Download emboj201038-sup

A
ChIP efficiency
(%input)
0.15
H2AX
0.1
0.05
0
4OHT
Primers
AsiSI site
B
mock
-
+
Prox
- +
Dist
chr22:
19180307
- +
- +
Prox
- +
- +
- +
Dist
Prox
Prox
chr21:
21292316
Chr6
chr6:
chr6:
90404606 135861040
Chr1
Gene
density
0.4
Log2
0
(H2AX/input)
-0.4
0.4
Log2
(mock/input) 0
-0.4
C
Chr6 p11.2
Chr1 q32.1
0.3
0.2
Log2(H2AX/input)
Log2(H2AX/input)
0.3
0.2
0.1
0
-0.1
0.1
0
-0.1
-0.2
-0.2
53 MB
55 MB
57 MB
200 MB
202 MB
204 MB
206MB
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.
A
B
0.3
Log2(gH2AX Abcam/input)
Log2(gH2AX Upstate /input)
0.35
0.25
0.15
0.05
-8
-4
0
4
0.2
0.1
0
8
-8
Distance from the AsiSI site (kb)
C
-4
0
4
8
Distance from the AsiSI site (kb)
Log2(gH2AX Epitomics /input)
0.4
0.3
0.2
0.1
0
-8
-4
0
4
8
Distance from the AsiSI site (kb)
Figure S2: gH2AX is depleted around AsiSI sites.
A. The log2 gH2AX/input signal (average of two gH2AX ChIP-chips after 4OHT treatment,
performed with the Upstate 07-164 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 2212-1).
Chr 1_6
Chr 6_4
Log2(gH2AX/H2AX)
Log2(gH2AX/input)
0.8
0.6
Log2
Log2
0.6
0.4
0.4
0.2
0.2
88 500 000
0.8
89 500 000
Chr 1_8
90 500 000
Log2(gH2AX/H2AX)
Log2(gH2AX/input)
30 000 000
0.8
0.4
32 000 000
Chr 6_5
Log2(gH2AX/H2AX)
Log2(gH2AX/input)
0.4
0.2
0.2
37 000 000
108 500 000
109 500 000
Chr 1_12
38 000 000
39 000 000
110 500 000
Log2(gH2AX/H2AX)
Log2(gH2AX/input)
0.8
0.6
Chr 6_7
Log2(gH2AX/H2AX)
Log2(gH2AX/input)
0.6
Log2
Log2
31 000 000
0.6
Log2
Log2
0.6
0.8
Log2(gH2AX/H2AX)
Log2(gH2AX/input)
0.8
0.4
0.2
0.4
0.2
228 000 000
229 000 000
230 000 000
89 500 000
90 500 000
91 500 000
Figure S3: The gH2AX profile is very similar when analyzed over H2AX or input.
Detailed views around selected AsiSI sites (indicated by arrows) of the gH2AX
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 gH2AX antibody) is shown. Note the strong similarity
between the two profiles.
Abcam ab2893
Upstate 07-164
Epitomics 2212-1
Log2(gH2AX/input)
Chr 1_6
Abcam ab2893
Upstate 07-164
1
0.6
0.2
89 000 000
90 000 000
91 000 000
Chr 1_8
Log2(gH2AX/input)
Chr 1_8
1
0.6
0.2
109 000 000
110 000 000
111 000 000
Figure S4: The gH2AX profile is consistent between three gH2AX antibodies.
Detailed views, around selected AsiSI sites (indicated by arrows), of the gH2AX
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.
U20S
T98G_G2
T98G_G1
88 500 000
89 500 000
Log2 gH2AX/input
chr1_6
0.8
0.4
0
0.8
0.4
0
0.8
0.4
0
chr6_7
U20S
T98G_G2
T98G_G1
90 000 000
90 500 000
91 000 000
92 000 000
chr6_4
Log2 gH2AX/input
Log2 gH2AX/input
0.8
0.4
0
0.8
0.4
0
0.8
0.4
0
0.8
0.4
0
0.8
0.4
0
0.8
U20S
T98G_G2
T98G_G1
0.4
0
30 000 000
31 000 000
32 000 000
Figure S5: gH2AX profiles are consistent between cell lines and cell cycle phases.
Detailed views of gH2AX 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.
B
A
C
0.35
0.25
0.15
0.05
Log2(gH2AX Epitomics/input)
gH2AX Upstate
Log2(gH2AX Upstate/input)
0.28
0.24
0.2
0.16
0.12
0.08
-8
-4
0
4
-8
8
Distance from the TSS (kb)
-4
0
4
0.25
0.2
0.15
0.1
-8
-4
0
4
8
E
14
0.15
10
gH2AX
Pol II
0.05
6
2
-8
-4
0
4
Distance from the TSS (kb)
8
PolII enrichment (ChIP-seq)
0.25
H2AX
0.08
18
Log2(gH2AX/H3)
0.3
Distance from the TSS (kb)
Distance from the TSS (kb)
D
gH2AX Epitomics
0.05
8
Log2(H2AX/input)
Log2(gH2AX Abcam/input)
gH2AX Abcam
0.04
0
-0.04
-8
-4
0
4
8
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.
4
Log2(pol II/input)
Log2(pol II/input)
A
2
0
(
+
Strand (-)
)
4
2
0
67 600 000
67 800 000
4
Log2(pol II/input)
Log2(pol II/input)
Strand (+)
2
0
(
+
Strand (-)
)
154 700 000
154 900 000
120 250 000
120 450 000
4
2
0
Strand (+)
119 500 000
119 300 000
B
Log2(pol II/input)
0.5
0.4
0.3
0.2
0.1
-8
-4
0
4
8
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.
A
B
0.05
Pol II -4OHT
Log2(Pol II/input)
Log2 (Pol II/ input)
4
0.02
3
2
1
0
-0.02
-5
-1
0
+5
Distance from the border (kb)
0.5
0
0.5
1
1.5
Log2(gH2AX/H2AX)
-2.5
D
1.5
0.9
Mean log2( gH2AX/H3)
on genes
Mean log2( gH2AX/input)
on genes
C
0.5
-0.5
1.5
-0.5
Mean log2( Pol II/input)
on genes
3.5
0.7
0.5
0.3
0.1
-1 -0.1 0
1
2
3
-0.3
-0.5
Mean log2( Pol II/input)
on genes
Figure 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.
A
9.82
RNA(+) -4OHT
Transcription on (-) strand
Transcription on (+) strand
9.77
9.65
9.52
-5
0
+5
Distance from the border (kb)
RNA(-) -4OHT
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
8
13
-0.4
Mean sense RNA on genes
18
Mean Log2(gH2AX/input) on genes
1
Mean log2( gH2AX/H3) on genes
Mean log2( gH2AX/H2AX) on genes
B
1.5
1
0.5
0
8
13
-0.5
Mean Sense RNA on genes
Figure 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.
18
% of cleveage efficiency
100
90
-OHT
80
+OHT
70
60
50
40
30
20
10
0
1
chr1_6
2
chr1_8
3
chr6_7
4
chr22_ctrl
5
gapdh
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
17
15
13
11
9
7
7
9
11
13
15
17
Mean sense RNA – 4OHT
Figure S11: Gene Transcription in gH2AX 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 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.
transcription
factory
gH2AX
foci
gH2AX
foci
Figure 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.
4OHT
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
chr
N°
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6
6
6
6
6
6
6
6
6
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
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
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 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.
N°
chr1_1
Left
boundary
Right
boundary
AsiSI position (hg18)
8856659
10458126
9572031-9634451
symmetry
left
right
Domain
(ratio right/left
spreading spreading
size (bp)
spreading
(bp)
(bp)
distance)
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 110684622
109838221-110120612110329096
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 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.
Primers used for the Q-PCR
chr22:19180307_dist_FW: CCCATCTCAACCTCCACACT
chr22:19180307_dist_REV CTTGTCCAGATTCGCTGTGA
chr22:19180307_prox_FW :CCTTCTTTCCCAGTGGTTCA
chr22:19180307_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: GATTGGCTATGGGTGTGGAC
chr1_6:89231183_REV CATCCTTGCAAACCAGTCCT
chr1_8:109838221_FW CCCTGGAGGTAGGTCTGGT
chr1_8:109838221_REV CGCACACTCACTGGTTCCT
chr6_12_90404906_FW TGCCGGTCTCCTAGAAGTTG
chr6_12_90404906_REV GCGCTTGATTTCCCTGAGT
chr6:101505264_FW : ACCTGGGATGGGACATATCA
chr6:101505264_REV: TACCAAGCCTGTCCCTGAAC
chr6: 40663811_FW: CAAACACACTCCCCCGTACT
chr6: 40663811_REV: CTGGGTTTTCTCCACTGCTG
chr1:3092903_FW CGAGATCCAAGGAAGTCGTG
chr1:3092903 _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