The molecular basis for stability of heterochromatin-mediated silencing in mammals
© Hiragami-Hamada et al; licensee BioMed Central Ltd. 2009
Received: 20 May 2009
Accepted: 04 November 2009
Published: 04 November 2009
The archetypal epigenetic phenomenon of position effect variegation (PEV) in Drosophila occurs when a gene is brought abnormally close to heterochromatin, resulting in stochastic silencing of the affected gene in a proportion of cells that would normally express it. PEV has been instrumental in unraveling epigenetic mechanisms. Using an in vivo mammalian model for PEV we have extensively investigated the molecular basis for heterochromatin-mediated gene silencing. Here we distinguish 'epigenetic effects' from other cellular differences by studying ex vivo cells that are identical, apart from the expression of the variegating gene which is silenced in a proportion of the cells. By separating cells according to transgene expression we show here that silencing appears to be associated with histone H3 lysine 9 trimethylation (H3K9me3), DNA methylation and the localization of the silenced gene to a specific nuclear compartment enriched in these modifications. In contrast, histone H3 acetylation (H3Ac) and lysine 4 di or tri methylation (H3K4me2/3) are the predominant modifications associated with expression where we see the gene in a euchromatic compartment. Interestingly, DNA methylation and inaccessibility, rather than H3K9me3, correlated most strongly with resistance to de-repression by cellular activation. These results have important implications for understanding the contribution of specific factors involved in the establishment and maintenance of gene silencing and activation in vivo.
Interphase eukaryotic nuclei contain two forms of chromatin : densely DNA-stained regions termed heterochromatin and more diffusely stained regions called euchromatin. In contrast to euchromatin, heterochromatin is rich in repetitive DNA elements, poor in transcriptionally active genes, highly resistant to nuclease digestion and the DNA replicates late in S-phase . In mammals, constitutive heterochromatin is enriched with specific chromatin modifications including histone H3 lysine 9 (H3K9) trimethylation (me3) [3–5], H4K20me3 [6–8] and DNA methylation [9–12], all of which have been implicated in gene silencing. These modifications may occur in a coordinated manner. For instance, mice deficient in Suv39h, a H3K9 histone methyltransferase (HMTase), have reduced DNA methylation at their pericentric repeats , indicating the interdependence between these modifications. Furthermore, the relationship between H3K9me3 and DNA methylation has been implicated in the regulation of genes involved in early development and across species [13, 14].
The gene-repressive effect of heterochromatin was first demonstrated by position effect variegation (PEV)  in Drosophila, where a normally euchromatic white+ gene (responsible for red eye pigmentation) was silenced in a proportion of eye cells when the gene was placed abnormally close to a block of pericentric heterochromatin. Similar phenomena were observed in organisms ranging from yeast to mice when a reporter gene was inserted within pericentric or telomeric regions of chromosomes [16–18]. The extent of heterochromatin-induced silencing effects can be modulated by the dosage of chromatin modifiers. This was elegantly shown in the Saccharomyces cerevisiae telomere position effect (TPE)[19, 20]. In addition, mutations in genes encoding homologues of Suv39 [17, 21–23] or a structural chromatin component [24–27], heterochromatin protein 1 (HP1) [22, 28–30], led to reduced silencing of variegating reporter genes in Drosophila and Schizosaccharomyces pombe. On the other hand, enhanced/increased silencing of a variegating reporter gene was observed with over-expression of Su(var)3-9  or of an HP1 homologue in Drosophila, S. pombe and mice [29, 31, 32].
Biochemical analyses in Drosophila and murine PEV revealed that repressed variegating genes have a chromatin structure and/or nucleosome organization pattern comparable to constitutive heterochromatin of pericentric regions [18, 33, 34]. Consistent with the spreading hypothesis for heterochromatin formation, chromosomal rearrangement affecting two reporter genes resulted in silencing of the reporter proximal to the rearrangement breakpoint whenever the reporter distal to the breakpoint was silenced [35, 36]. This was accompanied by 'compaction' visualized as the darkening of polytene chromosome bands adjacent to the rearrangement breakpoint, suggesting the spreading of heterochromatin over the breakpoint. Based on these earlier observations, together with the sensitivity of PEV to the dosage of chromatin modifiers, it has been thought that PEV results from the stochastic spreading of heterochromatin-forming factors or heterochromatic chromatin marks from a nearby heterochromatic region into the variegating gene, in the absence of dominant cis-acting boundary elements [37–39]. The spreading of heterochromatin may occur in a linear manner or in trans, where an interaction between a variegating gene and heterochromatin on the same or another chromosome occurs [40, 41]. In a landmark study, Harmon and Sedat demonstrated the correlation between silencing in Drosophila PEV and the localization of the reporter to heterochromatin . However, there is little molecular evidence for the spreading of 'heterochromatic' chromatin modifications over a repressed variegating gene in vertebrates.
Here, we investigated the chromatin modifications associated with a variegating transgene and dissected out their roles in chromatin compaction and stable gene silencing, using the variegating human CD2 (hCD2) transgenic mouse system [18, 31]. Our results revealed that repressed variegating hCD2 transgenes are indeed associated with known heterochromatic chromatin modifications, including H3K9me3 and DNA methylation, and positioned within or close to a repressive nuclear domain. However, DNA methylation was the key modification that accompanied the formation of an inaccessible chromatin structure and more stable gene silencing upon cellular activation and through cell division.
hCD2 transgenic mice as a model for mammalian PEV
Repressed hCD2 transgenes exhibit histone modification patterns similar to pericentric heterochromatin
Pericentric heterochromatin is enriched with H3K9me3 and contains few modifications associated with transcriptional activation such as H3K9/K4Ac or H3K4me2/3. In order to test whether repressed hCD2 transgenes have a histone modification pattern comparable with pericentric heterochromatin, we performed chromatin immunoprecipitation (ChIP) on chromatin prepared from sorted hCD2+ and/or hCD2- T cells from CD2 1.3B and 1.3A14 transgenics, using antibodies against various histone modifications indicated in Figure 1C. Enrichment for histone modifications along the 8kb hCD2 transgene locus was analysed by quantitative polymerase chain reaction (PCR) using primer pairs for six different regions of the transgene (Figure 1B). Controls were the β-actin and CD3ε genes, which are transcribed in T cells, and the 5' long terminal repeats (LTR) of intracisternal A particle (IAP), which is a transposon element present in many repressed copies in the murine genome. For each transgenic line, β-actin and CD3ε showed enrichment for H3K4me2 and H3K9/K14Ac whereas IAP was mainly enriched with H3K9me2/3, as anticipated (Figure 1D). hCD2 transgenes in hCD2- T cells from both CD2 1.3B and CD2 1.3A14 transgenic mice were enriched with H3K9me3 and were markedly depleted in H3K4me2 and H3K9/K14Ac (Figure 1C). In contrast, hCD2 transgenes in hCD2+ T cells showed high levels of H3K4me2 and H3K9/K14Ac (Fig. 1C). H3K9me1/2 was detected at the transgene with similar levels in hCD2+ and hCD2- T cells (Figure 1C). These modifications are not predominant marks for pericentric heterochromatin [3–5]. Thus, the repressed hCD2 transgene in both CD2 1.3B and CD2 1.3A14 transgenics have a histone modification pattern similar to that of pericentric heterochromatin. However, levels of H3K4me2 and H3K9/K14Ac were much lower along the repressed transgenes in CD2 1.3B than those in CD2 1.3A14 transgenic mice (Additional file 1, Figure S3A). In CD2 1.3A14 transgenic mice, the H3K9me3 mark was preferentially enriched at the coding and 3' regulatory region, compared with the distal and proximal promoter regions (Additional file 1, Figure S3B). This suggests that the transgene in CD2 1.3B and CD2 1.3A14 transgenic mice may be repressed by a mechanism involving H3K9me3, but the mode of acquisition or maintenance of this mark may differ between the two lines, as may the nucleosome occupancy which would be interesting to assess in more detail.
Key regulatory regions of repressed hCD2 transgenes are marked by DNA methylation
A repressed hCD2 transgene is preferentially located at a repressive nuclear domain
Absence of the promoter but not the enhancer DNase I hypersensitivity correlates with the repressed status of the hCD2 transgene
The stability of hCD2 transgene repression correlates with chromatin conformation at the enhancer
Discussion and conclusion
Epigenetic differences between expressed and repressed hCD2 transgenes
Distinct roles of H3K9me3 and DNA methylation in hCD2 transgene silencing
The differences in the levels/patterns of repressive chromatin modifications/chromatin compaction along the repressed hCD2 transgene and the stability of hCD2 repression in two different hCD2 transgenic lines (CD2 1.3A14 and CD2 1.3B) suggested an unequal contribution of DNA methylation and H3K9me3 to maintenance of hCD2 repression. Although, we have not yet studied nucleosomal occupancy directly, our results suggest that a higher level of DNA methylation rather than H3K9me3, correlates with compaction of the hCD2 regulatory regions and more stable hCD2 repression. This is consistent with recent studies which indicate that DNA methylation appears to prevent reactivation of GFP transgenes  and developmentally regulated genes  in mammalian cells. However, it must be noted that the H3K9 HMTase, Suv39h, is an important factor in the establishment of repression of the hCD2 transgene (S Uribe-Lewis, unpublished data). Furthermore, it will be interesting to study other histone modifications such as H3K27me3 and H4K20me3 in the future. Taken together, different chromatin modifications might contribute differentially during the initiation and maintenance stages of hCD2 repression.
Open enhancer conformation and instability of hCD2 silencing
Conformation of the enhancer chromatin appears to correlate with the stability of hCD2 repression during T cell activation (Figure 4 and 5). It is possible that the open enhancer conformation in CD2 1.3A14 transgenic mice might lead to greater hCD2 derepression as trans-activators, which become up-regulated during T cell activation, may bind to the enhancer and promote expression of a previously repressed hCD2 transgene. Alternatively, insertion into the major satellite sequences might lead to an increased spreading of the heterochromatic state into the CD2 1.3B transgene making it more resistant to derepression than the CD2 1.3A14 transgene array, which integrated near to pericentric heterochromatin. Another possibility is that the CD2 1.3A14 array is more subject to the effect of transgene copy number whereas the pericentric array is affected more by the satellite repeats . Furthermore, in accordance with recent descriptions of H3K9 demethylases [58–60], previously acquired H3K9me3 at the enhancer may not be enough to keep the hCD2 transgene in a repressed state if an H3K9 demethylase is recruited to the region by an activator complex. In the case of CD2 1.3B transgenic mice, compaction of the enhancer (and promoter) might prevent the binding of trans-activators and further recruitment of activating chromatin modifiers making the hCD2 transgene more resistant to derepression. In addition, as revealed by cryo-FISH, embedding the repressed hCD2 transgene within the highly heterochromatic environment in this transgenic line might maintain a local abundance of heterochromatic factors high, which competitively prevent binding of trans-activators to the transgene. However, even in the CD2 1.3B transgenic line, the maintenance of hCD2 transgene repression is incomplete and significant hCD2 derepression occurs with prolonged T cell activation. Interestingly, it has been reported that over-activation of the JAK signalling pathway counteracts heterochromatic silencing [61, 62]. As the constitutive heterochromatin is not completely inaccessible to molecules and is maintained by dynamic processes [63–66], the continuous presence of a high level of trans-activators might eventually compete with heterochromatic factors and gain access to binding sites on the hCD2 transgene to activate hCD2 expression . Future work will address the role of a growing number of histone modifications that may also participate in these processes. In conclusion, our studies on mammalian PEV have enabled us to dissect ex vivo potential epigenetic factors associated with maintenance of gene silencing through cell division and indicate that DNA methylation correlates most strongly with both the maintenance of inaccessibility and the transmission of gene-silencing information to the daughter cell population.
Materials and methods
All hCD2 transgenic mice lines were generated as previously described [18, 31]. Transgenic mice that are heterozygous for the hCD2 transgene were used for all the experiments. The project was given ethical approval by Imperial College London and the UK Home Office.
Peripheral lymphocytes were obtained from mesenteric lymph nodes and spleens of transgenic mice. T cells were isolated from the mixed population using Dynal T cell negative selection kit (Dynal, UK). hCD2+ T cells were obtained by positive selection using MACS hCD2 microbeads (Miltenyi Biotec). The flow-through containing hCD2- T cells was further purified by incubation with Dynal hCD2 magnetic beads (Dynal, UK) to deplete any contamination with hCD2+ T cells. Alternatively, peripheral T cells were stained with an antibody cocktail containing phycoerythrin (PE)-conjugated anti-hCD2 (BD Pharmingen), tri-colour (TC)-conjugated anti-CD4 and anti-CD8 antibodies (Caltag), and sorted using a fluorescence-activated cell sorter (FACS) DIVA (Becton Dickenson).
For cell culture, all the sorting procedures were carried out under sterile conditions. Sorted hCD2- peripheral T cells were resuspended in T-cell medium (Iscove's Modified Dulbecco's Medium/10% fetal calf serum [Sigma Aldrich]/1% penicillin/streptomycin/0.05% β-mercaptoethanol/20 U/ml recombinant IL-2 [Roche], all from GIBCO, unless indicated). For T-cell stimulation, 96-or 24-well plates were coated with 10 μg/ml anti-TCRβ chain and 2 μg/ml anti-CD28 co-stimulatory molecules (BD Pharmingen) in phosphate buffered saline (PBS). Cells were cultured at 1 × 106 or 4 × 106 cells/well in 96- or 24-well plates, respectively with or without T cell stimulation. To measure the extent of cell proliferation, cells were loaded with 1 μM carboxyfluorescin diacetate succinimidylester (CFSE; Molecular Probes) in PBS prior to the culture.
For ex vivo T cells, 1 × 105 cells were stained with PE-conjugated anti-hCD2 (1/200) and TC-conjugated anti-CD4 or CD8 (1/400) antibodies. For cultured T cells, 1 × 106 T cells were stained with PE-conjugated hCD2, biotin-conjugated anti-CD69 (1/100) and allophycocyanin (APC)-conjugated CD4 or CD8 (1/100; all from BD Pharmingen) or with PE-conjugated hCD2, TC-conjugated CD4/CD8 and APC-conjugated CD25 (1/100; BD Pharmingen). For CD69 staining, cells were incubated with cychrome-conjugated streptavidin (1/400; BD Pharmingen) after staining with the primary antibody. Acquisition and analysis were carried out using FACS Calibur and Cell Quest software (Becton Dickinson). The cut-off point of hCD2 expression level was set according to hCD2 fluorescence of CBA/Ca; >98% of T cells from the negative control fell below the cut-off point.
ChIP was performed on sonicated chromatin prepared from sorted T cells using standard protocols. Chromatin was prepared from 5 × 106 sorted T cells. Cells were fixed with 1% formaldehyde in X-linking buffer (50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) pH8.0, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA], 0.5 mM ethylene glycol tetraacetic acid [EGTA]) for 5 min. The X-linking reaction was quenched by addition of 125 mM glycine/PBS. This was followed by cell lysis and nuclear lysis in appropriate buffers. X-linked chromatin was then sonicated on ice to produce 300-500 bp chromatin fragments. For each immunoprecipitation (IP) sample, 2 μg chromatin was diluted to a final volume of 375 μl in ice-cold IP buffer (1.1% Triton-X100, 0.01% SDS, 1.2 mM EDTA, 167 mM NaCl, 20 mM Tris-HCl at pH8.1, 10 mM Na butyrate, 0.1 mM phenylmethanesulphonylfluoride, 0.1 mM Benzamidine, 1/1000 Protease inhibitors cocktail [Sigma-Aldrich, UK]) and was incubated with 10 μg anti-dimethyl histone H3K4 (Abcam), 10 μg anti-acetyl histone H3 (Upstate Biotechnology), 10 μg normal rabbit IgG (Santa Cruz) or 5 μg anti-mono-, di- or tri-methyl histone H3K9 (from Dr T Jenuwein) for 3 h at 4°C on a rotator. 100 μl BSA-blocked protein A-agarose beads (Upstate Biotechnology) was added to each tube and incubated for further 1.5 h as before. After the IP, the beads were washed twice each with high salt, low salt and lithium chloride wash buffers. The washed beads were resuspended in an elution buffer and heated at 65°C for 30 min. Eluates were applied to spin-X columns (Fisher Scientific) and spun at 6 k rpm for 2 min to remove the protein A beads. Input and bound fractions were treated with RNase A and proteinase K (both from Roche). IP-ed DNA was purified using Qiagen PCR clean-up kit (Qiagen, UK). Purified IP-ed DNA was diluted five times and purified input DNA was diluted 100 times in sterile H2O. For PCR with 'control' primers (IAP, β-actin and CD3ε), the reaction was performed with 5 μl DNA, 1× PCR buffer (Sigma), 2 mM MgCl (Sigma), 0.4 mM dNTPs (Fermentas), 0.2 μM each forward and reverse primer and 2.5 U Taq polymerase (Sigma) in a total volume of 20 μl. The PCR cycle was run as following: the initial incubation at 94°C for 5 min, 28 cycles (IAP) or 40 cycles (β-actin and CD3ε) of denaturing (94°C for 40 s), annealing (60°C for 40 s) and extension (72°C for 36 s) and the final extension at 72°C for 10 min. The PCR products were analysed by electrophoresis of the products on 1.5% TBE agarose gels. For real-time PCR analysis using hCD2 primers, the reaction was carried out with 4 μl DNA, 0.4 μM each forward and reverse primer and 1× SYBR Jump-Start reaction mix (Sigma) in a total volume of 20 μl. PCR cycles were set on and run through Opticon 1 or Opticon 2 program (MJ Research) connected to a PCR cycler. The PCR cycle consisted of the initial activation of Taq polymerase at 94°C for 2 min, 40 cycles of denaturing (94°C for 30 s), annealing (60°C for 30 s) and extension (72°C for 30 s). The fluorescence intensity was read at 75, 78, 80 and 82°C after each cycle. The analysis of resulting C (t) values was performed using the Opticon1/2 program. Threshold was set at 0.1, at which PCR amplification curves were still in a linear range on a logarithmic scale. C(t) values for samples and those for serial dilutions of input DNA were read at this point. Standard calibration curves were generated by plotting C(t) values against log (arbitrary DNA concentration). This showed a linear correlation between C(t) and log(DNA concentration) with R2 value >0.95. The arbitrary DNA concentration of each sample was calculated using equations generated from the calibration curves. The resulting DNA concentration of samples was normalized against input and enrichments for histone modifications are presented as percentage input (1/20). The details of the antibodies and primers used in the ChIP assays are shown in Additional file 2, Tables S1 and S2.
Restriction enzyme digest/Metabisulfite sequencing
DNA was isolated from cells using a standard phenol-chloroform extraction and ethanol precipitation protocol. For methylation-sensitive restriction digest, DNA was digested with Bgl II and Hha I (New England Biolab). Completion of genomic DNA digestion was controlled with λ DNA (or a plasmid) digestion mixed with each reaction mix. Digested genomic DNA was analysed by Southern blotting using a [32P]dCTP-labelled hCD2 probe and analysed using a Phosphorimager (Amersham Pharmacia) and Image Quant software (Molecular Probes). For metabisulfite sequencing, DNA was digested with Afl II and/or Xba I. Digested DNA was denatured by heating and treatment with NaOH and then treated with sodium bisulfite (Sigma). Nested PCR was performed using an Expand High Fidelity PCR kit (Roche) to amplify fully converted DNA templates. PCR products were cloned into a TA-cloning vector (Invitrogen). Approximately 30 clones were sequenced to determine the methylation status for each sample. Only the clones that showed >97% C->T conversion at non-CpG sites were used for analysis.
DNase I hypersensitivity assay
For 3D FISH, 1-2 × 105 cells were stuck to poly-L-lysine-covered coverslips and fixed with 0.1% glutaraldehyde in fix/permeabilization buffer (20 mM KH2PO4, 130 mM NaCl, 20 mM KCl, 10 mM EGTA, 2 mM MgCl2, 0.1% Triton-X100) for 30 min at RT. Fixation was quenched by treatment with Na borohydride. Cells were re-fixed with a mixture of EGS and sulfo-EGS before in situ denaturation of genomic DNA. hCD2 probe and γ-satellite probe were labelled using digoxigenin (DIG)-Nick Translation Mix (Roche) or with fluorescein-dUTP using Nick Translation Mix (Roche), respectively. Labelled probes were mixed with mouse-Cot1 DNA and salmon sperm DNA in FISH hybridization mix (50%(v/v) formamide, 2 × SSC, 10% dextran sulfate, pH7.2). Genomic DNA was denatured in situ by treatment with CAPS:NaOH solution (pH12.85-13.10) for 2 min and hybridized with heat-denatured probes for overnight-48 h at 38°C in a humidified chamber. Cells were washed extensively with 1-2× SSC/0.05% Tween-20. Signal from DIG-labelled hCD2 probe was visualized by treatment with sheep anti-DIG antibody (GIBCO), followed by rhodamine-conjugated donkey anti-sheep IgG antibody (Jackson Laboratories). After washes with 4 × SSC/0.05% Tween-20, cells were stained with DAPI (100 ng/ml) and mounted in Vectashield. Images were acquired and analysed using a DeltaVision System and Soft RoRx software (Applied Precisions). The experiments were conducted in a blind manner.
Ultracryosectioning and FISH was performed essentially as described in . Resting T cells from hCD2 1.3B lymph nodes were fixed in 4% and 8% paraformaldehyde in 250 mM HEPES (pH 7.6; 10 min and 2 h, respectively) . Cell pellets were embedded in 2.1 M sucrose in PBS and frozen in liquid nitrogen. Cryosections were cut using an UltraCut UCT 52 ultracryomicrotome (Leica, Milton Keynes, UK) with 200 nm in thickness, captured in sucrose drops, and transferred to coverslips for cryo-FISH.
Probes used to label pericentromeric clusters (γ- satellite plasmid; from Dr N Dillon) and human CD2 (hCD2-cos1 construct; from Dr D Kioussis) were labelled with DIG and biotin by nick translation (Roche), respectively, and separated from unincorporated nucleotides using microBioSpin P-30 chromatography columns (BioRad, UK). Hybridization mixtures contained 50% deionized formamide, 2 × SSC, 10% dextran sulphate, 50 mM phosphate buffer (pH 7.0), 1 μg/μl human Cot1 DNA, 2 μg/μl salmon sperm DNA, 1 μl nick-translated γ- satellite and 4 μl hCD2-cos1 DNA (in 6 μl hybridization buffer). Probes were denatured at 70°C for 10 min, and reannealed at 37°C for 30 min before hybridization. Cryosections were rinsed (three times in PBS, incubated (30 min) in 20 mM glycine in PBS, rinsed (three times) in PBS, permeabilized (10 min) with 0.2% Triton X-100 +0.2% saponin in PBS, and then washed (three times) in PBS. After washing with PBS, cryosections were incubated (1 h, 37°C) with 250 μg/ml RNase A, treated (10 min) with 0.1 M HCl, dehydrated in ethanol (50% to 100% series, 3 min each), denatured (12 min, 80°C) in 70% deionized formamide, 2×SSC and dehydrated as above. Hybridization was carried out at 37°C in a moist chamber for >40 h. Posthybridization washes were as follows: 50% formamide in 2×SSC (42°C; three times over 25 min), 0.1× SSC (60°C, three times over 30 min) and 4×SSC with 0.1% Tween-20 (42°C, 10 min). Sections were incubated (30 min) with casein-blocking solution (Vector Laboratories) containing 2.6% NaCl, 0.5% BSA, 0.1% fish skin gelatin, pH 7.5-8.0). The γ-satellite signal was amplified (2 h) with sheep Fab fragments anti-DIG (Roche), washed (three times over 1 h), incubated (1 h) with Cy3-conjugated donkey antibodies anti-sheep IgG (Jackson Laboratories). The hCD2 signal was amplified (1 h) with AlexaFluor488 Neutravidin (Molecular Probes, Eugene, OR), washed (three times over 1 h), incubated (1 h) with biotinylated goat anti-avidin antibodies (Vector), washed (three times over 1 h), incubated (1 h) with AlexaFluor488 Neutravidin, all in casein-blocking solution. Nuclei were counterstained with 2 μM TOTO-3 (Molecular Probes) in PBS/0.05% Tween-20, washed (four times) in PBS and mounted in VectaShield.
Hybridization of non-transgenic cells with hCD2 probes showed no detectable signals.
Images (TIFF files) were acquired on a confocal laser scanning microscope (Leica TCS SP2; 100× objective, NA 1.4, Milton Keynes, UK), equipped with Argon (488), HeNe (543 nm) and HeNe (633 nm) lasers, and pinhole equivalent to 1 Airy disk. Images (TIFF files) from the different channels were collected sequentially to prevent bleedthrough.
Students t-test (unpaired, two-tailed), Fisher's exact test and Chi-squared test were carried out using GraphPad Software http://www.graphpad.com and Mann-Whitney's U-test was performed using VassarStats http://faculty.vassar.edu/lowry/utest.html. Also, non-parametric Kolmogorov-Smirnov test was carried out using an online calculator http://www.physics.csbsju.edu/stats/KS-test.html. Statistical methods used in particular analyses are stated in the main text or figure legends.
carboxyfluorescin diacetate succinimidyl ester
ethylenediamine tetraacetic acid
ethylene glycol tetraacetic acid
fluorescence-activated cell sorter
fluorescence in in situ hybridization
histone H3 lysine 9
heterochromatin protein 1
intracicternal A particle
locus control region
long terminal repeats
phosphate buffered saline
polymerase chain reaction
position effect variegation
T cell receptor β
telomeric position effect.
We would like to thank Dr Thomas Jenuwein (IMP, Vienna) for his generous gift of anti-mono-, di- and trimethylated H3K9 antibodies and the MRC (UK) for their support. We are also grateful to the Members of Gene Control Mechanisms and Disease Group for helpful discussions and Nataliya Elina for reading the manuscript.
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