Presentation on theme: "ChIP efficiency (%input)"— Presentation transcript:
1 ChIP efficiency (%input) - +4OHTProxPrimersDist0.10.150.05ChIP efficiency (%input)mockH2AX-+chr22:chr21:chr6:chr6:AsiSI siteABChr6Chr1Genedensity-0.40.4Log2(H2AX/input)Log2(mock/input)C0.2-0.2Log2(H2AX/input)0.10.3-0.10.3Chr6 p11.2Chr1 q184.108.40.206Log2(H2AX/input)-0.1-0.253 MB MB MB200 MB MB MB MBFigure S1: H2AX distribution on human chromosomes.A, ChIP was performed on AsiSI-ER-U20S cells after 4OHT treatment using an anti-H2AX antibody (black bars) or no antibody (mock, white bars), followed by real time Q-PCR amplification with the indicated primers to assess H2AX distribution. A representative experiment is shown. B, Global H2AX (black, top) and mock (dark grey, bottom) profiles are shown across chromosomes 1 and 6. Enrichment is expressed as log2 relative to the input, and smoothed using a sliding window of 500 probes. A representative experiment is shown. The low enrichment of H2AX observed by ChIP-chip, is not due to low ChIP efficiency (since we could detect high levels of H2AX when analysing H2AX ChIP by Q-PCR) but reflects a general incorporation of H2AX along chromosome arms (as ChIP-chip experiments do not assess the absolute level of a protein on chromatin, but rather its change in distribution along the genome). Note however that, we can observe a increased presence of H2AX in regions harboring high gene density (light grey, upper panel). C, Detailed view of H2AX distribution across two genomic regions. The pericentromeric region of chr6p (left panel) is depleted in H2AX, whereas the q32.1 cytogenetic band of the chr1 (right panel) is enriched.
2 A B C Figure S2: H2AX is depleted around AsiSI sites. 0.350.3Log2(gH2AX Upstate /input)0.250.150.050.2Log2(gH2AX Abcam/input)0.1Distance from the AsiSI site (kb)Distance from the AsiSI site (kb)C0.4Log2(gH2AX Epitomics /input)0.30.20.1Distance from the AsiSI site (kb)Figure S2: H2AX is depleted around AsiSI sites.A. The log2 gH2AX/input signal (average of two gH2AX ChIP-chips after 4OHT treatment, performed with the Upstate gH2AX antibody) was calculated using a 1000 bp sliding window and is shown over a 20kb window centered on all AsiSI sites contained in gH2AX domains. B. Same as in A except that the log2 gH2AX /input was obtained using a different gH2AX antibody (Abcam ab2893). C. Same as in A except that the log2 gH2AX /input was obtained using a third gH2AX antibody (Epitomics ).
3 Chr 1_6Chr 6_4Log2(gH2AX/H2AX)Log2(gH2AX/input)Log2(gH2AX/H2AX)Log2(gH2AX/input)0.20.40.60.8Log20.80.6Log220.127.116.11.40.60.8Log2Chr 1_18.104.22.168.8Log2Chr 6_5Log2(gH2AX/H2AX)Log2(gH2AX/input)Log2(gH2AX/H2AX)Log2(gH2AX/input)0.20.40.60.8Log22.214.171.124.8Log2Chr 1_12Log2(gH2AX/H2AX)Log2(gH2AX/input)Chr 6_7Log2(gH2AX/H2AX)Log2(gH2AX/input)Figure S3: The H2AX profile is very similar when analyzed over H2AX or input.Detailed views around selected AsiSI sites (indicated by arrows) of the H2AX enrichment over H2AX (in light red) or input (in dark red), expressed as log2 and smoothed using a 500 probe sliding window. ChIP-chip analysis was performed using chromatin from AsiSI-ER-U20S cells treated with 4OHT. A representative experiment (performed with the Upstate gH2AX antibody) is shown. Note the strong similarity between the two profiles.
4 Abcam ab2893UpstateChr 1_6EpitomicsAbcam ab28931UpstateLog2(gH2AX/input)0.60.2Chr 1_8Chr 1_810.6Log2(gH2AX/input)0.2Figure S4: The H2AX profile is consistent between three gH2AX antibodies.Detailed views, around selected AsiSI sites (indicated by arrows), of the H2AX enrichment over input obtained with the Upstate gH2AX antibody (in red), with the Abcam gH2AX antibody (in black), or with the Epitomics antibody (in orange) expressed as log2 and smoothed using a 500 probe sliding window. ChIP-chip analyses was performed using chromatin from AsiSI-ER-U20S cells treated with 4OHT. Representative experiments are shown. Note the strong similarity between the three profiles.
5 chr1_6chr6_126.96.36.199.8U20SU20S0.40.8Log2 gH2AX/input0.40.8T98G_G2Log2 gH2AX/inputT98G_G188.8.131.52.8T98G_G1T98G_G1chr6_40.40.8U20S0.40.8Log2 gH2AX/inputT98G_G20.40.8T98G_G1Figure S5: H2AX profiles are consistent between cell lines and cell cycle phases.Detailed views of H2AX enrichment over input (expressed as log2 and smoothed using a 500 probe sliding window), across several domains of chromosome 1 and 6. ChIP-chip was performed using chromatin from AsiSI-ER-U20S cells (dark red), or AsiSI-ER-T98G in G1 phase (orange), and AsiSI-ER-T98G in G2 phase (red) treated with 4OHT for 4 hours. Representative experiments (performed with the Epitomics antibody) are shown. Arrows indicate AsiSI site positions.
6 ABCgH2AX AbcamgH2AX UpstategH2AX Epitomics0.350.2184.108.40.206.250.2Log2(gH2AX Epitomics/input)0.20.15Log2(gH2AX Upstate/input)0.160.15Log2(gH2AX Abcam/input)0.120.10.050.080.05Distance from the TSS (kb)Distance from the TSS (kb)Distance from the TSS (kb)DEH2AX181410620.080.250.150.05Log2(gH2AX/H3)0.04PolII enrichment (ChIP-seq)gH2AXPol IILog2(H2AX/input)-0.04Distance from the TSS (kb)Distance from the TSS (kb)Figure S6: Profiles of gH2AX and H2AX across transcription start sites (TSS).A, The 368 genes contained within the gH2AX domains were oriented with respect to transcription start sites (with the transcribed region on the right). The log2 gH2AX/input signal obtained with the gH2AX antibody from Abcam was calculated using a 200 bp sliding window and is shown over a 20kb window centered on the TSS. B, Same as in A, except that the log2 gH2AX/input signal was obtained with the gH2AX antibody from Upstate. C, Same as in A, except that the log2 gH2AX/input signal was obtained with the gH2AX antibody from Epitomics. D, Same as in A, except that the log2 gH2AX/H3 signal is plotted. E, Same as in A, except that the log2 H2AX/input signal is plotted.
7 A4242Log2(pol II/input)Log2(pol II/input)(+)Strand (+)Strand (-)4242Log2(pol II/input)Log2(pol II/input)(+)Strand (+)Strand (-)B0.50.40.30.20.1Log2(pol II/input)Distance to the TSS (kb)Figure S7: Pol II is enriched on genes and at gene promoters.A, Detailed view of PolII binding (in untreated AsiSI-ER-U20S) on selected genes from chromosome 1. Note that PolII can bind over the entire gene locus or can be restricted to the promoter region. B, 3072 genes, located on chromosome 1 and 6, were oriented with respect to transcription (with the transcribed sequence on the right) and the log2 PolII/input signal was calculated using a 200 base sliding window and is shown over a 20kb window centered on the TSS position. Note that, as expected (Barski et al, 2007), PolII is mainly enriched at promoters on a genome wide scale.
8 A B C D Figure S8: gH2AX and Pol II binding are mutually exclusive. 0.05Pol II -4OHT1234-1Log2 (Pol II/ input)0.02Log2(Pol II/input)-0.02-5+5Distance from the border (kb)0.511.5Log2(gH2AX/H2AX)CDMean log2( gH2AX/input)on genesMean log2( gH2AX/H3)on genesMean log2( Pol II/input)on genesMean log2( Pol II/input)on genesFigure S8: gH2AX and Pol II binding are mutually exclusive.A, The 534 “hole” borders previously identified were aligned and overlaid (right and mirror left borders are combined). The white part of the graph corresponds to gH2AX “holes” (as on Figure 6A). The profile of PolII over a 10kb window centered on the hole border and averaged using a 500 base window size is shown. Note that PolII levels are higher in gH2AX holes. B, The log2 (PolII/input) from two independent experiments was averaged, and for each probe encompassed by the previously defined gH2AX domains, the log2 PolII/input (y axis) was plotted against the log2 gH2AX/H2AX (x axis). The probes showing a high value for gH2AX/H2AX have a low value for PolII, and vice versa, indicating that PolII and gH2AX are mutually exclusive. C, The log2 (Pol II/input) (x axis) and log2 (gH2AX /input) (y axis) signals were averaged on each of the 368 genes encompassed in gH2AX domains (from the TSS to the end of the gene), and plotted against each other. Genes showing high Pol II value show low gH2AX level. D, Same as in C, except that the gH2AX/H3 signal is used in y axis.
9 A B Figure S9: High RNA levels and gH2AX are mutually exclusive . 9.77Distance from the border (kb)Transcription on (-) strandRNA(-) -4OHT9.829.609.71+5-5RNA(+) -4OHTTranscription on (+) strand9.659.52-5+5Distance from the border (kb)BMean log2( gH2AX/H2AX) on genesMean log2( gH2AX/H3) on genesMean Log2(gH2AX/input) on genesMean sense RNA on genesMean sense RNA on genesMean Sense RNA on genesFigure S9: High RNA levels and gH2AX are mutually exclusive .A, RNA were extracted from AsiSI-ER-U20S cells (without 4OHT), and reverse transcribed using a protocol that keeps strand information, in order to analyze (+) and (-) strand expression (see Material and Methods). cDNAs were hybridized on the Affymetrix Human Tilling 2.0 A array in order to generate high resolution strand specific expression maps. The 534 borders of gH2AX “holes” previously identified were aligned and overlaid (right and mirror left borders are combined). The profile of the RNA transcribed from the (+) strand (upper panel) and the (-) strand (lower panel), are shown over a 10kb window centered on the hole’s border, and averaged using a 500 base window size. As for PolII binding, RNA levels are increased in gH2AX holes. B, The sense RNA signal for each genes (obtained from the (-) or (+) strand signal depending on gene orientation, see Material and Methods), obtained by the strand specific expression profiling experiment were averaged on each of the 368 genes encompassed in gH2AX domains (from the TSS to the end of the gene). For each of these genes the log2 (gH2AX Upstate/H2AX) (left panel), the lod2 (gH2AX Upstate/H3) (middle panel), or log2 (gH2AX Upstate/input) (right panel) were averaged as well. gH2AX (y axis) and RNA value (x axis) were plotted against each other. As for Pol II binding, the genes showing high expression levels show low gH2AX levels, irrespective of the normalization against H2AX,H3, or input.
10 Figure S10: Cleavage efficiency on AsiSI sites % of cleveage efficiencyFigure S10: Cleavage efficiency on AsiSI sitesGenomic DNA was extracted before and after 4OHT treatment and assayed for cleavage at AsiSI sites as described in the Material and Methods section. In these experiments, an AsiSI linearized plasmid was added to each sample before performing ligation, as a normalization control. Pulled down DNA was analyzed by quantitative PCR amplification using primers close to three cleaved AsiSI sites, and two control (uncleaved) sequences. Cleavage efficiency (as a percentage) was calculated relative to the signal obtained with primers located on the AsiSI linearized plasmid. Data shown correspond to the mean and standard deviation from three independent experiments.
11 Mean sense RNA + 4OHTMean sense RNA – 4OHTFigure S11: Gene Transcription in gH2AX domains is not effected by DSB inductionRNA levels were assessed by strand expression profiling with or without 4OHT treatment (see Material and Methods). For each of the 368 genes located within gH2AX domains, sense expression was analyzed by averaging the signal over the gene from either the cDNA1 or cDNA2 array experiments, depending on each gene’s orientation.
12 Figure S12: Model of 3D gH2AX spreading. transcriptionfactorygH2AXfocigH2AXfociFigure S12: Model of 3D gH2AX spreading.The current model of chromosome organization in the nucleus is based on the existence of clusters of chromatin loops aggregated into 3-dimensional domains (Dorman et al, 2007). Large chromosomal domains may be delimited by elements (depicted in blue) that could therefore block the spreading of gH2AX. Inside gH2AX foci (in red), some loops could be withdraw from the foci, for example to be transcribed in transcription factories (in green), therefore leading to “holes” within the gH2AX domain (as seen when depicted linearly). In addition, some regions distant from the break (but still encompassed in the same large chromosomal domain) may be physically proximal to the break within the nucleus, and therefore covered by gH2AX. This model also explains how the state of gene transcription can be maintained even upon DSB induction and gH2AX focus formation.
13 Table S1: Positions of gH2AX-enriched domains. 4OHTchrN°Left boundaryRight boundaryAnnotationScore+11667769796Telomere2AsiSI34567891011Prox AsiSI12131415165015160606171819202122232425262728-737291153485Table S1: Positions of gH2AX-enriched domains.Domains were demarcated using the average of gH2AX over H2AX signal from two independent experiments by an in house algorithm (see Materials and Methods). Positions are according to the UCSC hg18 release.
14 symmetry (ratio right/left spreading distance) Left boundaryRight boundaryAsiSI position (hg18)Domain size (bp)left spreading (bp)right spreading (bp)symmetry (ratio right/left spreading distance)chr1_1chr1_2chr1_3chr1_4476384274792201592chr1_5554152476669chr1_6443932chr1_7chr1_8chr1_9chr1_10842946420890422056chr1_11646115338646307469chr1_12587535821608chr1_13693087133815559272chr6_1766953526394240559chr6_2chr6_3chr6_4841731492983chr6_5chr6_6619227918487chr6_7chr6_8487069chr6_9767909422008345901chr6_10625732790406Table S2: Final set of gH2AX domains used in our analyses.A select set of the previously identified gH2AX domains (Supplementary Table S1) were merged in cases where multiple domains corresponded to a single AsiSI site.These domains were used for the further studies (i.e., “holes” detection, and gH2AX signal across genes). Size and symmetry were however analyzed only for domains that contain a single AsiSI site. Note that domains can be quite asymmetrical relative to the DSB position.
15 Primers used for the Q-PCR chr22: _dist_FW: CCCATCTCAACCTCCACACTchr22: _dist_REV CTTGTCCAGATTCGCTGTGAchr22: _prox_FW :CCTTCTTTCCCAGTGGTTCAchr22: _prox_REV: GTGGTCTGACCCAGAGTGGTchr22: _dist_FW: TGGCTGGAACTGCTTTCTTTchr22: _dist_REV: GGTGAGTGAATGAGCTGCAAchr22: _prox_FW: ATGCCATGTGTCCTGATGAAchr22: _prox_REV CTGACTGGTGGCTTTTCCATchr1_6: _FW: GATTGGCTATGGGTGTGGACchr1_6: _REV CATCCTTGCAAACCAGTCCTchr1_8: _FW CCCTGGAGGTAGGTCTGGTchr1_8: _REV CGCACACTCACTGGTTCCTchr6_12_ _FW TGCCGGTCTCCTAGAAGTTGchr6_12_ _REV GCGCTTGATTTCCCTGAGTchr6: _FW : ACCTGGGATGGGACATATCAchr6: _REV: TACCAAGCCTGTCCCTGAACchr6: _FW: CAAACACACTCCCCCGTACT chr6: _REV: CTGGGTTTTCTCCACTGCTGchr1: _FW CGAGATCCAAGGAAGTCGTGchr1: _REV CCCCGGACACTTTAAAAGGAARV1_FW aaccaggaggccaaagagttARV1_REV ccaccacctcaggtatgcttSARS_FW ctggcctgtctacctgcttcSARS_REV ctggcagcatgattcaaagaCELSR2_FW gtgactcaaacccgtgtcctCELSR2_REV ctcacagtatggcccaaggtAMPD2_FW cgtagtgccccgtatgagttAMPD2_REV cgagtcactgtccgtcttcaC6ORF129_FW gaggagaagctgtcccagtgC6ORF129_REV atagacgagcgtcaggaggaZFAND3_FW ggaggaagccatcatgaaaaZFAND3_REV tggctggctaaagaaaggaaPrimers used for the Double Strand oligonucleotide in cleavage assay:FW CGC AAG CTT TAA-TAC-GAC-TCA-CTA-TAG-GGREV Biot-CC CTA TAG TGA GTC GTA TTA AAG CTT GCG ATTable S3: Sequence of primers used for Q-PCR amplification and Cleavage assay