Nature - USA (2020-06-25)

590 | Nature | Vol 582 | 25 June 2020

Article

PARP inhibition (Extended Data Fig. 10w), but it did not have any further
effect on the inherent PARP inhibitor sensitivity in mutant IDH1 cells
(demonstrating epistasis). By contrast, H3K9M expression rescued
the PARP inhibitor sensitivity in mIDH1 cells.
We next tested whether HDR might still be able to occur at sites
in which the H3K9me3 levels were low at baseline even in cells with
high 2HG. By analysis of published ChIP followed by high-throughput
sequencing (ChIP–seq) data generated from the IDH1(R132H) mutant
and wild-type astrocytes^28 (Extended Data Fig. 10x), we identified
three types of genomic sites in the mutant IDH1 and wild-type cells:
(1) hypomethylated at baseline in wild-type cells but hypermethyl-
ated in mutant IDH1 cells; (2) hypomethylated in both; and (3) hyper-
methylated in both, confirmed by ChIP (Fig. 4f). Using CRISPR–Cas9
to induce site-specific DSBs at these sites (Extended Data Fig. 10y),
we assayed for the recruitment of DNA repair factors via ChIP. In the
differentially H3K9 methylated site, we saw recruitment of HDR fac-
tors by ChIP in the wild-type but not mutant IDH1 astrocytes (Fig. 4g,
h, Extended Data Fig. 10z, aa). At the site with low H3K9me3 in both,
we observed recruitment of HDR factors in both cases. Notably, at
this site, an H3K9me3 signal could be produced at the DSB not only
in the wild-type astrocytes, but also in the IDH1 mutant ones (Fig. 4i).
At the site with high H3K9me3 in both, there was no H3K9me3 signal
above background in either the wild-type or mutant IDH1 cells and
recruitment of HDR factors was attenuated. These results support our
model that it is the pre-existing H3K9me3 hypermethylation (caused
by metabolite inhibition of KDM4B) that inhibits repair of DSBs by the
HDR pathway because the H3K9me3 signal cannot be generated. Con-
sistent with the need for an H3K9me3 spike, siRNA knockdown of the
H3K9 histone methyltransferase, SUV39H1, impaired HDR (Extended
Data Fig. 10ab–ad), consistent with a previous report^17.
The above experiments provide a direct link between
oncometabolite-induced HDR deficiency, KDM4B inhibition and
H3K9me3 status. They identify H3K9 methylation as the key regulatory

target in the pathway by which increased metabolites cause decreased

HDR. This pathway is characterized in normal cells by a rapid spike in

H3K9 trimethylation that coordinates recruitment of TIP60 and MRE11,

promoting ATM activation, licensing end-resection, and leading to the

downstream recruitment of RPA, BRCA1 and RAD51. In cells that over-

produce metabolites, much of the genome has high levels of H3K9me3,

and at such regions, this H3K9me3 signal is not properly induced. The

constitutively high levels of H3K9me3 prevent a hypermethylation

spike, which impairs HDR factor recruitment and end-resection, con-

ferring sensitivity to PARP inhibitors. These results reveal a pathway by

which metabolism influences DNA repair and may provide the basis for

therapeutic strategies for patients with metabolite-associated malig-

nancies.

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availability are available at https://doi.org/10.1038/s41586-020-2363-0.

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0

2

4

6

Differential

H3K9me3

DSB–+–+

IDH1 WT R132H

H3K9me3 high

–+–+

WT R132H

H3K9me3 low

–+–+

WT R132H

IP: TIP60

Genome site

Break occupancy (% input)

P = 0.0152

P = 0.23

P = 0.14P = 0.376

P = 0.0061P = 0.0399

Loci occupancy (% input)

Time after Cas9 electroporation (h)

0.0

0.2

0.4

0.6

IP: RAD51

Break occupancy (% input)

P = 0.0104P = 0.0005P = 0.0019

P = 0.31P = 0.22

P = 0.91

H3K9me3

No DSB

Differential

H3K9me3

H3K9me3

high

H3K9me3

low

Genome site

10

15

5

Loci occupancy (% input)

IDH1 WT

IDH1 R132H

P = 0.0057

P = –0.86

P = 0.85

+AGI5198

WT H3

SNU1079

(IDH1 R132C/+)

GRQM

H3K4

Mutants

GRQM

H3K9

Mutants

GRQM

H327

Mutants

GRQM

H3K36

Mutants

20

40

60

TIP60 foci-positive nuclei (%)

P = 0.0195

Time after DSB (h)

DSB ChIP:

WT H3 + DMSO

Relative br

eak occupancy

0

2

4

6

8

10

Time after DSB (h)

DSB ChIP:

H3K9M + DMSO

00 .5 11 .5 24624 00 .511.5 (^2462400) Time after DSB (h) .511.5 24624 00 .5 11 .5 24624
DSB ChIP:
WT H3 + 2HG
SUV39H1γH2A.X
H3K9me3TIP60
MRE11ATM
BRCA1RAD51RPA
SUV39H1γH2A.X
H3K9me3TIP60
MRE11ATM
BRCA1RAD51RPA
SUV39H1γH2A.X
H3K9me3TIP60
MRE11ATM
BRCA1RAD51RPA
SUV39H1γH2A.X
H3K9me3TIP60
MRE11ATM
BRCA1RAD51RPA
Time after DSB (h)
DSB ChIP:
H3K9M + 2HG
Differential
H3K9me3
DSB–+–+
IDH1 WT R132H
H3K9me3 high
–+–+
WT R132H
H3K9me3 low
–+–+
WT R132H
Genome site
IDH1 WT
IDH1 R132H
IDH1 WT
IDH1 R132H^23456
0
5
10
IP: H3K9me3
abcde
fg h i
Relative br
eak occupancy
0
2
4
6
8
10
Relative br
eak occupancy
0
2
4
6
8
10
Relative br
eak occupancy
0
2
4
6
8
10
Differential H3K9me3
WT IDH1IDH1 R132H
WT vs R132H (F = 80.84, df = 1)P = 0.0008
H3K9me3 lowWT IDH1
WT vs R132H IDH1 R132HP = 0.404
(F = 0.87, df = 1)
H3K9me3 highWT IDH1
WT vs R132H IDH1 R132HP = 0.733
(F = 0.12, df = 1)
Fig. 4 | Aberrant H3K9 methylation impairs HDR and mechanistically
underlies oncometabolite induced HDR deficiency. a, Quantification of
TIP60 foci-positive nuclei in SNU1079 cells after expression of the indicated H3
mutant constructs or after treatment with the mIDH1 inhibitor, AGI-5198.
b–e, Heat map representation of DSB–ChIP assays after transfection with
either wild-type H3 or H3K9M and treatment with either DMSO control or 2HG,
as indicated. f, Confirmation by ChIP of H3K9me3 levels in wild-type and
IDH1(R132H)-expressing astrocytes at selected loci identified by analysis of
published ChIP–seq data to show either differential H3K9me3 levels (high
H3K9me3 in IDH1(R132H)-expressing astrocytes and low in wild-type
astrocytes), low H3K9me3 levels in both cell lines, or high H3K9me3 levels in
both. g, h, ChIP analysis of TIP60 (g) and R AD51 (h) recruitment 12 h after
Cas9–guide RNA nucleofection to generate site-specific DSBs at the loci
profiled in f in the wild-type IDH1 and IDH1(R132H) astrocyte cell lines. i, ChIP
analysis over time of H3K9me3 levels at the same loci as in f–h after Cas9–guide
RNA nucleofection. Data are mean ± s.e.m. from three biological replicates.
P values were determined by two-tailed unpaired t-test, df = 4 (a, f–h) or
A N OVA (i).