TRIM28 Functions As the SUMO E3 Ligase for PCNA in Prevention of Transcription Induced DNA Breaks

TRIM28 functions as the SUMO E3 ligase for PCNA in prevention of transcription induced DNA breaks

Min Lia, Xiaohua Xua, Chou-Wei Changa, and Yilun Liua,1

aDepartment of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope, Duarte, CA 91010-3000

Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved August 17, 2020 (received for review March 3, 2020) In human cells, the DNA replication factor proliferating cell nuclear SUMO2-PCNA is important for resolving TRC (17), which, if antigen (PCNA) can be conjugated to either the small ubiquitinlike not resolved, is a major cause of DNA breaks and instability at modifier SUMO1 or SUMO2, but only SUMO2-conjugated PCNA is common fragile sites (CFSs) (25, 26). SUMO2-PCNA achieves induced by transcription to facilitate resolution of transcription– TRC resolution by dissociating active RNAPII (RNAPIIo) via replication conflict (TRC). To date, the SUMO E3 ligase that pro- chromatin remodeling by enriching the histone chaperones vides substrate specificity for SUMO2-PCNA conjugation in re- CAF1 and facilitates chromatin transaction (FACT) at the rep- sponse to TRC remains unknown. Using a proteomic approach, lication fork (17). CAF1 deposits new histones that contain re- we identified TRIM28 as the E3 ligase that catalyzes SUMO2-PCNA pressive histone marks at the replication fork, and FACT conjugation. In vitro, TRIM28, together with the RNA polymerase II removes parental histones ahead of the replication fork (27, 28). (RNAPII)-interacting protein RECQ5, promotes SUMO2-PCNA con- Through this mechanism, SUMO2-PCNA promotes chromatin jugation but inhibits SUMO1-PCNA formation. This activity re- remodeling to establish compact chromatin structure and de- quires a PCNA-interacting protein (PIP) motif located within the stabilize RNAPIIo from TRC sites to allow replication fork bromodomain of TRIM28. In cells, TRIM28 interaction with PCNA progression (17). Because CAF1A, the catalytic subunit of on human chromatin is dependent on both transcription and CAF1, interacts with SUMO2 but not SUMO1 in vitro (29), this RECQ5, and SUMO2-PCNA level correlates with TRIM28 expression. preference for SUMO2 binding likely explains why TRC spe- As a consequence, TRIM28 depletion led to RNAPII accumulation at cifically induces SUMO2-PCNA conjugation to facilitate CAF1- TRC sites, and expression of a TRIM28 PIP mutant failed to sup- mediated chromatin remodeling. press TRC-induced DNA breaks.

The distinct functions of SUMO1-PCNA and SUMO2-PCNA BIOCHEMISTRY indicate that these modifications are mediated by different sets TRIM28 | PCNA | SUMO2 | RECQ5 | DNA replication of enzymes. In general, SUMOylation is initiated by the conjugation of a SUMO protein to the E1 ligase Sae1/2, which CNA is a DNA replication factor that forms a homotrimeric transfers SUMO to the E2 ligase UBC9. A SUMO E3 ligase then Pring on DNA and interacts with and anchors DNA poly- simultaneously binds SUMO-UBC9 and the target substrate to merases at the replication fork to enhance their processivity. In promote SUMO conjugation of the substrate (30). Although addition to its role in normal DNA replication, PCNA also or- cells contain only one SUMO E1 and E2, there are several E3 chestrates several cellular processes to regulate the cell cycle and ligases, which provide substrate specificity. Most likely, a distinct maintain genomic stability. These functions depend on distinct SUMO E3 ligase and/or its cofactor(s) is required to provide the posttranslational modifications of PCNA lysine residue 164 specificity needed to conjugate SUMO2 but not SUMO1 to (K164). For example, in undamaged cells, PCNA is mono- PCNA in a transcription-dependent manner. Even though the ubiquitinated at K164 by the ubiquitin E3 ligase CRL4(CDT2) to antagonize the ubiquitin hydrolase USP1 (1) and facilitate Significance proteasome-dependent degradation of p21 and CDT1 to ensure proper cell cycle progression (2). In cells treated with DNA PCNA is an essential protein in DNA replication and repair, and damaging agents, monoubiquitination of PCNA K164, mediated these functions rely on multiple posttranslational modifica- by the ubiquitin E3 ligase RAD18, recruits error-prone DNA tions, including small ubiquitin-like modifiers SUMO1 and polymerases to the stalled replication fork and bypasses DNA SUMO2. SUMO2-conjugated PCNA has a distinct function in lesions (3). K164 can also be polyubiquitinated to recruit maintaining genomic stability from SUMO1-conjugated PCNA. ZRANB3 for DNA damage bypass through template switching Therefore, different SUMO E3 ligases are needed to provide – (4 6). Posttranslational modification of PCNA K164 is not lim- the specificity to covalently attach either SUMO1 or SUMO2 to ited to ubiquitination (7). In both yeast and human cells, PCNA in response to different replication stress. However, even SUMOylation of PCNA K164 recruits antirecombination heli- though SUMO-conjugated PCNA molecules have been ob- cases (i.e., Srs2, PARI, FBH1, and RTEL) to the replication fork served in human cells since 2012, to date, the SUMO E3 ligases – to suppress homologous recombination (8 14). for PCNA have yet to be identified. This paper reports a SUMO Unlike yeast, which only contains one SUMO gene, there are E3 ligase that directly conjugates SUMO2 to PCNA with high – four SUMO paralogs in human cells (i.e., SUMO1 4). Both specificity to prevent transcription induced DNA breaks in SUMO1- and SUMO2-conjugated PCNA are found in human human cells. cell extracts (12, 15, 16). SUMO1-PCNA recruits the helicase PARI to suppress unwanted homologous recombination at Author contributions: M.L. and Y.L. designed research; M.L., X.X., and C.-W.C. performed stalled replication forks (12, 15). However, whether SUMO2- research; M.L. contributed new reagents/analytic tools; M.L. and Y.L. analyzed data; and PCNA plays a redundant role to SUMO1-PCNA in suppressing Y.L. wrote the paper. homologous recombination for maintaining replication fork The authors declare no competing interest. stability was not clear until our recent study, which showed that This article is a PNAS Direct Submission. SUMO2-conjugated PCNA but not SUMO1-conjugated PCNA Published under the PNAS license. was induced by transcription during DNA replication (17). 1To whom correspondence may be addressed. Email: [email protected]. SUMO2-PCNA conjugation is dependent on RECQ5 (17), a This article contains supporting information online at https://www.pnas.org/lookup/suppl/ DNA helicase that functions as a tumor suppressor and is a doi:10.1073/pnas.2004122117/-/DCSupplemental. member of the RNAPII complex (18–24). Transcription-induced

www.pnas.org/cgi/doi/10.1073/pnas.2004122117 PNAS Latest Articles | 1of9 Downloaded by guest on September 23, 2021 presence of SUMO1- and SUMO2-conjugated PCNA in human A cell extracts has been reported since 2012 (12, 15, 16), to date, E the E3 ligases for these different PCNA SUMO modifications remain unclear. In the current study, we describe our identification of TRIM28, also known as KRAB-associated protein 1 (KAP1), as B the E3 ligase that specifically conjugates SUMO2 but not SUMO1 to PCNA. TRIM28 interacts with PCNA through its PIP motif to promote SUMO2 conjugation, and this interaction depends on both transcription and RECQ5, which may act as a sensor for TRC by interacting with both components of the replication and transcriptional complexes when they are in F proximity. Our identification of TRIM28 as the SUMO2-PCNA E3 ligase is further supported by our demonstration that TRIM28 depletion leads to the accumulation of RNAPII and C DNA damage at TRC sites, and that DNA breaks formed in the absence of TRIM28 are suppressed by the expression of a SUMO2-PCNA fusion protein to bypass the requirement for TRIM28 in SUMO2-PCNA conjugation. Results TRIM28-PCNA Interaction Takes Place during DNA Replication and Is Dependent on Transcription. The SUMO E3 ligase that promotes SUMO2 conjugation of PCNA is expected to interact with PCNA. Therefore, we searched for the presence of any known SUMO E3 ligase(s) in our mass spectrometric analysis of FLAG- tagged PCNA protein complexes purified from human chromatin prepared from HEK293T cells (17). Our mass spectrometric D GH analysis detected the presence of the E3 ligases RANBP2 (20 peptides) and TRIM28 (16 peptides) in the purified FLAG- PCNA complex (Fig. 1A) as well as limited PIAS1 (1 peptide) and PIAS2 (1 peptide). Western blot analysis of the purified FLAG-PCNA complex confirmed the presence of the two top candidates, RANBP2 and TRIM28 (SI Appendix, Fig. S1A). Each of these SUMO E3 ligases is capable of conjugating SUMO2 to its substrate and is a potential candidate for the SUMO2-PCNA conjugation reaction (31–34). RECQ5 is a critical factor for preventing TRC-induced DNA breaks in multiple human cell lines, including HEK293T cells, Fig. 1. TRIM28-PCNA interaction on human chromatin is dependent on and the level of SUMO2-PCNA on human chromatin is de- transcription. (A) Molecular weight (MW) and number of peptides detected pendent on RECQ5 (17). Importantly, SUMO2 conjugation of by mass spectrometry for each of the indicated proteins that copurified with PCNA requires a direct interaction between RECQ5 and PCNA FLAG-tagged PCNA isolated from the chromatin-bound (CB) fraction of (17). Therefore, to narrow down the potential SUMO E3 ligase HEK293T cells as performed previously (17). (B) MW and number of peptides detected by mass spectrometry for each of the indicated proteins that candidates, we determined which of these SUMO E3 ligases was copurified with FLAG-tagged RECQ5 isolated from the CB fraction of also associated with RECQ5 in an abundant amount on human HEK293T cells. (C) Western blot analysis of the indicated proteins in the CB chromatin. For this, we immunopurified FLAG-tagged RECQ5 fractions (Left) and the FLAG-RECQ5 complexes immunopurified from CB from the CB fraction of HEK293T cells and identified RECQ5- fractions (Right) prepared from HEK293T cells with or without exogenously associated proteins using mass spectrometry (SI Appendix, Table expressed FLAG-RECQ5. (D) Western blot analysis of the indicated proteins in S1). In addition to known RECQ5-interacting proteins the CB fractions (Left) and the endogenous TRIM28 protein complexes (i.e., RNAPII [represented by RPB1–3 and 9], PCNA, and top- immunopurified using an α-TRIM28 antibody from CB fractions (Right) pre- oisomerase I) (18, 24, 35, 36), we found that TRIM28 was the pared from HEK293T cells. (E and F) Western blot analysis of the indicated most abundant SUMO E3 ligase candidate that copurified with proteins in the CB fractions (E) and the FLAG-PCNA complexes immunopurified from CB fractions (F) prepared from HEK293T cells with or without RECQ5 and there was also a limited RANBP2 associated with B exogenously expressed FLAG-PCNA and with or without DRB treatment. (G) RECQ5 (Fig. 1 ). Western blot analysis of the FLAG-RECQ5 Western blot analysis of the indicated proteins in whole cell extract (WCE) of complexes purified from the CB fraction confirmed the associ- HEK293T cells containing an inducible TRIM28 shRNA construct with or ation of RECQ5 with TRIM28 (Fig. 1C). In contrast, RANBP2 without DOX treatment. Tubulin and MCM7 were used as loading controls. was present at a much lower level in the purified FLAG-RECQ5 (H) Western blot analysis of the indicated proteins associated with tran- complexes (Fig. 1C). Using an antibody specific to TRIM28, we scriptionally active open chromatin (CB:RNA+; Left) and proteins bound to further validated that the interactions were observed among transcriptionally low or inactive DNA regions (CB:RNA-; Right) prepared from endogenous TRIM28, PCNA, and RECQ5 proteins (Fig. 1D). HEK293T cells shown in G. MCM7 was used as a loading control. SUMO2-PCNA conjugation takes place during DNA replication and is induced by RNAPII-dependent transcription (17). Indeed, similar to DNA replication factors FEN1 and MCM2–7 (represented by Western blot for MCM7), we found that PCNA interaction primarily takes place in the transcriptionally β TRIM28 was enriched in the CB FLAG-PCNA complex during active chromatin fraction, treatment with 5,6-dichloro-1- -D-ri- the S phase (SI Appendix, Fig. S1 B and C). We further asked if bofuranosylbenzimidazole (DRB), which inhibits RNAPIIo RECQ5-TRIM28-PCNA interaction is regulated by transcrip- (Fig. 1E) (37), weakened PCNA interaction with RECQ5 and tion. Consistent with our previous observation that RECQ5- TRIM28 but not replication factors, such as MCM2–7 helicase

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.2004122117 Li et al. Downloaded by guest on September 23, 2021 (represented by Western blot for MCM2 and MCM7; Fig. 1F and A SI Appendix,Fig.S1D and E). These decreased protein–protein interactions among PCNA, TRIM28, and RECQ5 correlated with a reduced SUMO2-PCNA level on chromatin (Fig. 1F). The ability of TRIM28 to copurify with both RECQ5 and the PCNA-containing replisome in a transcription-dependent manner suggested that TRIM28 is the E3 ligase for SUMO2-PCNA conjugation. Indeed, when we treated HEK293T cells stably in- tegrated an inducible short hairpin RNA (shRNA) construct specific to TRIM28 with doxycycline (DOX) to induce TRIM28 shRNA and to knockdown (KD) expression of TRIM28 (Fig. 1G), we found that the level of SUMO2-PCNA, which was primarily found in the transcriptionally active chromatin fraction (i.e., CB:RNA+), was reduced in TRIM28 KD cells compared to control cells (Fig. 1H). These analyses indicated that SUMO2- PCNA conjugation is dependent on TRIM28.

TRIM28 Conjugates SUMO2 but Not SUMO1 to PCNA. Next, we wished to determine if TRIM28 is the E3 ligase that specifically catalyzes the conjugation reaction of SUMO2-PCNA but not B SUMO1-PCNA. For this, we performed in vitro SUMOylation assays using purified recombinant Strep-tagged PCNA and either His-SUMO1 or His-SUMO2 in the presence of Sae1/2 E1 ligase and UBC9 E2 ligase. We found that Sae1/2 and UBC9 alone (in the absence of RECQ5 or TRIM28) were capable of conjugating either SUMO1 or SUMO2 to PCNA (Fig. 2 A, Bottom, lanes b and f). Importantly, the level of SUMO2-PCNA was greatly enhanced by the addition of either purified recombinant RECQ5 or TRIM28 with further increase when both RECQ5 and BIOCHEMISTRY TRIM28 were present in the reaction (Fig. 2A, compare lane f to lanes g–i). In contrast to SUMO2-PCNA conjugation, we found C that the presence of TRIM28 or RECQ5 not only did not enhance SUMO1-PCNA conjugation, but also exhibited an inhib- itory effect on SUMO1-PCNA conjugation, despite the same experimental conditions (Fig. 2A, compare lane b to lanes c–e). We further confirmed that the SUMO2 conjugation of PCNA took place at the K164 residue of PCNA as wild type (WT) PCNA, but not a mutant in which K164 was mutated to arginine (R) was SUMOylated in RECQ5- and TRIM28-dependent manners (Fig. 2B). In addition, we showed that WT TRIM28 (Fig. 2C, lanes d–f) but not SUMO E3 ligase defective C651F mutant (Fig. 2C, lanes g–i) (38) stimulated SUMO2-PCNA conjugation. Interestingly, at higher concentration, TRIM28 C651F mutant further inhibited the stimulative effect by RECQ5 (Fig. 2C, compare lanes c and i). This result concludes that TRIM28 SUMO E3 ligase activity is required for the conjugation of SUMO2 to PCNA. To validate the specificity of TRIM28 on SUMO2 but not SUMO1 conjugation of PCNA, we immunopurified FLAG- D TRIM28 protein complexes from the CB fraction of HEK293T cells stably expressing FLAG-TRIM28 proteins and performed in vitro SUMOylation reactions. We found that, similar to the results of reactions using purified recombinant proteins (Fig. 2A), FLAG-TRIM28 complexes purified from human chromatin also stimulated SUMO2 conjugation of PCNA while inhibiting the formation of SUMO1-PCNA (Fig. 2D). To summarize, we identified TRIM28 as the SUMO E3 ligase that

Fig. 2. TRIM28 and RECQ5 catalyze SUMO2-PCNA conjugation but inhibit SUMO1-PCNA formation. (A) Western blot analysis using anti-StrepII antibody to detect recombinant StrepII-PCNA after in vitro SUMOylation assays Sae1/2, and UBC9. (C) Western blot analysis using anti-StrepII antibody to in the presence or absence of His-tagged SUMO1 or SUMO2, RECQ5, and detect recombinant StrepII-PCNA after in vitro SUMOylation assays in the TRIM28. Long exposure (Bottom) shows the formation of SUMO1-PCNA in presence of His-tagged SUMO2, RECQ5 and increasing amounts of recombi- the absence of a SUMO E3 ligase; this conjugation decreased in the presence nant TRIM28 WT (d–f) or C651F mutant (g–i). (D) Western blot analysis using of TRIM28 or RECQ5. All reactions contained Sae1/2 and UBC9. (B) Western anti-StrepII antibody to detect recombinant StrepII-PCNA after in vitro blot analysis using anti-StrepII antibody to detect recombinant StrepII-PCNA SUMOylation assays in the presence of His-tagged SUMO1 or SUMO2 and WT (Left) and K164R mutant (Right) after in vitro SUMOylation assays in the increasing amounts of FLAG-TRIM28 protein complex purified from the CB presence or absence of RECQ5 and TRIM28. All reactions contained SUMO2, fraction of HEK293T cells.

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D E

Fig. 3. Identification of the PIP motif of TRIM28 as important for SUMO2-PCNA conjugation. (A) Diagram of human TRIM28. The PHD domain and bromodomain (light gray box) important for its SUMO E3 ligase function, PIP1 and PIP2 residues are shown. (B) Western blot analysis of the indicated proteins in the CB fractions (Left; input) and in the FLAG-TRIM28 complexes immunopurified from CB fractions (Right) prepared from HEK293T cells expressing FLAG- tagged WT, PIP1 mutant, or PIP2 mutant TRIM28 proteins. (C) Western blot analysis using anti-StrepII antibody to detect recombinant StrepII-PCNA (Bottom) after in vitro SUMOylation assays in the presence of RECQ5 and increasing amounts of WT, PIP1, or PIP2 mutant TRIM28 proteins (Top). (D) Western blot analysis of the indicated proteins in the WCE of TRIM28-shRNA-containing HEK293T cells with or without DOX treatment and expressing indicated FLAG vector (V), FLAG-TRIM28, or FLAG-PCNA constructs. Actin was used as a loading control. (E) Average tail moments (horizontal lines) measured using neutral comet assay in the cells shown in D. Each dot represents the tail moment of a single cell. At least, 150 cells were analyzed per experiment. The graph is representative of one experiment; the result was reproduced in two independent comet assays.

works with RECQ5 to promote SUMO2-PCNA conjugation but form in reactions containing the TRIM28 PIP2 mutant (Fig. 3C, inhibits SUMO1-PCNA conjugation. lanes f–h). In contrast, the TRIM28 PIP1 mutant induced SUMO2 conjugation of PCNA in a concentration-dependent PIP Motif of TRIM28 Is Required for SUMO2-PCNA Conjugation and manner (Fig. 3C, lanes i–k) similar to the WT TRIM28 Suppresses Transcription-Induced DNA Breaks. We next determined (Fig. 3C, lanes c–e). Taken together, these results suggest that the residues on TRIM28 that are involved in direct binding to TRIM28 interacts with PCNA via its PIP2 motif and that this PCNA (i.e., the substrate) and are important for subsequent motif, located within the bromodomain of TRIM28, is required conjugation to SUMO2. PCNA-interacting proteins are known for SUMO2 conjugation of PCNA. to bind to PCNA via a PIP motif (39). We identified two PIP SUMO2-PCNA is important for resolving TRC to prevent motifs within TRIM28: PIP1 among residues 342–348 and PIP2 DNA breaks (17). To demonstrate that TRIM28 as the SUMO among residues 786–792 (Fig. 3A), then, performed mutagenesis E3 ligase for SUMO2-PCNA is required for preventing TRC- to make TRIM28 PIP1 and PIP2 mutants. When we expressed induced DNA breaks, we transiently expressed WT, PIP1 mu- TRIM28 PIP1 or PIP2 mutants as FLAG-tagged proteins in tant, or PIP2 mutant TRIM28 as FLAG-tagged proteins in HEK293T cells, we found that PCNA copurified with the PIP1 TRIM28 shRNA KD cells (Fig. 3 D, Top). We then measured mutant (Fig. 3B). In contrast, PCNA showed reduced interaction DNA break frequencies in these cells using neutral comet assays. with the PIP2 mutant, suggesting that PIP2 is responsible for After DOX induction to deplete TRIM28, we observed an TRIM28 interaction with PCNA. In addition, MCM2-7 was expected increase in tail moments (indicative of DNA breaks); copurified with TRIM28 (Fig. 1D), but the amount of MCM2–7 this increase was suppressed by the expression of either the WT associated with the TRIM28 PIP2 mutant was also reduced or the PIP1 mutant TRIM28 (Fig. 3E, comparing columns 1–4 (Fig. 3B), suggesting that TRIM28 interacts with other compo- from the left). In contrast, the increase in tail moments was nents of the replisome through PCNA. PIP2 is located within the sustained after complementation with the PIP2 mutant (Fig. 3E, bromodomain of TRIM28 (Fig. 3A); together with an upstream third column from the right). To determine if TRIM28-mediated PHD finger, this bromodomain has been shown to possess SUMO2 conjugation of PCNA is required for suppressing DNA SUMO E3 ligase activity (38, 40). Therefore, to determine if breaks in TRIM28 KD cells, we expressed the PCNA K164R TRIM28 binding to PCNA via its PIP2 motif is required for mutant (i.e., KR; Fig. 3D, third lane from the left), and con- conjugating SUMO2 onto PCNA, we purified TRIM28 PIP1 and firmed that the PCNA KR mutant failed to suppress DNA PIP2 mutants as recombinant proteins from Escherichia coli and breaks by TRIM28 KD (Fig. 3E, last column from the right). In tested the ability of each to promote SUMO2 conjugation of contrast, in TRIM28 KD cells expressing a SUMO2-PCNA KR PCNA in vitro. Indeed, we found that SUMO2-PCNA failed to fusion protein (i.e., S2-KR; Fig. 3D, fourth lane from the left),

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.2004122117 Li et al. Downloaded by guest on September 23, 2021 AB Therefore, to validate the role of TRIM28 in facilitating SUMO2 conjugation of PCNA, we analyzed RNAPII occupancy at FRA7K, a known CFS located within the IMMP2L gene, in TRIM28 shRNA KD cells. To do this, we performed chromatin immunoprecipitation (ChIP) using anti-RNAPIIo antibody (4H8) in HEK293T cells with or without TRIM28 shRNA induction (Fig. 4 A and B). After reversing the crosslinks between proteins and DNA, we measured the amount of RNAPIIo bound C to FRA7K by qPCR using primers complementing different regions of the IMMP2L gene. We found that RNAPIIo accumu- lated within the FRA7K region of the IMMP2L gene, but there was no significant accumulation of RNAPIIo in the IMMP2L coding gene region outside of FRA7K (Fig. 4C). The significant accumulation of RNAPIIo within FRA7K was accompanied by an increased level of γH2AX in the same cells, indicative of DNA damage (Fig. 4D). Importantly, the accumulation of RNAPIIo (Fig. 4E) and the increased γH2AX level (Fig. 4F) were suppressed by overexpressing PCNA S2-KR to bypass the requirement of TRIM28. We further confirmed that the elevated RNAPIIo levels were observed at other CFS loci (i.e., FRA3B, D FRA16D, and FRA7I) after TRIM28 KD, and RNAPIIo accumulation was suppressed in all fragile sites by overexpressing PCNA S2-KR (SI Appendix, Fig. S2). On the other hand, overexpressing PCNA KR showed little or reduced efficiency in suppressing RNAPIIo accumulation at CFSs (SI Appendix, Fig. S2). To summarize, these data together support a role for TRIM28 in TRC resolution via SUMO2 conjugation of PCNA.

RNAPIIo-Associated RECQ5 Promotes TRIM28-PCNA Interaction for BIOCHEMISTRY EF SUMO2-PCNA Conjugation. In cells, SUMO2-PCNA conjugation requires both TRIM28 and RECQ5 (Fig. 1H) (17). Indeed, overexpressing RECQ5 or TRIM28 (Fig. 5A) enhanced SUMO2-PCNA conjugation in the CB:RNA+ fraction (Fig. 5B) (17). Although PIAS1 has been shown to contribute to SUMOylation of PCNA in human cells (41), overexpressing PIAS1 (Fig. 5A) had little effect on the SUMO2-PCNA level on chromatin (Fig. 5B), confirming the specificity of SUMO2- PCNA by TRIM28. To confirm that RECQ5 and TRIM28 Fig. 4. TRIM28 deficiency leads to the accumulation of RNAPIIo and γH2AX function synergistically on chromatin to promote SUMO2- at CFS. (A) Western blot analysis of the indicated proteins in formaldehyde- PCNA conjugation, we transiently KD TRIM28 using siRNA treated TRIM28-shRNA-containing HEK293T cells with or without DOX and suppressed the enhancement by RECQ5 overexpression treatment. Tubulin was used as a loading control. (B) Western blot analysis (Fig. 5C). The enhanced SUMO2-PCNA conjugation by α of RNAPIIo that was immunopurified using an -RNAPII phosphor-CTD 4H8 TRIM28 overexpression was also abolished after RECQ5 KD antibody or a control immunoglobulin G antibody from cells shown in A. The using a siRNA specific to RECQ5 (Fig. 5D). blot was probed using an α-RNAPII A10 antibody. (C) Schematic (Top)of regions containing DNA breaks associated with FRA7K in the IMMP2L gene. We next wished to determine the role of RECQ5 in promoting The immunopurified RNAPIIo shown in B was used for ChIP analysis (Bottom) TRIM28-mediated SUMO2-PCNA conjugation. Previously, we of RNAPIIo occupancy at the indicated regions of the IMMP2L gene using identified a novel PIP-like (PIP-L; Fig. 5E) motif at the C ter- primers derived from the indicated regions of the gene. Each bar represents minus of RECQ5, and this motif is important for PCNA inter- the average value ±SD calculated from triplicate qPCR reactions per one action and SUMO2-PCNA conjugation (17). Indeed, the representative experiment. Indicated P values were calculated using t test overexpressed RECQ51–542 fragment lacking the PIP-L motif for statistically significant differences. (D) ChIP analysis of γH2AX occupancy (Fig. 5 F, Top) failed to enhance SUMO2-PCNA formation at the indicated regions of the IMMP2L gene using an α-γH2AX antibody F Bottom ± (Fig. 5 , ). Overexpressing the RECQ5450–991 fragment using the same cells shown in A. Each bar represents the average value SD lacking the N-terminal superfamily helicase domain 2 (SF2; calculated from triplicate qPCR reactions per one representative experiment. E F Bottom Indicated P values were calculated using t test for statistically significant Fig. 5 ) enhanced SUMO2-PCNA formation (Fig. 5 , ), differences. (E and F) ChIP analysis of RNAPIIo (E) and γH2AX (F) occupancy indicating that RECQ5 helicase activity is not required for at FRA7K (IMMP2L3b) of the IMMP2L gene using TRIM28-shRNA-containing SUMO2-PCNA formation. Indeed, overexpressing the HEK293T cells with or without DOX treatment and with or without RECQ5 ATPase-defective D157A (DA) mutant efficiently in- expressing PCNA S2-KR fusion protein. duced SUMO2-PCNA conjugation (Fig. 5 F, Bottom). RECQ5 also interacts with inactive RNAPII (RNAPIIa) through the KIX domain and RNAPIIo via the SET2-RPB1-interction (SRI) do- which functionally mimics PCNA that has been SUMO2 conju- main (Fig. 5E) (18, 20, 42, 43). Interestingly, overexpressing the gated at K164 (17), suppressed the increase in tail moments in RECQ51–899 fragment, which interacts with PCNA and RNA- the TRIM28 KD cells (Fig. 3E, second column from the right), PIIa but not RNAPIIo (Fig. 5G), failed to enhance SUMO2- confirming that S2-KR but not KR PCNA could bypass the re- PCNA formation (Fig. 5 F, Bottom). This result indicated that quirement for TRIM28 to suppress DNA breaks. SUMO2-PCNA conjugation requires RECQ5 interactions with both PCNA and RNAPIIo. TRIM28 Prevents RNAPIIo Accumulation at CFSs. SUMO2-PCNA TRIM28 copurified with WT RECQ5 and all of the truncation resolves TRC by destabilizing RNAPIIo from TRC sites (17). mutants, including RECQ51–542 and RECQ5450–991 fragments

Li et al. PNAS Latest Articles | 5of9 Downloaded by guest on September 23, 2021 (Fig. 5G), suggesting a potential direct interaction between to the replication complex and increasing H3K9me2/3 re- TRIM28 and residues 450–542 of RECQ5. Alternatively, pressive marks (17). TRIM28 is a component of the transcriptional elongation com- In addition to the association with RNAPII, RECQ5 also in- plex to regulate RNAPIIo release from the pausing site (44), and teracts with PCNA (35, 36), and this interaction is required for we found that RNAPIIo copurified with TRIM28 (Fig. 1D). It is SUMO2 conjugation of PCNA (17). In the present study, we possible that TRIM28 interacts either directly with the N ter- further revealed the mechanism by which RECQ5 promotes minus of RECQ5 or indirectly through their mutual interactions SUMO2 conjugation of PCNA at transcriptionally active chro- with RNAPIIo or both (Fig. 5E, gray arrows). Indeed, when we matin. We showed that RNAPIIo-associated RECQ5 mediates immunopurified the FLAG-TRIM28 complex from the CB SUMO2-PCNA conjugation. We suggest that RECQ5 acts as a fractions of the control and RECQ5 KD cells, we found that sensor to promote TRIM28-dependent SUMO2 conjugation of RECQ5 depletion only weakened but not abolished TRIM28- PCNA when RECQ5 simultaneously interacts with components RNAPIIo interaction on chromatin (Fig. 5H). Importantly, we of replisome (i.e., PCNA) and transcriptional elongation com- found that PCNA and MCM2–7 failed to copurify with TRIM28 plex (i.e., RNAPIIo and TRIM28) when they are in proximity. in RECQ5-depleted cells (Fig. 5H), and we further confirmed Alternatively, additional TRIM28 may be recruited to TRC for this effect by immunopurifying FLAG-PCNA in the control and SUMO2-PCNA conjugation through a direct interaction with RECQ5 KD cells (Fig. 5I). In contrast, the interaction of PCNA the N terminus of RECQ5. Interestingly, our previous mass with MCM2–7 helicase was not affected after RECQ5 depletion spectrometric analysis revealed that components of the RNAPII (Fig. 5I). Most likely, RECQ5 promotes the interaction between are the most abundant proteins copurified with RECQ5 on hu- TRIM28 and its substrate PCNA as part of the replisome. In- man chromatin (18). Our new mass spectrometric analysis of the deed, when we incubated equal amounts of purified recombinant RECQ5 complexes purified from the CB fraction of HEK293T cells TRIM28 with Strep-PCNA immobilized on Strep-Tactin beads pretreated with NEM, a ubiquitin and SUMO hydrolase inhibitor, in the presence of increasing amounts of RECQ5, we found that revealed that the associations of replication factors including the amount of TRIM28 pulled down by Strep-PCNA was directly MCM2–7 and FACT histone chaperone with RECQ5 were greatly proportional to the amount of RECQ5 present in the reaction stabilized by NEM as indicated by the high number of the peptides (SI Appendix, Fig. S3). Taken together, our results identify derived from these proteins (SI Appendix,TableS1). It would be a multiple interactions among RECQ5, RNAPIIo, PCNA, and great interest to determine if the interactions of RECQ5 with these TRIM28 on human chromatin (Fig. 5E), and this RECQ5- enzymes are dependent on the SUMOylation or ubiquitination of RNAPIIo-PCNA-TRIM28 complex is crucial for SUMO2- RECQ5 or the partner proteins. PCNA conjugation. The contribution of TRIM28 to TRC resolution suggests that, similar to RECQ5, TRIM28 may function as a tumor suppressor. Discussion Indeed, TRIM28 haploinsufficiency has been linked to Wilms SUMO modification of the DNA replication factor PCNA was tumor (51). Interestingly, CFS instability within the WWOX gene, first observed in human cells in 2012 (12, 15, 16). However, which can be suppressed by SUMO2-PCNA (17), has also been identifying the SUMO E3 ligase(s) that catalyzes this reaction showntobeassociatedwithWilmstumor(52).Itwouldbeofgreat has been difficult, due to the fact that, in vitro, PCNA can be interest to establish animal models to determine if TRIM28 in its efficiently SUMOylated in the absence of a SUMO E3 ligase, role as the E3 ligase for SUMO2-PCNA directly contributes to leaving the requirement for an E3 ligase that facilitates SUMO Wilms tumor pathogenesis in patients with TRIM28 haploinsufficiency. conjugation of PCNA in human cells ambiguous (45, 46). Using an unbiased proteomic approach, our study identified TRIM28 Materials and Methods as the SUMO E3 ligase that promotes SUMO2-specific conju- Plasmids. TRIM28 complementary DNA (cDNA) was PCR-amplified from a gation of PCNA on human chromatin. Consistent with the role HeLa cDNA library, then, cloned into p3xFLAG CMV7.1 (Sigma-Aldrich) be- of SUMO2-PCNA conjugation in resolving TRC (17), TRIM28 tween the HindIII and the EcoRI sites to generate an N-terminal FLAG- deficiency led to an accumulation of spontaneous DNA breaks, tagged TRIM28 mammalian expression construct. The TRIM28 cDNA was which could be suppressed by exogenously expressing SUMO2- also cloned into pTXB1 (NEB) between the NdeI and the EcoRI sites to generate a C-terminal chitin-binding domain-tagged TRIM28 bacterial ex- PCNA fusion protein to bypass the requirement for TRIM28 in pression construct. The TRIM28 PIP motif mutants were generated by mu- SUMOylating PCNA. Importantly, PIP2 mutation, which dis- tagenesis using the WT plasmid as the template and the following primers: rupted the interaction of TRIM28 with PCNA and impaired PIPM1: 5′-CCAAGATCCAGAAGCACGCGGAGCACGCTCTGCGCGCTGCCTCTTGGG SUMO2-PCNA conjugation, failed to suppress DNA breaks in CTCTGG-3′ and PIPM2: 5′-TGCAGTCCATCATCGGCGCGCAGCGCGCCGCCGAGA TRIM28 KD cells, arguing that TRIM28 plays a significant role CGCGCATGAACG-3′. The primer for generating the TRIM28 C651F mutant as the E3 ligase for SUMO2-PCNA in genome maintenance. In was: 5′-GTTTCCACCTGGACTTTCA CCTGCCGGCCCT-3′. PCNA K164R muta- addition to the PIP2 motif, phosphorylation of TRIM28 serine genesis was performed as previously described (17). Complementary oligo- 473 (S473) has been shown to enhance TRIM28 association with nucleotides containing shRNA targeting TRIM28 was dimerized and cloned the replication complex to reestablish heterochromatin after into the pLKO-Tet-On vector. The target sequence for the TRIM28 shRNA construct was: 5′-CCTGGCTCTGTTCTCTGTCCT-3′. pET11-SUMO1 and pET11- DNA replication (47). It would be a great interest in the future SUMO2 were kindly provided by Dr. Yuan Chen (City of Hope) and used for to explore potential crosstalk between S473 phosphorylation and expression and purification of bacterial His-SUMO1 and His-SUMO2. The TRIM28-dependent SUMO2-PCNA conjugation. StrepII-PCNA, pTXB1-RECQ5, pBiFC-VN173-PCNA WT, VN173-PCNA K164R SUMO2-PCNA resolves TRC by recruiting CAF1 and FACT (PCNA KR), VN173-SUMO2-PCNA K164R (S2-KR), pCMV-FLAG-PIAS1, and to promote histone exchanges to establish repressive chromatin pCMV-FLAG-RECQ5 constructs were generated during our previous study and destabilize RNAPII from the TRC site to facilitate rep- (17). All plasmid sequences were confirmed by DNA sequencing. lication fork progression (17). Of particular relevance to our study, TRIM28 has been shown to functionally interact with Antibodies. Rabbit α-TRIM28 (no. 2,521; 1:5,000) was from ProSci Incorpo- CAF1 for retroviral gene silencing (48). TRIM28 also con- rated. Mouse α-PCNA PC10 (sc-56, 1:5,000), mouse α-tubulin (sc-8,035; α α tributes to transcriptional gene silencing via the recruitment 1:3,000), rabbit -H3 (sc-10,809), goat -actin (sc-1,616; 1:1,000), mouse α-RanPB2 (sc-74,518; 1:1,000), mouse α-His (sc-8,036; 1:1,000), mouse RECQ5 of a H3K9 deacetylase (CHD3) and a H3K9 methyltransfer- (sc-515,050), and mouse α-RNAPII A10 (sc-17,798, 1:1,000) were from Santa ase (SETDB1) to promote an inactive chromatin structure Cruz Biotechnology. Mouse α-RNAPII phospho-CTD (phospho S5; 4H8; (49, 50). These functions of TRIM28, coupled with its newly C49,196; 1:5,000) was from Lifespan Biosciences, Inc. Rabbit α-GAPDH (no. demonstrated role as the SUMO E3 ligase for PCNA, are 2,118; 1:5,000) and rabbit α-PIAS1 (no. 3,550; 1:1,000) were from Cell Signaling. consistent with the role of SUMO2-PCNA in recruiting CAF1 Rabbit α-MCM7 (ab52,489; 1:5,000) was from Abcam. Mouse α-NWSHPQFEK

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Fig. 5. TRIM28-PCNA interaction on human chromatin is dependent on RNAPIIo-associated RECQ5. (A) Western blot analysis of the indicated proteins in WCE prepared from HEK293T cells overexpressing the indicated FLAG constructs. Tubulin was used as a loading control. (B) Western blot analysis of the indicated proteins in CB:RNA+ (Left) and CB:RNA fractions (Right) of HEK293T cells overexpressing the indicated FLAG proteins. The ratio of the active form of RNAPII (RNAPIIo) relative to the inactive form of RNAPII (RNAPIIa) was used as the fractionation control for CB:RNA+ and CB:RNA- fractions. (C) Western blot analysis of the indicated proteins in WCE prepared from HEK293T cells with or without overexpression of FLAG-RECQ5 and treated with control or TRIM28 siRNA. (D) Western blot analysis of the indicated proteins in the CB:RNA+ fraction prepared from HEK293T cells with or without overexpression of FLAG-TRIM28 and treated with control or RECQ5 siRNA. (E) Diagram of human RECQ5. The SF2, KIX, PIP-L, and SRI domains are shown. Double arrows indicate protein–protein interactions among TRIM28, PCNA, RNAPIIo, and different domains of RECQ5. (F) Western blot analysis of the indicated proteins in WCE prepared from HEK293T cells with or without overexpression of FLAG-RECQ5 WT and mutants. (G) Western blot analysis of the indicated proteins in the FLAG-RECQ5 WT or mutant complexes immunopurified from CB fractions prepared from HEK293T cells with or without exogenously expressed FLAG-RECQ5 WT or mutant proteins as shown in F.(H) Western blot analysis of the indicated proteins in the CB fractions (Top) and the FLAG-TRIM28 complexes immunopurified from CB fractions (Bottom) prepared from HEK293T cells with or without exogenously expressed FLAG-TRIM28 and treated with control or RECQ5 siRNA. (I) Western blot analysis of the indicated proteins in the CB fractions (Top) and the FLAG-PCNA complexes immunopurified from CB fractions (Bottom) prepared from HEK293T cells with or without exogenously expressed FLAG-PCNA and treated with control or RECQ5 siRNA.

tag (StrepII tag; A01,732; 1:3,000) was from GeneScript. Rabbit α-FLAG activated cell sorter analysis was carried out using a standard propidium (F7,425; 1:5,000) was from Sigma-Aldrich. Mouse α-γH2AX (NP002,096) for iodide method. ChIP was from EMD Millipore. Rabbit α-Trim28 for ChIP (15,202-1-AP) was from Proteintech. Rabbit α-PCNA (1:2,000) was kindly provided by Dr. Robert Cell Fractionation and Immunoprecipitation. Cells were lysed (30 min on ice) in Hickey (City of Hope). Rabbit α-RECQ5 (1:3,000) was generated during our three volumes of cytoplasmic buffer (10 mM 2-amino-2-hydroxymethyl-1,3- · previous study (20). propanediol-Cl [Tris Cl] pH 7.5, 0.34 M sucrose, 3 mM CaCl2, 2 mM MgCl2, 0.1 mM [ethylenedinitrilo]tetraacetic acid [EDTA], 1 mM dithiothreitol [DTT], Cell Culture, Cell Transfection, and Cell Cycle Synchronization. HEK293T cells and 0.5% Nonidet P-40, 40 mM NEM) containing protease and phosphatase inhibitors. The nuclear pellet was collected by centrifugation (2,400 × g, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supple- 5 min). Nuclei were then resuspended in three volumes of nuclear buffer mented with 10% fetal bovine serum and streptomycin/penicillin (1,00 U − (20 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [Hepes] pH 7.5, mL 1). TRIM28 siRNA (sc-38550) was purchased from Santa Cruz. RECQ5 1.5 mM MgCl , 1 mM EDTA, 150 mM KCl, 0.1% Nonidet P-40, 1 mM DTT, and ′ 2 stealth small interfering RNA (siRNA) 5 -UAGACUUGGCAAUAUUCCAAUGGG 10% glycerol) and homogenized with a 21G1/2 needle. The intact chromatin ′ C-3 was purchased from Invitrogen. Plasmids and siRNAs were transfected pellet was collected after centrifugation (18,000 × g, 30 min). To obtain the using continuum transfection reagent (GEMINI). When DRB and DOX were CB fraction, the chromatin pellet was incubated with two volumes of nu- μ −1 used, the concentrations were 50 M and 25 ng mL , respectively. For clease buffer (20 mM Hepes pH 7.5, 1.5 mM MgCl2, 1 mM EDTA, 150 mM KCl, − synchronization, cells were cultured in DMEM with 50 ng/mL nocodazole for 10% glycerol, and 0.5 U μL 1 benzonase) overnight at 4 °C, and the super- 22 h and, then, released by washing 2× with complete DMEM. Fluorescence- natant was collected as the CB fraction. Alternatively, to obtain separate

Li et al. PNAS Latest Articles | 7of9 Downloaded by guest on September 23, 2021 CB:RNA+ and CB:RNA fractions, the chromatin pellet was first incubated 40, and 1% sodium deoxycholate). After washing, the beads were resus- with RNase A in RNase A buffer (50 mM Tris·Cl pH 8.0, 10 mM EDTA, 150 mM pended in 200 μL TE buffer, and formaldehyde cross-links were reversed in − NaCl, and RNase A 10 μgmL 1) for, at least, 2 h to overnight at 4 °C. The the presence of 0.5% SDS overnight at 70 °C. DNA was purified using phe- supernatant was collected as the CB:RNA+ fraction. The remaining pellet nol/chloroform and ethanol precipitation. qPCR primers against IMMP2L was then digested with benzonase for, at least, 2 h to overnight at 4 °C in were previously described (17). qPCR was performed using an ABI 7500 fast the nuclease buffer, and the solubilized proteins were collected as the real-time PCR system and SYBR Green. Enrichment was calculated using the CB:RNA fraction. To immunopurify FLAG-tagged protein complexes, chro- comparative Ct method. The IMMP2L and WWOX primers used for qPCR are matin extracts were incubated overnight with M2-agarose (Sigma-Aldrich) as described previously (17). The qPCR primers for FHIT4 are as follows: 5′- at 4 °C. After binding of the protein complexes, beads were washed ex- TGGCATATTGGACAGGGGAGGT-3′ and 5′-CTAGTGCGGGACCTGGCACA-3′. tensively with FLAG-A binding buffer (10 mM Hepes pH 7.9, 1.5 mM MgCl2, The qPCR primers for CNTNAP2 are as follows: 5′-GTGCTGGGGGATGGTCTC 0.3 M NaCl, 10 mM KCl, 0.2% Triton X-100, and 10% glycerol). The purified CA-3′ and 5′-CCAGTTTCTCCCTGTGTCGCTGT-3′. FLAG-tagged protein complexes were eluted by using either sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) loading buffer or In Vitro SUMOylation and Pull-Down Assays. In vitro SUMOylation was carried FLAG elution A buffer (10 mM Hepes pH 7.9, 0.2 M NaCl, 0.2 mM EDTA, out for 4 h at 37 °C using a SUMOylation kit (Enzo Life Sciences, Inc.). For −1 0.05% Triton X-100, 0.3 mg mL FLAG peptide, and 10% glycerol). All each reaction presented in Fig. 2A, StrepII-PCNA (200 nM) was incubated subsequent mass spectrometry analyses were conducted by the Taplin Mass with the following proteins: SUMO E1 (25 nM), SUMO E2 (50 nM), SUMO1 or Spectrometry Facility at Harvard University. To purify endogenous TRIM28 SUMO2 (300 nM), TRIM28 (1 nM), and RECQ5 (1 nM). Similar in vitro protein complexes, CB fractions were incubated overnight with a TRIM28 SUMOylation assays were performed to generate data presented in Figs. 2D antibody conjugated to protein A/G agarose beads at 4 °C. After binding of and 3C, except that the TRIM28 complex was prepared from the CB fraction the protein complexes, beads were washed extensively with IP buffer of HEK293T cells expressing FLAG-TRIM28. For pull-down assay, StrepII- −1 (20 mM Hepes pH 8.0, 1.5 mM MgCl2, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM bound and StrepII-PCNA-bound chitin beads were blocked with 1 mg mL EDTA, 1 mM DTT, and 10% glycerol) before analysis by Western blotting. bovine serum albumin in FLAG-binding buffer (10 mM Hepes pH 7.9, 1.5 mM

MgCl2, 250 mM NaCl, 0.1% Triton X-100, and 10% glycerol) for 30 min at ChIP. For RNAPIIo and γH2AX ChIP analyses, cells were fixed with 1% 4 °C. TRIM28 and RECQ5 were then added to the chitin beads and incubated formaldehyde for 10 min at room temperature, and the reaction was stop- for 2 h at 4 °C. The bound proteins were washed extensively with FLAG- ped by the addition of 0.125 M glycine. Cells were resuspended in buffer I (5 binding buffer, boiled in SDS sample buffer, separated by SDS/PAGE, and mM Pipes pH 8.0, 85 mM KCl, and 0.5% Nonidet P-40), followed by ho- analyzed using Western blots. mogenization using a Dounce homogenizer. The chromatin pellet was iso- × lated by centrifugation at 2,350 g for 5 min at 4 °C. The pellets were Statistics and Reproducibility. For all experiments, unless stated otherwise, resuspended in ChIP lysis buffer (1.0% SDS, 10 mM EDTA, and 50 mM Tris pH representative analyses from a minimum of three independent experiments 8.0) plus protease inhibitors, and chromatin was sheared by sonication to are shown. All in vitro analyses using purified recombinant proteins were < generate DNA fragments of 1 kb. Chromatin was diluted 10 times in ChIP performed, at least, three times using two different sets of purified proteins. dilution buffer (16.7 mM Tris pH 8.0, 0.01% SDS, 1.1% Triton X-100, 1.2 mM For qPCR, each value represents mean ± SD calculated from triplicate qPCR EDTA, and 167 mM NaCl) plus protease inhibitors, then, precleared with reactions for one representative experiment. P values were calculated using protein A/G beads (Thermo Scientific) for 1 h at 4 °C. Precleared samples two-tailed Student’s t tests for statistically significant differences. were incubated overnight at 4 °C with antibodies. For RNAPIIo ChIP, the Additional methods and materials for protein purification and neutral beads were washed sequentially twice with low salt buffer A (0.1% SDS, comet assays are available in the SI Appendix. 1.0% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, and 0.15 M NaCl), high salt buffer A (0.1% SDS, 1.0% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, Data Availability. All study data are included in the article and the and 0.5 M NaCl), LiCl buffer A (0.25 M LiCl, 1.0% Nonidet P-40, 1% sodium SI Appendix. deoxycholate, 1 mM EDTA, and 10 mM Tris pH 8.0), and TE buffer. The RNAPIIo–DNA complexes were eluted using 300 μL elution buffer (0.1 M sodium bicarbonate and 1.0% SDS) for 15 min at room temperature, reverse ACKNOWLEDGMENTS. We thank Dr. Sarah Wilkinson for her comments and expert editing of this paper. This work was supported by NIH Grant R01 cross-linked by adding 20 μL 5 M NaCl, and incubated overnight at 65 °C. The CA225843 to Y.L. Research reported in this publication included work DNA was digested using RNase A and proteinase K and purified by phenol- performed in the Analytical Cytometry Core and Drug Discovery & Structural chloroform and ethanol precipitation. For γH2AX ChIP, beads were washed Biology Core supported by the NCI of NIH under Grant P30CA033572. The once in dialysis buffer (2 mM EDTA, 50 mM Tris pH 8, and 0.2% sarkosyl) and content is solely the responsibility of the authors and does not necessarily four times in wash buffer (100 mM Tris pH 8.8, 500 mM LiCl, 1% Nonidet P- represent the official views of the NIH.

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