleotide 299ccacccaaca tgctctcttt 2030020DNAArtificial SequenceOligonucleotide 300ccatcattta ccagccctgt

2030120DNAArtificial SequenceOligonucleotide 301gctcattgcc ctgcagatag 2030220DNAArtificial SequenceOligonucleotide 302tgatcctcca gtaaccgcat 2030320DNAArtificial SequenceOligonucleotide 303agacctgcat ttccttccct 2030420DNAArtificial SequenceOligonucleotide 304actgtggctc tgttgccttt 2030520DNAArtificial SequenceOligonucleotide 305ccctcagacc cacttcacat 2030620DNAArtificial SequenceOligonucleotide 306cccttctgca cgtctcttct 2030725DNAArtificial SequenceOligonucleotide 307cagtgtctat tcagaaacca cagaa 2530821DNAArtificial SequenceOligonucleotide 308cgtgagttca ggcctactgt c 2130921DNAArtificial SequenceOligonucleotide 309catgcagaga agatcaccat t 2131020DNAArtificial SequenceOligonucleotide 310gggccaagtc ctcaagataa 2031122DNAArtificial SequenceOligonucleotide 311tggcaattat ccattgtcat tt 2231220DNAArtificial SequenceOligonucleotide 312tgtttaagag gatgcctggg 2031326DNAArtificial SequenceOligonucleotide 313ttagtaatct ttggaacatc tgaaca 2631420DNAArtificial SequenceOligonucleotide 314ggcactgaat acacgatggg 2031520DNAArtificial SequenceOligonucleotide 315ttattgccta acccagtgcc 2031620DNAArtificial SequenceOligonucleotide 316tcctatgtca acctggaccc 2031721DNAArtificial SequenceOligonucleotide 317tcaagccttt ctgcctctac a 2131822DNAArtificial SequenceOligonucleotide 318gacacgtgat agcacctaac ca 2231920DNAArtificial SequenceOligonucleotide 319acatatgccg gagttggaaa 2032020DNAArtificial SequenceOligonucleotide 320tctgggctat ctccgtgatt 2032120DNAArtificial SequenceOligonucleotide 321gcactctcca gatccaggtg 2032220DNAArtificial SequenceOligonucleotide 322ctggctgagt cccactcttc 2032320DNAArtificial SequenceOligonucleotide 323aaccacattc ccaaggacaa 2032420DNAArtificial SequenceOligonucleotide 324ggtcgggtag aaggaacctc 2032520DNAArtificial SequenceOligonucleotide 325gtccccactt aggaatccgt 2032618DNAArtificial SequenceOligonucleotide 326gctgctgcct ccagtgtc 1832718DNAArtificial SequenceOligonucleotide 327actcgaagca ccgcactc 1832818DNAArtificial SequenceOligonucleotide 328gaggggaccc ccagtgac 1832920DNAArtificial SequenceOligonucleotide 329agcaggcttc tcgctgttac 2033021DNAArtificial SequenceOligonucleotide 330ggagagaaag agctgcagtg a 2133120DNAArtificial SequenceOligonucleotide 331accaccggaa cgtaactgaa 2033220DNAArtificial SequenceOligonucleotide 332cgaaacatca tggttagggg 2033320DNAArtificial SequenceOligonucleotide 333ggattttgcc cactgtgaac 2033420DNAArtificial SequenceOligonucleotide 334cgggcatcta tcatgtgttg 2033520DNAArtificial SequenceOligonucleotide 335ttcctatttg ctagcgtggg 2033620DNAArtificial SequenceOligonucleotide 336atggaacagc tggaagagga 2033720DNAArtificial SequenceOligonucleotide 337gggcaaagca atcaataacc 2033820DNAArtificial SequenceOligonucleotide 338ttctccccta cctgccctat 2033920DNAArtificial SequenceOligonucleotide 339tacccctgct gagtacagcc 2034020DNAArtificial SequenceOligonucleotide 340acctccaggc ttccttccta 2034120DNAArtificial SequenceOligonucleotide 341gaggagtcag cagggcatag 2034220DNAArtificial SequenceOligonucleotide 342agcttggctt tagttgcctg 2034320DNAArtificial SequenceOligonucleotide 343aggggcagag agaaggtcat 2034419DNAArtificial SequenceOligonucleotide 344aggtcttggg catctcacc 1934520DNAArtificial SequenceOligonucleotide 345tttggagcag gacctaatgg 2034620DNAArtificial SequenceOligonucleotide 346accactcaag ctagagccca 2034720DNAArtificial SequenceOligonucleotide 347ctgcctgcag tgacatcatc 2034819DNAArtificial SequenceOligonucleotide 348ctgcccatgt cctgtctgt 1934920DNAArtificial SequenceOligonucleotide 349gccaatgtga gggagttgat 2035020DNAArtificial SequenceOligonucleotide 350catgggtatg cacacaggac 2035120DNAArtificial SequenceOligonucleotide 351ctggacttgg gctcacagat 2035220DNAArtificial SequenceOligonucleotide 352gctgagtggg aaacaactcc 2035320DNAArtificial SequenceOligonucleotide 353tctgacctcc accaggattc 2035420DNAArtificial SequenceOligonucleotide 354taggagtgag cgtgtggttg 2035520DNAArtificial SequenceOligonucleotide 355agatgggctg gtaccctctt 2035620DNAArtificial SequenceOligonucleotide 356gtagctggcg aaaccagaag 2035720DNAArtificial SequenceOligonucleotide 357ttctggagag aggcaagcag 2035820DNAArtificial SequenceOligonucleotide 358tcctcacaca tgttgctggt 2035920DNAArtificial SequenceOligonucleotide 359caagcacaca ggcagatgtt 2036020DNAArtificial SequenceOligonucleotide 360caatggctcc ccattctcta 2036120DNAArtificial SequenceOligonucleotide 361ggatgtccat ggagcagaat 2036220DNAArtificial SequenceOligonucleotide 362ctttgcttct gccactcctc 2036320DNAArtificial SequenceOligonucleotide 363attcagacac agcccagtcc 2036420DNAArtificial SequenceOligonucleotide 364ccccacttta caccctgcta 2036520DNAArtificial SequenceOligonucleotide 365gggtgacctt ggataagggt 2036620DNAArtificial SequenceOligonucleotide 366gagacaggag aggcaggatg 2036720DNAArtificial SequenceOligonucleotide 367atcatccaac tcagcaaggg 2036819DNAArtificial SequenceOligonucleotide 368gggcttagag aggcaccag 1936920DNAArtificial SequenceOligonucleotide 369attgaggtgg ccacttatgc 2037020DNAArtificial SequenceOligonucleotide 370tcgtagtgct gtggtgaagg 2037120DNAArtificial SequenceOligonucleotide 371tggcccacac tcttagcttt 2037220DNAArtificial SequenceOligonucleotide 372aacatgtcag aggacccagc 2037320DNAArtificial SequenceOligonucleotide 373aagggaagcc aggtctcagt 2037420DNAArtificial SequenceOligonucleotide 374cttcccaccc cttaggtctc 2037520DNAArtificial SequenceOligonucleotide 375agtaggggtg tgggtgacag 2037620DNAArtificial SequenceOligonucleotide 376gccacaggaa aagctaacca 2037720DNAArtificial SequenceOligonucleotide 377gttggctgtg gctcctaaag 2037820DNAArtificial SequenceOligonucleotide 378cgcactcctc tcgtctaacc 2037920DNAArtificial SequenceOligonucleotide 379ccttgtcttt gctacctggc 2038020DNAArtificial SequenceOligonucleotide 380tccccatcac cactgtagaa 2038120DNAArtificial SequenceOligonucleotide 381ccattccccc tttaccctta 2038220DNAArtificial SequenceOligonucleotide 382gcaactcagg tccagacaca 2038320DNAArtificial SequenceOligonucleotide 383ataaactgcc agccaacagc 2038420DNAArtificial SequenceOligonucleotide 384gagtttgcaa gaccccactc 2038520DNAArtificial SequenceOligonucleotide 385cgctgcactg taagatccct 2038620DNAArtificial SequenceOligonucleotide 386aatgcaacct gcttacctgg 2038720DNAArtificial SequenceOligonucleotide 387acgtatgctg atggggaaac 2038821DNAArtificial SequenceOligonucleotide 388caactctttc gtggatctgg a 2138920DNAArtificial SequenceOligonucleotide 389aggtgggttt acctgaggct 2039020DNAArtificial SequenceOligonucleotide 390gtgggttttt cacgcctcta 2039120DNAArtificial SequenceOligonucleotide 391atctcccact tgcctttcct 2039220DNAArtificial SequenceOligonucleotide 392ccattcctaa tcccaccctt 2039320DNAArtificial SequenceOligonucleotide 393tgtcctgcac accctgtaga 2039420DNAArtificial SequenceOligonucleotide 394ggtcctggaa tgcctcacta 2039520DNAArtificial SequenceOligonucleotide 395gcatcttcgt gtgagcttga 2039620DNAArtificial SequenceOligonucleotide 396tgggaagaga caagccatct 2039720DNAArtificial SequenceOligonucleotide 397gaaagtaagg caggggctct 2039820DNAArtificial SequenceOligonucleotide 398cccaacccct taccaagaat 2039920DNAArtificial SequenceOligonucleotide 399tacaggttca aggtttgggg 2040019DNAArtificial SequenceOligonucleotide 400atgcatcagc aagcaggac 1940120DNAArtificial SequenceOligonucleotide 401gcctgcttgt ggacctactc 2040220DNAArtificial SequenceOligonucleotide 402agagcggaaa agccatacct 2040320DNAArtificial SequenceOligonucleotide 403acagcctcct taggctccag 2040420DNAArtificial SequenceOligonucleotide 404aacacttgac cgctagcacc 2040520DNAArtificial SequenceOligonucleotide 405gttgaggctg tgctgtcaaa 2040620DNAArtificial SequenceOligonucleotide 406agccgatgca agcctaacta 2040720DNAArtificial SequenceOligonucleotide 407cactgcttca ccccataggt 2040820DNAArtificial SequenceOligonucleotide 408atggcaaatg ctctgaggtc 2040920DNAArtificial SequenceOligonucleotide 409acacgaatag aggcacccag 2041018DNAArtificial SequenceOligonucleotide 410aaccctgaga tctgcccc 1841120DNAArtificial SequenceOligonucleotide 411ttttcaggag cagaggagga 2041220DNAArtificial SequenceOligonucleotide 412agcggaggga aaactgatct 2041320DNAArtificial SequenceOligonucleotide 413cctcaaagaa tgctgaaggg 2041420DNAArtificial SequenceOligonucleotide 414ccttctgagt cctcccacag 2041520DNAArtificial SequenceOligonucleotide 415tgaccttgaa ggatggaagg 2041620DNAArtificial SequenceOligonucleotide 416cccaggaccc aaaatcttct 20

Claims

1. A single cell assay for determining the effect of chromosomal contact on the transcriptional activity of at least one gene of interest in a cell, comprising: (i) at least one site-specific nuclease for inducing a double stranded break in at least one region of chromatin or DNA engaged in chromosomal contact in the cell; (ii) an immunofluorescent probe or a recombinant protein expressing a fluorescent label for detecting the site of the double stranded break; and (iii) at least one fluorescent oligonucleotide probe capable of hybridising to at least one target mRNA sequence produced by the transcription of at least one gene of interest, wherein the presence or absence of fluorescence and the relative intensity of fluorescence of the at least one fluorescent oligonucleotide probe hybridised to the target mRNA sequence is indicative of the effect of the chromosomal contact on the transcriptional activity of the at least one gene of interest.

2. The single cell assay of claim 1, wherein transcriptional activity of the at least one gene of interest may be abrogated by a mechanism selected from the group consisting of: (i) recruitment of proteins involved in a repair process of the double stranded break, which when bound to the chromatin or DNA obstruct the chromosomal contact, (ii) enhancement of mobility of the region of chromatin or DNA containing the double stranded break, which results in a reduction in capacity of a chromatin or DNA loop to engage in chromosomal contact; and (iii) loss of structural integrity of a chromatin or DNA loop, which results in abrogation of chromosomal contact.

3. The single cell assay of claim 1, wherein the chromosomal contact may be an intergenic contact, an intragenic contact or both intergenic and intragenic contact.

4. The single cell assay of claim 1, wherein the double stranded break perturbs the chromatin or DNA engaged in chromosomal contact and perturbs transcriptional activity of the at least one gene of interest.

5. The single cell assay of claim 4, wherein the region of chromatin or DNA that is perturbed comprises a gene or regulatory element selected from the group consisting of: (i) an enhancer and/or a promoter, (ii) a site within a chromatin or DNA loop that engages in intra- or inter-chromosomal contact, and (iii) a regulatory site within a chromatin or DNA loop which determines the loop structure.

6. The single cell assay of claim 1, wherein the site specific nuclease is selected from the group consisting of a meganuclease, a zinc finger nuclease, a TALE nuclease, a BUD1 nuclease, and a CrispR/Cas9 nuclease.

7. The single cell assay of claim 1, wherein the site-specific nuclease is delivered to the cell by transfection of the cell with a vector encoding the site-specific nuclease and wherein the site-specific nuclease is endogenously expressed in the cell or wherein the site-specific nuclease is exogenously expressed and delivered to the cell.

8. The single cell assay of claim 1, wherein the immunofluorescent probe is an antibody which binds to at least one of the proteins involved in the cellular repair process of a double strand break in the cell.

9. The single cell assay of claim 1, wherein alocation of the hybridized fluorescent oligonucleotide probe in the cell is indicative of achromosomal location of gene expression for the target mRNA sequences and alocation of the hybridised immunofluorescent probe is indicative of the location of the double stranded break.

10. The single cell assay of claim 1, wherein location of the hybridized fluorescent oligonucleotide probe in the cell is observable by a technique selected from the group consisting of: diffraction limited imaging techniques, sub-diffraction limit image resolution and imaging in three dimensions, wherein detection of chromosomal location of gene expression for the target mRNA sequences and diffraction limited or sub-diffractive location of the hybridised immunofluorescent probe is indicative of location of the double stranded break.

11. The single cell assay of claim 1, wherein the cell is a eukaryotic cell or a prokaryotic cell.

12. The single cell assay of claim 5, wherein the gene or regulatory element is an enhancer and/or a promoter.

13. The single cell assay of claim 5, wherein the gene or regulatory element is a site within a chromatin or DNA loop that engages in intra- or inter-chromosomal contact.

14. The single cell assay of claim 5, wherein the gene or regulatory element is a regulatory site within a chromatin or DNA loop which determines the loop structure.

15. The single cell assay of claim 6, wherein the site specific nuclease is a meganuclease.

16. The single cell assay of claim 6, wherein the site specific nuclease is a zinc finger nuclease.

17. The single cell assay of claim 6, wherein the site specific nuclease is a TALE nuclease.

18. The single cell assay of claim 6, wherein the site specific nuclease is a BUD1 nuclease.

19. The single cell assay of claim 6, wherein the site specific nuclease is a CrispR/Cas9 nuclease.