Presentation on theme: "Chr6 p11.2 B A Gene density Log2 (H2AX/input) Log2 (mock/input) -0.4 0 0.4 -0.4 0 0.4 C 53 MB 55 MB 57 MB 200 MB 202 MB 204 MB 206MB 0 0.2 -0.2 Log2(H2AX/input)"— Presentation transcript:
Chr6 p11.2 B A Gene density Log2 (H2AX/input) Log2 (mock/input) C 53 MB 55 MB 57 MB 200 MB 202 MB 204 MB 206MB Log2(H2AX/input) Chr1 q OHT Prox Primers Dist ChIP efficiency (%input) mock H2AX ProxDistProx chr22: chr21: chr6: chr6: AsiSI site Log2(H2AX/input) Figure 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. Chr6Chr1
B Log2( H2AX Abcam/input) Distance from the AsiSI site (kb) A Distance from the AsiSI site (kb) Figure S2: H2AX is depleted around AsiSI sites. A. The log2 H2AX/input signal (average of two H2AX ChIP-chips after 4OHT treatment, performed with the Upstate H2AX antibody) was calculated using a 1000 bp sliding window and is shown over a 20kb window centered on all AsiSI sites contained in H2AX domains. B. Same as in A except that the log2 H2AX /input was obtained using a different H2AX antibody (Abcam ab2893). C. Same as in A except that the log2 H2AX /input was obtained using a third H2AX antibody (Epitomics ). C Distance from the AsiSI site (kb) Log2( H2AX Epitomics /input) Log2( H2AX Upstate /input)
Log Log Log Log Log Log2 Log 2 ( H2AX/H2AX) Log 2 ( H2AX/input) Log 2 ( H2AX/H2AX) Log 2 ( H2AX/input) Log 2 ( H2AX/H2AX) Log 2 ( H2AX/input) Log 2 ( H2AX/H2AX) Log 2 ( H2AX/input) Log 2 ( H2AX/H2AX) Log 2 ( H2AX/input) Log 2 ( H2AX/H2AX) Log 2 ( H2AX/input) Chr 1_6 Chr 1_12 Chr 1_8 Chr 6_7 Chr 6_5 Chr 6_ 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 H2AX antibody) is shown. Note the strong similarity between the two profiles.
Log2( H2AX/input) Chr 1_6 Chr 1_8 Abcam ab2893 Upstate Abcam ab2893 Upstate Figure S4: The H2AX profile is consistent between three H2AX antibodies. Detailed views, around selected AsiSI sites (indicated by arrows), of the H2AX enrichment over input obtained with the Upstate H2AX antibody (in red), with the Abcam H2AX 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 Chr 1_ Log2( H2AX/input) Epitomics
Figure 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 Log2 H2AX/input chr1_6chr6_7 U20S T98G_G2 T98G_G1 U20S T98G_G2 T98G_G1 Log2 H2AX/input U20S T98G_G2 T98G_G1 Log2 H2AX/input chr6_4
Figure S6: Profiles of H2AX and H2AX across transcription start sites (TSS). A, The 368 genes contained within the H2AX domains were oriented with respect to transcription start sites (with the transcribed region on the right). The log2 H2AX/input signal obtained with the H2AX 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 H2AX/input signal was obtained with the H2AX antibody from Upstate. C, Same as in A, except that the log2 H2AX/input signal was obtained with the H2AX antibody from Epitomics. D, Same as in A, except that the log2 H2AX/H3 signal is plotted. E, Same as in A, except that the log2 H2AX/input signal is plotted. A Log2( H2AX Abcam/input) H2AX Abcam Distance from the TSS (kb) CB Log2( H2AX Upstate/input) Distance from the TSS (kb) H2AX Upstate Log2( H2AX Epitomics/input) Distance from the TSS (kb) H2AX Epitomics Distance from the TSS (kb) PolII enrichment (ChIP-seq) Log2( H2AX/H3) H2AX Pol II D Log2(H2AX/input) Distance from the TSS (kb) E H2AX
B Log2(pol II/input) A (+) (+) Strand (+) Strand (-) Distance to the TSS (kb) Log2(pol II/input) (+) (+) Strand (+) Strand (-) 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 Log2(pol II/input) Log2(pol II/input) 4 2 0
A Distance from the border (kb) Pol II -4OHT Log2 (Pol II/ input) B Log2( H2AX/H2AX) Log2(Pol II/input) Figure S8: H2AX 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 H2AX 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 H2AX holes. B, The log2 (PolII/input) from two independent experiments was averaged, and for each probe encompassed by the previously defined H2AX domains, the log2 PolII/input (y axis) was plotted against the log2 H2AX/H2AX (x axis). The probes showing a high value for H2AX/H2AX have a low value for PolII, and vice versa, indicating that PolII and H2AX are mutually exclusive. C, The log2 (Pol II/input) (x axis) and log2 ( H2AX /input) (y axis) signals were averaged on each of the 368 genes encompassed in H2AX domains (from the TSS to the end of the gene), and plotted against each other. Genes showing high Pol II value show low H2AX level. D, Same as in C, except that the H2AX/H3 signal is used in y axis. Mean log2( H2AX/input) on genes Mean log2( Pol II/input) on genes C Mean log2( H2AX/H3) on genes Mean log2( Pol II/input) on genes D
Mean log2( H2AX/H3) on genes Mean sense RNA on genes A B Figure S9: High RNA levels and H2AX 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 H2AX 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 holes border, and averaged using a 500 base window size. As for PolII binding, RNA levels are increased in H2AX 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 H2AX domains (from the TSS to the end of the gene). For each of these genes the log2 ( H2AX Upstate/H2AX) (left panel), the lod2 ( H2AX Upstate/H3) (middle panel), or log2 ( H2AX Upstate/input) (right panel) were averaged as well. H2AX (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 H2AX levels, irrespective of the normalization against H2AX,H3, or input. Distance from the border (kb) Transcription on (+) strand RNA(+) -4OHT Distance from the border (kb) Transcription on (-) strand RNA(-) -4OHT Mean log2( H2AX/H2AX) on genes Mean sense RNA on genes Mean Sense RNA on genes Mean Log2( H2AX/input) on genes
% of cleveage efficiency Figure S10: Cleavage efficiency on AsiSI sites Genomic 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.
Mean sense RNA + 4OHT Mean sense RNA – 4OHT Figure S11: Gene Transcription in H2AX domains is not effected by DSB induction RNA levels were assessed by strand expression profiling with or without 4OHT treatment (see Material and Methods). For each of the 368 genes located within H2AX domains, sense expression was analyzed by averaging the signal over the gene from either the cDNA1 or cDNA2 array experiments, depending on each genes orientation.
transcription factory H2AX foci H2AX foci Figure S12: Model of 3D H2AX 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 H2AX. Inside H2AX 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 H2AX 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 H2AX. This model also explains how the state of gene transcription can be maintained even upon DSB induction and H2AX focus formation.
4OHT chrN° Left boundary Right boundary AnnotationScore Telomere AsiSI AsiSI AsiSI AsiSI AsiSI AsiSI AsiSI AsiSI AsiSI Prox AsiSI AsiSI AsiSI AsiSI AsiSI Telomere AsiSI AsiSI AsiSI AsiSI AsiSI Prox AsiSI AsiSI AsiSI AsiSI AsiSI AsiSI Telomere Telomere Prox AsiSI Telomere Telomere Table S1: Positions of H2AX-enriched domains. Domains were demarcated using the average of H2AX over H2AX signal from two independent experiments by an in house algorithm (see Materials and Methods). Positions are according to the UCSC hg18 release.
N° Left boundary Right boundary AsiSI position (hg18) Domain size (bp) left spreading (bp) right spreading (bp) symmetry (ratio right/left spreading distance) chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr1_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ chr6_ Table S2: Final set of H2AX domains used in our analyses. A select set of the previously identified H2AX 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 H2AX 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.
Primers used for the Q-PCR chr22: _dist_FW: CCCATCTCAACCTCCACACT chr22: _dist_REV CTTGTCCAGATTCGCTGTGA chr22: _prox_FW :CCTTCTTTCCCAGTGGTTCA chr22: _prox_REV: GTGGTCTGACCCAGAGTGGT chr22: _dist_FW: TGGCTGGAACTGCTTTCTTT chr22: _dist_REV: GGTGAGTGAATGAGCTGCAA chr22: _prox_FW: ATGCCATGTGTCCTGATGAA chr22: _prox_REV CTGACTGGTGGCTTTTCCAT chr1_6: _FW: GATTGGCTATGGGTGTGGAC chr1_6: _REV CATCCTTGCAAACCAGTCCT chr1_8: _FW CCCTGGAGGTAGGTCTGGT chr1_8: _REV CGCACACTCACTGGTTCCT chr6_12_ _FW TGCCGGTCTCCTAGAAGTTG chr6_12_ _REV GCGCTTGATTTCCCTGAGT chr6: _FW : ACCTGGGATGGGACATATCA chr6: _REV: TACCAAGCCTGTCCCTGAAC chr6: _FW: CAAACACACTCCCCCGTACT chr6: _REV: CTGGGTTTTCTCCACTGCTG chr1: _FW CGAGATCCAAGGAAGTCGTG chr1: _REV CCCCGGACACTTTAAAAGGA ARV1_FW AACCAGGAGGCCAAAGAGTT ARV1_REV CCACCACCTCAGGTATGCTT SARS_FW CTGGCCTGTCTACCTGCTTC SARS_REV CTGGCAGCATGATTCAAAGA CELSR2_FW GTGACTCAAACCCGTGTCCT CELSR2_REV CTCACAGTATGGCCCAAGGT AMPD2_FW CGTAGTGCCCCGTATGAGTT AMPD2_REV CGAGTCACTGTCCGTCTTCA C6ORF129_FW GAGGAGAAGCTGTCCCAGTG C6ORF129_REV ATAGACGAGCGTCAGGAGGA ZFAND3_FW GGAGGAAGCCATCATGAAAA ZFAND3_REV TGGCTGGCTAAAGAAAGGAA Primers used for the Double Strand oligonucleotide in cleavage assay: FW CGC AAG CTT TAA-TAC-GAC-TCA-CTA-TAG-GG REV Biot-CC CTA TAG TGA GTC GTA TTA AAG CTT GCG AT Table S3: Sequence of primers used for Q-PCR amplification and Cleavage assay