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)

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chr21: 21292316

chr6:90404606

chr6: 135861040 AsiSI site

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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.

Chr6 Chr1

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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 07-164 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 2212-1).

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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 07-164 H2AX antibody) is shown. Note the strong similarity between the two profiles.

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Chr 1_6

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Abcam ab2893Upstate 07-164

Abcam ab2893Upstate 07-164

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.

1

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109 000 000 110 000 000 111 000 000

Log2

(H

2AX/

inpu

t)Epitomics 2212-1

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.

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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.

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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.

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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.

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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 hole’s 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.

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Figure 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 sitesandtwo 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.

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Figure S11: Gene Transcription in H2AX 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 H2AX 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.

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

chr N°Left

boundaryRight

boundaryAnnotation Score

+ 1 1 1667 769796 Telomere 0.21065718+ 1 2 8856659 10458126 AsiSI 0.1815597+ 1 3 13330738 15001758 AsiSI 0.11594671+ 1 4 19176113 20477024 AsiSI 0.17012137+ 1 5 25170848 25647232 AsiSI 0.12276864+ 1 6 40193077 41223898 AsiSI 0.20742705+ 1 7 88787251 90495397 AsiSI 0.36496324+ 1 8 91621723 92216354 AsiSI 0.14798611+ 1 9 108792296 110684622 AsiSI 0.31235426+ 1 10 202097763 202793777 AsiSI 0.15728331+ 1 11 202903944 203068866 Prox AsiSI 0.1146983+ 1 12 206062519 206905465 AsiSI 0.10589541+ 1 13 221760623 222406738 AsiSI 0.13346664+ 1 14 228483320 229892463 AsiSI 0.24552357+ 1 15 240620585 241313672 AsiSI 0.17528072+ 6 16 5015 160606 Telomere 0.13853534+ 6 17 15710622 16477575 AsiSI 0.11982131+ 6 18 20147573 20707680 AsiSI 0.10790963+ 6 19 26312797 28189029 AsiSI 0.12851565+ 6 20 30371674 31706388 AsiSI 0.26693177+ 6 21 36827745 37905795 AsiSI 0.22907432+ 6 22 38256019 38889459 Prox AsiSI 0.1109641+ 6 23 49406313 50944027 AsiSI 0.16586761+ 6 24 89662279 91549544 AsiSI 0.22628273+ 6 25 135373970 136997035 AsiSI 0.13061982+ 6 26 144227252 144995161 AsiSI 0.20552018+ 6 27 149304066 150720204 AsiSI 0.12267045+ 6 28 170729773 170896781 Telomere 0.2488081- 1 1 1667 737291 Telomere 0.22899012- 1 2 222110520 222287958 Prox AsiSI 0.12393546- 6 3 5015 153485 Telomere 0.14786207- 6 4 170727437 170896781 Telomere 0.20948009

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

boundaryRight

boundaryAsiSI position (hg18)

Domain size (bp)

left spreading

(bp)

right spreading

(bp)

symmetry (ratio

right/left spreading distance)

chr1_1 8856659 10458126 9572031-9634451

chr1_2 13330738 15001758 13948767-14015273-14797808

chr1_3 19176113 20477024 19684740-19845677

chr1_4 25170848 25647232 25445640 476384 274792 201592 0.733616699

chr1_5 40193077 41223898 40747229 1030821 554152 476669 0.860177352

chr1_6 88787251 90495397 89231183 1708146 443932 1264214 2.847764973

chr1_7 91621723 92216354 91970661-92144412-92156699

chr1_8 108792296 110684622109838221-110120612-

110329096

chr1_9 202097763 203068866 202098047-202647074

chr1_10 206062519 206905465 206483409 842946 420890 422056 1.00277032

chr1_11 221760623 222406738 222099269 646115 338646 307469 0.907936311

chr1_12 228483320 229892463 229070855 1409143 587535 821608 1.398398393

chr1_13 240620585 241313672 240754400 693087 133815 559272 4.179441767

chr6_1 15710622 16477575 16237016 766953 526394 240559 0.456994191

chr6_2 20147573 20707680 20320298-20664300

chr6_3 26312797 28189029 26768137-27253344-27769878

chr6_4 30371674 31706388 31213405 1334714 841731 492983 0.58567761

chr6_5 36827745 38889459 37184118-37429778

chr6_6 49406313 50944027 50025540 1537714 619227 918487 1.48327996

chr6_7 89662279 91549544 89764157-90404906

chr6_8 135373970 136997035 135861039 1623065 487069 1135996 2.332310207

chr6_9 144227252 144995161 144649260 767909 422008 345901 0.819655078

chr6_10 149304066 150720204 149929798 1416138 625732 790406 1.263170175

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

chr22:19180307_dist_FW: CCCATCTCAACCTCCACACTchr22:19180307_dist_REV CTTGTCCAGATTCGCTGTGA

chr22:19180307_prox_FW :CCTTCTTTCCCAGTGGTTCAchr22:19180307_prox_REV: GTGGTCTGACCCAGAGTGGT

chr22: 21292316_dist_FW: TGGCTGGAACTGCTTTCTTT chr22: 21292316_dist_REV: GGTGAGTGAATGAGCTGCAA

chr22: 21292316_prox_FW: ATGCCATGTGTCCTGATGAA chr22: 21292316_prox_REV CTGACTGGTGGCTTTTCCAT

chr1_6:89231183_FW: GATTGGCTATGGGTGTGGACchr1_6:89231183_REV CATCCTTGCAAACCAGTCCT

chr1_8:109838221_FW CCCTGGAGGTAGGTCTGGTchr1_8:109838221_REV CGCACACTCACTGGTTCCT

chr6_12_90404906_FW TGCCGGTCTCCTAGAAGTTGchr6_12_90404906_REV GCGCTTGATTTCCCTGAGT

chr6:101505264_FW : ACCTGGGATGGGACATATCAchr6:101505264_REV: TACCAAGCCTGTCCCTGAAC

chr6: 40663811_FW: CAAACACACTCCCCCGTACT chr6: 40663811_REV: CTGGGTTTTCTCCACTGCTG

chr1:3092903_FW CGAGATCCAAGGAAGTCGTGchr1:3092903 _REV CCCCGGACACTTTAAAAGGA

ARV1_FW AACCAGGAGGCCAAAGAGTTARV1_REV CCACCACCTCAGGTATGCTT

SARS_FW CTGGCCTGTCTACCTGCTTCSARS_REV CTGGCAGCATGATTCAAAGA

CELSR2_FW GTGACTCAAACCCGTGTCCTCELSR2_REV CTCACAGTATGGCCCAAGGT

AMPD2_FW CGTAGTGCCCCGTATGAGTTAMPD2_REV CGAGTCACTGTCCGTCTTCA

C6ORF129_FW GAGGAGAAGCTGTCCCAGTGC6ORF129_REV ATAGACGAGCGTCAGGAGGA

ZFAND3_FW GGAGGAAGCCATCATGAAAAZFAND3_REV TGGCTGGCTAAAGAAAGGAA

Primers 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 AT

Table S3: Sequence of primers used for Q-PCR amplification and Cleavage assay

Documents

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)