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0. 15 Ch. IP efficiency (%input) A H 2 AX 0. 1 0. 05 0. 15 Ch. IP efficiency (%input) A H 2 AX 0. 1 0. 05 0 4 OHT Primers Asi. SI site B mock - + Prox - + Dist chr 22: 19180307 - + Prox - + - + Dist Prox chr 21: 21292316 Chr 6 chr 6: 90404606 135861040 Chr 1 Gene density 0. 4 Log 2 0 (H 2 AX/input) -0. 4 Log 2 (mock/input) 0 -0. 4 C Chr 6 p 11. 2 0. 3 0. 2 Chr 1 q 32. 1 0. 2 Log 2(H 2 AX/input) 0. 3 0. 1 0 -0. 1 -0. 2 53 MB 55 MB 57 MB 200 MB 202 MB 204 MB 206 MB Figure S 1: H 2 AX distribution on human chromosomes. A, Ch. IP was performed on Asi. SI-ER-U 20 S cells after 4 OHT treatment using an anti-H 2 AX antibody (black bars) or no antibody (mock, white bars), followed by real time Q-PCR amplification with the indicated primers to assess H 2 AX distribution. A representative experiment is shown. B, Global H 2 AX (black, top) and mock (dark grey, bottom) profiles are shown across chromosomes 1 and 6. Enrichment is expressed as log 2 relative to the input, and smoothed using a sliding window of 500 probes. A representative experiment is shown. The low enrichment of H 2 AX observed by Ch. IP-chip, is not due to low Ch. IP efficiency (since we could detect high levels of H 2 AX when analysing H 2 AX Ch. IP by Q-PCR) but reflects a general incorporation of H 2 AX along chromosome arms (as Ch. IP-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 H 2 AX in regions harboring high gene density (light grey, upper panel). C, Detailed view of H 2 AX distribution across two genomic regions. The pericentromeric region of chr 6 p (left panel) is depleted in H 2 AX, whereas the q 32. 1 cytogenetic band of the chr 1 (right panel) is enriched.

A B 0. 3 Log 2( H 2 AX Abcam/input) Log 2( H 2 A B 0. 3 Log 2( H 2 AX Abcam/input) Log 2( H 2 AX Upstate /input) 0. 35 0. 25 0. 15 0. 05 -8 -4 0 4 0. 2 0. 1 0 8 -8 Distance from the Asi. SI site (kb) C -4 0 4 8 Distance from the Asi. SI site (kb) Log 2( H 2 AX Epitomics /input) 0. 4 0. 3 0. 2 0. 1 0 -8 -4 0 4 8 Distance from the Asi. SI site (kb) Figure S 2: H 2 AX is depleted around Asi. SI sites. A. The log 2 H 2 AX/input signal (average of two H 2 AX Ch. IP-chips after 4 OHT treatment, performed with the Upstate 07 -164 H 2 AX antibody) was calculated using a 1000 bp sliding window and is shown over a 20 kb window centered on all Asi. SI sites contained in H 2 AX domains. B. Same as in A except that the log 2 H 2 AX /input was obtained using a different H 2 AX antibody (Abcam ab 2893). C. Same as in A except that the log 2 H 2 AX /input was obtained using a third H 2 AX antibody (Epitomics 2212 -1).

Chr 1_6 0. 8 Chr 6_4 Log 2( H 2 AX/H 2 AX) Log Chr 1_6 0. 8 Chr 6_4 Log 2( H 2 AX/H 2 AX) Log 2( H 2 AX/input) 0. 8 0. 6 Log 2 0. 6 0. 4 0. 2 88 500 000 89 500 000 90 500 0. 8 Chr 1_8 Log 2( H 2 AX/H 2 AX) Log 2( H 2 AX/input) 30 000 31 000 32 000 0. 8 0. 4 Log 2( H 2 AX/H 2 AX) Log 2( H 2 AX/input) 0. 4 0. 2 37 000 38 000 39 000 108 500 000 109 500 000 110 500 000 Chr 1_12 Log 2( H 2 AX/H 2 AX) Log 2( H 2 AX/input) 0. 8 0. 6 Chr 6_7 Log 2( H 2 AX/H 2 AX) Log 2( H 2 AX/input) 0. 6 Log 2 Chr 6_5 0. 6 Log 2 0. 6 0. 8 Log 2( H 2 AX/H 2 AX) Log 2( H 2 AX/input) 0. 4 0. 2 228 000 229 000 230 000 89 500 000 90 500 000 91 500 000 Figure S 3: The H 2 AX profile is very similar when analyzed over H 2 AX or input. Detailed views around selected Asi. SI sites (indicated by arrows) of the H 2 AX enrichment over H 2 AX (in light red) or input (in dark red), expressed as log 2 and smoothed using a 500 probe sliding window. Ch. IP-chip analysis was performed using chromatin from Asi. SI-ER-U 20 S cells treated with 4 OHT. A representative experiment (performed with the Upstate 07 -164 H 2 AX antibody) is shown. Note the strong similarity between the two profiles.

Abcam ab 2893 Upstate 07 -164 Epitomics 2212 -1 Log 2( H 2 AX/input) Abcam ab 2893 Upstate 07 -164 Epitomics 2212 -1 Log 2( H 2 AX/input) Chr 1_6 Abcam ab 2893 Upstate 07 -164 1 0. 6 0. 2 89 000 90 000 91 000 Chr 1_8 Log 2( H 2 AX/input) 1 0. 6 0. 2 109 000 110 000 111 000 Figure S 4: The H 2 AX profile is consistent between three H 2 AX antibodies. Detailed views, around selected Asi. SI sites (indicated by arrows), of the H 2 AX enrichment over input obtained with the Upstate H 2 AX antibody (in red), with the Abcam H 2 AX antibody (in black), or with the Epitomics antibody (in orange) expressed as log 2 and smoothed using a 500 probe sliding window. Ch. IP-chip analyses was performed using chromatin from Asi. SI-ER-U 20 S cells treated with 4 OHT. Representative experiments are shown. Note the strong similarity between the three profiles.

U 20 S T 98 G_G 2 T 98 G_G 1 88 500 000 U 20 S T 98 G_G 2 T 98 G_G 1 88 500 000 89 500 000 90 500 000 Log 2 H 2 AX/input chr 1_6 0. 8 0. 4 0 chr 6_7 U 20 S T 98 G_G 2 T 98 G_G 1 90 000 91 000 92 000 chr 6_4 Log 2 H 2 AX/input 0. 8 0. 4 0 0. 8 U 20 S T 98 G_G 2 T 98 G_G 1 0. 4 0 30 000 31 000 32 000 Figure S 5: H 2 AX profiles are consistent between cell lines and cell cycle phases. Detailed views of H 2 AX enrichment over input (expressed as log 2 and smoothed using a 500 probe sliding window), across several domains of chromosome 1 and 6. Ch. IP-chip was performed using chromatin from Asi. SI-ER-U 20 S cells (dark red), or Asi. SI-ER-T 98 G in G 1 phase (orange), and Asi. SI-ER-T 98 G in G 2 phase (red) treated with 4 OHT for 4 hours. Representative experiments (performed with the Epitomics antibody) are shown. Arrows indicate Asi. SI site positions.

B A C 0. 35 0. 25 0. 15 0. 05 -8 -4 0 B A C 0. 35 0. 25 0. 15 0. 05 -8 -4 0 4 0. 28 0. 24 0. 2 0. 16 0. 12 0. 08 8 Log 2( H 2 AX Epitomics/input) H 2 AX Upstate Log 2( H 2 AX Upstate/input) Log 2( H 2 AX Abcam/input) H 2 AX Abcam Distance from the TSS (kb) -8 -4 0 4 0. 25 0. 2 0. 15 0. 1 -8 -4 0 4 8 Distance from the TSS (kb) E 0. 15 10 H 2 AX Pol II 0. 05 6 2 -8 -4 0 4 Distance from the TSS (kb) 8 Log 2(H 2 AX/input) 14 Pol. II enrichment (Ch. IP-seq) 0. 25 H 2 AX 0. 08 18 Log 2( H 2 AX/H 3) 0. 3 0. 05 8 Distance from the TSS (kb) D H 2 AX Epitomics 0. 04 0 -0. 04 -8 -4 0 4 8 Distance from the TSS (kb) Figure S 6: Profiles of H 2 AX and H 2 AX across transcription start sites (TSS). A, The 368 genes contained within the H 2 AX domains were oriented with respect to transcription start sites (with the transcribed region on the right). The log 2 H 2 AX/input signal obtained with the H 2 AX antibody from Abcam was calculated using a 200 bp sliding window and is shown over a 20 kb window centered on the TSS. B, Same as in A, except that the log 2 H 2 AX/input signal was obtained with the H 2 AX antibody from Upstate. C, Same as in A, except that the log 2 H 2 AX/input signal was obtained with the H 2 AX antibody from Epitomics. D, Same as in A, except that the log 2 H 2 AX/H 3 signal is plotted. E, Same as in A, except that the log 2 H 2 AX/input signal is plotted.

4 Log 2(pol II/input) A 2 0 ( + Strand (-) ) 4 2 4 Log 2(pol II/input) A 2 0 ( + Strand (-) ) 4 2 0 67 600 000 154 700 000 4 2 0 ( + Strand (-) ) 154 900 000 120 250 000 67 800 000 Log 2(pol II/input) Strand (+) 120 450 000 4 2 0 Strand (+) 119 500 000 119 300 000 B Log 2(pol II/input) 0. 5 0. 4 0. 3 0. 2 0. 1 -8 -4 0 4 8 Distance to the TSS (kb) Figure S 7: Pol II is enriched on genes and at gene promoters. A, Detailed view of Pol. II binding (in untreated Asi. SI-ER-U 20 S) on selected genes from chromosome 1. Note that Pol. II 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 log 2 Pol. II/input signal was calculated using a 200 base sliding window and is shown over a 20 kb window centered on the TSS position. Note that, as expected (Barski et al, 2007), Pol. II is mainly enriched at promoters on a genome wide scale.

A B 0. 05 Pol II -4 OHT Log 2(Pol II/input) Log 2 (Pol A B 0. 05 Pol II -4 OHT Log 2(Pol II/input) Log 2 (Pol II/ input) 4 0. 02 -5 3 2 1 0 -1 0 +5 Distance from the border (kb) 0. 5 0 0. 5 1 1. 5 -2. 5 D 1. 5 0. 9 Mean log 2( H 2 AX/H 3) on genes C Mean log 2( H 2 AX/input) on genes Log 2( H 2 AX/H 2 AX) 0. 5 -0. 5 1. 5 -0. 5 Mean log 2( Pol II/input) on genes 3. 5 0. 7 0. 5 0. 3 0. 1 -1 -0. 1 1 3 -0. 5 Mean log 2( Pol II/input) on genes Figure S 8: H 2 AX 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 H 2 AX “holes” (as on Figure 6 A). The profile of Pol. II over a 10 kb window centered on the hole border and averaged using a 500 base window size is shown. Note that Pol. II levels are higher in H 2 AX holes. B, The log 2 (Pol. II/input) from two independent experiments was averaged, and for each probe encompassed by the previously defined H 2 AX domains, the log 2 Pol. II/input (y axis) was plotted against the log 2 H 2 AX/H 2 AX (x axis). The probes showing a high value for H 2 AX/H 2 AX have a low value for Pol. II, and vice versa, indicating that Pol. II and H 2 AX are mutually exclusive. C, The log 2 (Pol II/input) (x axis) and log 2 ( H 2 AX /input) (y axis) signals were averaged on each of the 368 genes encompassed in H 2 AX domains (from the TSS to the end of the gene), and plotted against each other. Genes showing high Pol II value show low H 2 AX level. D, Same as in C, except that the H 2 AX/H 3 signal is used in y axis.

A 9. 82 RNA(+) -4 OHT Transcription on (-) strand Transcription on (+) strand A 9. 82 RNA(+) -4 OHT Transcription on (-) strand Transcription on (+) strand 9. 77 9. 65 9. 52 -5 0 +5 Distance from the border (kb) RNA(-) -4 OHT 9. 71 9. 60 -5 0 +5 Distance from the border (kb) 0. 8 0. 6 0. 4 0. 2 0 -0. 2 8 10 12 14 16 18 Mean sense RNA on genes 1 0. 8 0. 6 0. 4 0. 2 0 -0. 2 -0. 4 8 13 Mean sense RNA on genes 18 Mean Log 2( H 2 AX/input) on genes 1 Mean log 2( H 2 AX/H 3) on genes Mean log 2( H 2 AX/H 2 AX) on genes B 1. 5 1 0. 5 0 8 13 -0. 5 Mean Sense RNA on genes Figure S 9: High RNA levels and H 2 AX are mutually exclusive. A, RNA were extracted from Asi. SI-ER-U 20 S cells (without 4 OHT), and reverse transcribed using a protocol that keeps strand information, in order to analyze (+) and (-) strand expression (see Material and Methods). c. DNAs 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 H 2 AX “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 10 kb window centered on the hole’s border, and averaged using a 500 base window size. As for Pol. II binding, RNA levels are increased in H 2 AX 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 H 2 AX domains (from the TSS to the end of the gene). For each of these genes the log 2 ( H 2 AX Upstate/H 2 AX) (left panel), the lod 2 ( H 2 AX Upstate/H 3) (middle panel), or log 2 ( H 2 AX Upstate/input) (right panel) were averaged as well. H 2 AX (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 H 2 AX levels, irrespective of the normalization against H 2 AX, H 3, or input. 18

% of cleveage efficiency Figure S 10: Cleavage efficiency on Asi. SI sites Genomic % of cleveage efficiency Figure S 10: Cleavage efficiency on Asi. SI sites Genomic DNA was extracted before and after 4 OHT treatment and assayed for cleavage at Asi. SI sites as described in the Material and Methods section. In these experiments, an Asi. SI 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 Asi. SI sites, and two control (uncleaved) sequences. Cleavage efficiency (as a percentage) was calculated relative to the signal obtained with primers located on the Asi. SI linearized plasmid. Data shown correspond to the mean and standard deviation from three independent experiments.

Mean sense RNA + 4 OHT 17 15 13 11 9 7 7 12 Mean sense RNA + 4 OHT 17 15 13 11 9 7 7 12 17 Mean sense RNA – 4 OHT Figure S 11: Gene Transcription in H 2 AX domains is not effected by DSB induction RNA levels were assessed by strand expression profiling with or without 4 OHT treatment (see Material and Methods). For each of the 368 genes located within H 2 AX domains, sense expression was analyzed by averaging the signal over the gene from either the c. DNA 1 or c. DNA 2 array experiments, depending on each gene’s orientation.

transcription factory H 2 AX foci Figure S 12: Model of 3 D H transcription factory H 2 AX foci Figure S 12: Model of 3 D H 2 AX 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 H 2 AX. Inside H 2 AX 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 H 2 AX 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 H 2 AX. This model also explains how the state of gene transcription can be maintained even upon DSB induction and H 2 AX focus formation.

 4 OHT + + + + + + + - chr N° 1 4 OHT + + + + + + + - chr N° 1 1 1 1 6 6 6 6 1 1 6 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 1 2 3 4 Left boundary 1667 8856659 13330738 19176113 25170848 40193077 88787251 91621723 108792296 202097763 202903944 206062519 221760623 228483320 240620585 5015 15710622 20147573 26312797 30371674 36827745 38256019 49406313 89662279 135373970 144227252 149304066 170729773 1667 222110520 5015 170727437 Right boundary 769796 10458126 15001758 20477024 25647232 41223898 90495397 92216354 110684622 202793777 203068866 206905465 222406738 229892463 241313672 160606 16477575 20707680 28189029 31706388 37905795 38889459 50944027 91549544 136997035 144995161 150720204 170896781 737291 222287958 153485 170896781 Annotation Score Telomere Asi. SI Asi. SI Prox Asi. SI Telomere Asi. SI Prox Asi. SI Telomere Prox Asi. SI Telomere 0. 21065718 0. 1815597 0. 11594671 0. 17012137 0. 12276864 0. 20742705 0. 36496324 0. 14798611 0. 31235426 0. 15728331 0. 1146983 0. 10589541 0. 13346664 0. 24552357 0. 17528072 0. 13853534 0. 11982131 0. 10790963 0. 12851565 0. 26693177 0. 22907432 0. 1109641 0. 16586761 0. 22628273 0. 13061982 0. 20552018 0. 12267045 0. 2488081 0. 22899012 0. 12393546 0. 14786207 0. 20948009 Table S 1: Positions of H 2 AX-enriched domains. Domains were demarcated using the average of H 2 AX over H 2 AX signal from two independent experiments by an in house algorithm (see Materials and Methods). Positions are according to the UCSC hg 18 release.

Left boundary N° chr 1_1 Right boundary Asi. SI position (hg 18) 8856659 10458126 Left boundary N° chr 1_1 Right boundary Asi. SI position (hg 18) 8856659 10458126 9572031 -9634451 symmetry left right Domain (ratio right/left spreading size (bp) spreading (bp) distance) chr 1_2 13330738 15001758 13948767 -14015273 -14797808 chr 1_3 19176113 20477024 19684740 -19845677 chr 1_4 25170848 25647232 25445640 476384 274792 201592 0. 733616699 chr 1_5 40193077 41223898 40747229 1030821 554152 476669 0. 860177352 chr 1_6 88787251 90495397 89231183 1708146 443932 1264214 2. 847764973 chr 1_7 91621723 92216354 91970661 -92144412 -92156699 chr 1_8 108792296 110684622 109838221 -110120612110329096 chr 1_9 202097763 203068866 202098047 -202647074 chr 1_10 206062519 206905465 206483409 842946 420890 422056 1. 00277032 chr 1_11 221760623 222406738 222099269 646115 338646 307469 0. 907936311 chr 1_12 228483320 229892463 229070855 1409143 587535 821608 1. 398398393 chr 1_13 240620585 241313672 240754400 693087 133815 559272 4. 179441767 chr 6_1 15710622 16477575 16237016 766953 526394 240559 0. 456994191 chr 6_2 20147573 20707680 20320298 -20664300 chr 6_3 26312797 28189029 26768137 -27253344 -27769878 chr 6_4 30371674 31706388 31213405 1334714 492983 0. 58567761 chr 6_5 36827745 38889459 37184118 -37429778 chr 6_6 49406313 50944027 50025540 1537714 918487 1. 48327996 chr 6_7 89662279 91549544 89764157 -90404906 chr 6_8 135373970 136997035 135861039 1623065 487069 1135996 2. 332310207 chr 6_9 144227252 144995161 144649260 767909 422008 345901 0. 819655078 chr 6_10 149304066 150720204 149929798 1416138 625732 790406 1. 263170175 841731 619227 Table S 2: Final set of H 2 AX domains used in our analyses. A select set of the previously identified H 2 AX domains (Supplementary Table S 1) were merged in cases where multiple domains corresponded to a single Asi. SI site. These domains were used for the further studies (i. e. , “holes” detection, and H 2 AX signal across genes). Size and symmetry were however analyzed only for domains that contain a single Asi. SI site. Note that domains can be quite asymmetrical relative to the DSB position.

Primers used for the Q-PCR chr 22: 19180307_dist_FW: CCCATCTCAACCTCCACACT chr 22: 19180307_dist_REV CTTGTCCAGATTCGCTGTGA chr Primers used for the Q-PCR chr 22: 19180307_dist_FW: CCCATCTCAACCTCCACACT chr 22: 19180307_dist_REV CTTGTCCAGATTCGCTGTGA chr 22: 19180307_prox_FW : CCTTCTTTCCCAGTGGTTCA chr 22: 19180307_prox_REV: GTGGTCTGACCCAGAGTGGT chr 22: 21292316_dist_FW: TGGCTGGAACTGCTTT chr 22: 21292316_dist_REV: GGTGAATGAGCTGCAA chr 22: 21292316_prox_FW: ATGCCATGTGTCCTGATGAA chr 22: 21292316_prox_REV CTGACTGGTGGCTTTTCCAT chr 1_6: 89231183_FW: GATTGGCTATGGGTGTGGAC chr 1_6: 89231183_REV CATCCTTGCAAACCAGTCCT chr 1_8: 109838221_FW CCCTGGAGGTCTGGT chr 1_8: 109838221_REV CGCACACTGGTTCCT chr 6_12_90404906_FW TGCCGGTCTCCTAGAAGTTG chr 6_12_90404906_REV GCGCTTGATTTCCCTGAGT chr 6: 101505264_FW : ACCTGGGACATATCA chr 6: 101505264_REV: TACCAAGCCTGTCCCTGAAC chr 6: 40663811_FW: CAAACACACTCCCCCGTACT chr 6: 40663811_REV: CTGGGTTTTCTCCACTGCTG chr 1: 3092903_FW CGAGATCCAAGGAAGTCGTG chr 1: 3092903 _REV CCCCGGACACTTTAAAAGGA ARV 1_FW AACCAGGAGGCCAAAGAGTT ARV 1_REV CCACCACCTCAGGTATGCTT SARS_FW CTGGCCTGTCTACCTGCTTC SARS_REV CTGGCAGCATGATTCAAAGA CELSR 2_FW GTGACTCAAACCCGTGTCCT CELSR 2_REV CTCACAGTATGGCCCAAGGT AMPD 2_FW CGTAGTGCCCCGTATGAGTT AMPD 2_REV CGAGTCACTGTCCGTCTTCA C 6 ORF 129_FW GAGGAGAAGCTGTCCCAGTG C 6 ORF 129_REV ATAGACGAGCGTCAGGAGGA ZFAND 3_FW GGAGGAAGCCATCATGAAAA ZFAND 3_REV TGGCTAAAGGAA 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 S 3: Sequence of primers used for Q-PCR amplification and Cleavage assay