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• W2809592012 abstract "•Methylation of H3K4 by SETD1A maintains genome stability during replication stress•SETD1A and H3K4 methylation stabilize RAD51 nucleofilaments to protect nascent DNA•SETD1A-dependent H3K4 methylation enhances FANCD2-dependent histone remodeling•Histone mobility stabilizes RAD51 nucleofilaments to inhibit fork degradation Components of the Fanconi anemia and homologous recombination pathways play a vital role in protecting newly replicated DNA from uncontrolled nucleolytic degradation, safeguarding genome stability. Here we report that histone methylation by the lysine methyltransferase SETD1A is crucial for protecting stalled replication forks from deleterious resection. Depletion of SETD1A sensitizes cells to replication stress and leads to uncontrolled DNA2-dependent resection of damaged replication forks. The ability of SETD1A to prevent degradation of these structures is mediated by its ability to catalyze methylation on Lys4 of histone H3 (H3K4) at replication forks, which enhances FANCD2-dependent histone chaperone activity. Suppressing H3K4 methylation or expression of a chaperone-defective FANCD2 mutant leads to loss of RAD51 nucleofilament stability and severe nucleolytic degradation of replication forks. Our work identifies epigenetic modification and histone mobility as critical regulatory mechanisms in maintaining genome stability by restraining nucleases from irreparably damaging stalled replication forks. Components of the Fanconi anemia and homologous recombination pathways play a vital role in protecting newly replicated DNA from uncontrolled nucleolytic degradation, safeguarding genome stability. Here we report that histone methylation by the lysine methyltransferase SETD1A is crucial for protecting stalled replication forks from deleterious resection. Depletion of SETD1A sensitizes cells to replication stress and leads to uncontrolled DNA2-dependent resection of damaged replication forks. The ability of SETD1A to prevent degradation of these structures is mediated by its ability to catalyze methylation on Lys4 of histone H3 (H3K4) at replication forks, which enhances FANCD2-dependent histone chaperone activity. Suppressing H3K4 methylation or expression of a chaperone-defective FANCD2 mutant leads to loss of RAD51 nucleofilament stability and severe nucleolytic degradation of replication forks. Our work identifies epigenetic modification and histone mobility as critical regulatory mechanisms in maintaining genome stability by restraining nucleases from irreparably damaging stalled replication forks. To maintain genome stability during genome duplication, numerous cellular pathways have evolved to detect and repair structures and/or lesions that impair DNA replication. One key response to compromised replication (known as replication stress) involves the active reversal of stalled replication forks to form 4-way DNA junctions. This represents a critical step in stabilizing damaged forks (Neelsen and Lopes, 2015Neelsen K.J. Lopes M. Replication fork reversal in eukaryotes: from dead end to dynamic response.Nat. Rev. Mol. Cell Biol. 2015; 16: 207-220Crossref PubMed Scopus (284) Google Scholar) and involves homologous recombination (HR) factors such as RAD51 (Wang et al., 2015Wang A.T. Kim T. Wagner J.E. Conti B.A. Lach F.P. Huang A.L. Molina H. Sanborn E.M. Zierhut H. Cornes B.K. et al.A dominant mutation in human RAD51 reveals its function in DNA interstrand crosslink repair independent of homologous recombination.Mol. Cell. 2015; 59: 478-490Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, Zellweger et al., 2015Zellweger R. Dalcher D. Mutreja K. Berti M. Schmid J.A. Herrador R. Vindigni A. Lopes M. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells.J. Cell Biol. 2015; 208: 563-579Crossref PubMed Scopus (398) Google Scholar). However, despite the importance of fork reversal in protecting genome stability, it is also clear that the regressed arms of reversed forks are highly susceptible to nucleolytic degradation (Thangavel et al., 2015Thangavel S. Berti M. Levikova M. Pinto C. Gomathinayagam S. Vujanovic M. Zellweger R. Moore H. Lee E.H. Hendrickson E.A. et al.DNA2 drives processing and restart of reversed replication forks in human cells.J. Cell Biol. 2015; 208: 545-562Crossref PubMed Scopus (226) Google Scholar). Although controlled processing of these structures can help to maintain fork integrity and allow fork restart, uncontrolled degradation of nascent DNA leads to severe genome instability (Quinet et al., 2017Quinet A. Lemaçon D. Vindigni A. Replication fork reversal: players and guardians.Mol. Cell. 2017; 68: 830-833Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Components of the Fanconi anemia (FA) and HR pathways, including RAD51 (FANCR), FANCD2, BRCA1 (FANCS), and BRCA2 (FANCD1), play a vital role in protecting nascent DNA at reversed replication forks (Schlacher et al., 2011Schlacher K. Christ N. Siaud N. Egashira A. Wu H. Jasin M. Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11.Cell. 2011; 145: 529-542Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar, Schlacher et al., 2012Schlacher K. Wu H. Jasin M. A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2.Cancer Cell. 2012; 22: 106-116Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar, Somyajit et al., 2015Somyajit K. Saxena S. Babu S. Mishra A. Nagaraju G. Mammalian RAD51 paralogs protect nascent DNA at stalled forks and mediate replication restart.Nucleic Acids Res. 2015; 43: 9835-9855PubMed Google Scholar, Ying et al., 2012Ying S. Hamdy F.C. Helleday T. Mre11-dependent degradation of stalled DNA replication forks is prevented by BRCA2 and PARP1.Cancer Res. 2012; 72: 2814-2821Crossref PubMed Scopus (239) Google Scholar, Zadorozhny et al., 2017Zadorozhny K. Sannino V. Beláň O. Mlčoušková J. Špírek M. Costanzo V. Krejčí L. Fanconi-anemia-associated mutations destabilize RAD51 filaments and impair replication fork protection.Cell Rep. 2017; 21: 333-340Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Moreover, the deleterious resection of replication forks observed in the absence of these factors can be prevented by limiting fork reversal via depletion of “pro-reversal” factors; e.g., SMARCAL1 (Mijic et al., 2017Mijic S. Zellweger R. Chappidi N. Berti M. Jacobs K. Mutreja K. Ursich S. Ray Chaudhuri A. Nussenzweig A. Janscak P. Lopes M. Replication fork reversal triggers fork degradation in BRCA2-defective cells.Nat. Commun. 2017; 8: 859Crossref PubMed Scopus (189) Google Scholar, Taglialatela et al., 2017Taglialatela A. Alvarez S. Leuzzi G. Sannino V. Ranjha L. Huang J.W. Madubata C. Anand R. Levy B. Rabadan R. et al.Restoration of replication fork stability in BRCA1- and BRCA2-deficient cells by inactivation of SNF2-family fork remodelers.Mol. Cell. 2017; 68: 414-430.e8Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). However, despite intensive study, the mechanisms by which cells protect nascent DNA still remain poorly understood. It is imperative that we better understand these processes because restoring fork protection in tumor cells facilitates their ability to evade chemotherapy and acquire drug resistance (Ray Chaudhuri et al., 2016Ray Chaudhuri A. Callen E. Ding X. Gogola E. Duarte A.A. Lee J.E. Wong N. Lafarga V. Calvo J.A. Panzarino N.J. et al.Replication fork stability confers chemoresistance in BRCA-deficient cells.Nature. 2016; 535: 382-387Crossref PubMed Scopus (489) Google Scholar). Presently, it is unclear how reversed replication forks requiring protection are marked; this may involve the presence of specific factors, post-translational modification of the replication machinery and/or surrounding chromatin, and/or chromatin remodeling. In keeping with the premise that histone dynamics may play an important role in this process, members of the SNF2 family of remodeling ATPases promote fork degradation in the absence of protective factors (Taglialatela et al., 2017Taglialatela A. Alvarez S. Leuzzi G. Sannino V. Ranjha L. Huang J.W. Madubata C. Anand R. Levy B. Rabadan R. et al.Restoration of replication fork stability in BRCA1- and BRCA2-deficient cells by inactivation of SNF2-family fork remodelers.Mol. Cell. 2017; 68: 414-430.e8Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Moreover, the fork protection factor FANCD2 also remodels histones at sites of replication stress (Sato et al., 2012Sato K. Ishiai M. Toda K. Furukoshi S. Osakabe A. Tachiwana H. Takizawa Y. Kagawa W. Kitao H. Dohmae N. et al.Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair.EMBO J. 2012; 31: 3524-3536Crossref PubMed Scopus (51) Google Scholar). Interestingly, several chromatin modifiers have also been implicated in preventing fork degradation: the lysine methyltransferase (KMT) EZH2 regulates recruitment of MUS81 to stalled forks (Rondinelli et al., 2017Rondinelli B. Gogola E. Yücel H. Duarte A.A. van de Ven M. van der Sluijs R. Konstantinopoulos P.A. Jonkers J. Ceccaldi R. Rottenberg S. D’Andrea A.D. EZH2 promotes degradation of stalled replication forks by recruiting MUS81 through histone H3 trimethylation.Nat. Cell Biol. 2017; 19: 1371-1378Crossref PubMed Scopus (199) Google Scholar), whereas the KMTs KMT2C/KMT2D (MLL2/3) enhance MRE11-dependent fork processing (Ray Chaudhuri et al., 2016Ray Chaudhuri A. Callen E. Ding X. Gogola E. Duarte A.A. Lee J.E. Wong N. Lafarga V. Calvo J.A. Panzarino N.J. et al.Replication fork stability confers chemoresistance in BRCA-deficient cells.Nature. 2016; 535: 382-387Crossref PubMed Scopus (489) Google Scholar). In contrast, the yeast KMT Set1, a component of the evolutionarily conserved “complex proteins associated with Set1p” (COMPASS) that catalyzes methylation of lysine 4 of histone H3 (H3K4), is required in the response to replication stress (Faucher and Wellinger, 2010Faucher D. Wellinger R.J. Methylated H3K4, a transcription-associated histone modification, is involved in the DNA damage response pathway.PLoS Genet. 2010; 6 (e1001082)Crossref PubMed Scopus (112) Google Scholar). Recently, we identified BOD1L as a fork protection factor that protects nascent DNA from degradation by DNA2 (Higgs et al., 2015Higgs M.R. Reynolds J.J. Winczura A. Blackford A.N. Borel V. Miller E.S. Zlatanou A. Nieminuszczy J. Ryan E.L. Davies N.J. et al.BOD1L is required to suppress deleterious resection of stressed replication forks.Mol. Cell. 2015; 59: 462-477Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, Higgs and Stewart, 2016Higgs M.R. Stewart G.S. Protection or resection: BOD1L as a novel replication fork protection factor.Nucleus. 2016; 7: 34-40Crossref PubMed Scopus (10) Google Scholar). Here we demonstrate that BOD1L functionally interacts with the KMT SETD1A and that cells lacking SETD1A phenocopy those depleted of BOD1L. Furthermore, we show that SETD1A methylates H3K4 at stalled replication forks, which facilitates the mobilization of histones by FANCD2 and prevents replication fork degradation. Compromising H3K4 methylation abrogates FANCD2-dependent histone chaperone activity, leads to fork degradation, and mimics the inability of cells lacking SETD1A to recruit RAD51 to stalled forks. Our data therefore establish that SETD1A-dependent histone methylation and subsequent histone remodeling protect stalled forks from uncontrolled processing, thereby maintaining genome stability. We recently identified BOD1L as a factor that protects stalled replication forks from degradation (Higgs et al., 2015Higgs M.R. Reynolds J.J. Winczura A. Blackford A.N. Borel V. Miller E.S. Zlatanou A. Nieminuszczy J. Ryan E.L. Davies N.J. et al.BOD1L is required to suppress deleterious resection of stressed replication forks.Mol. Cell. 2015; 59: 462-477Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Previous studies (van Nuland et al., 2013van Nuland R. Smits A.H. Pallaki P. Jansen P.W. Vermeulen M. Timmers H.T. Quantitative dissection and stoichiometry determination of the human SET1/MLL histone methyltransferase complexes.Mol. Cell. Biol. 2013; 33: 2067-2077Crossref PubMed Scopus (150) Google Scholar) have suggested that BOD1L forms complexes with the KMTs SETD1A and SETD1B, two closely related members of the KMT2 family that catalyze H3K4 methylation (Bledau et al., 2014Bledau A.S. Schmidt K. Neumann K. Hill U. Ciotta G. Gupta A. Torres D.C. Fu J. Kranz A. Stewart A.F. Anastassiadis K. The H3K4 methyltransferase Setd1a is first required at the epiblast stage, whereas Setd1b becomes essential after gastrulation.Development. 2014; 141: 1022-1035Crossref PubMed Scopus (119) Google Scholar, Brici et al., 2017Brici D. Zhang Q. Reinhardt S. Dahl A. Hartmann H. Schmidt K. Goveas N. Huang J. Gahurova L. Kelsey G. et al.Setd1b, encoding a histone 3 lysine 4 methyltransferase, is a maternal effect gene required for the oogenic gene expression program.Development. 2017; 144: 2606-2617Crossref PubMed Scopus (35) Google Scholar, Lee and Skalnik, 2005Lee J.H. Skalnik D.G. CpG-binding protein (CXXC finger protein 1) is a component of the mammalian Set1 histone H3-Lys4 methyltransferase complex, the analogue of the yeast Set1/COMPASS complex.J. Biol. Chem. 2005; 280: 41725-41731Crossref PubMed Scopus (243) Google Scholar, Lee et al., 2007Lee J.H. Tate C.M. You J.S. Skalnik D.G. Identification and characterization of the human Set1B histone H3-Lys4 methyltransferase complex.J. Biol. Chem. 2007; 282: 13419-13428Crossref PubMed Scopus (182) Google Scholar). This suggested that SETD1A and/or SETD1B may function with BOD1L to regulate replication fork stability. We therefore first sought to confirm these interactions before investigating any potential role of these enzymes in fork protection. Interestingly, reciprocal immunoprecipitations confirmed that BOD1L interacts with SETD1A, but not with SETD1B, as was suggested previously (van Nuland et al., 2013van Nuland R. Smits A.H. Pallaki P. Jansen P.W. Vermeulen M. Timmers H.T. Quantitative dissection and stoichiometry determination of the human SET1/MLL histone methyltransferase complexes.Mol. Cell. Biol. 2013; 33: 2067-2077Crossref PubMed Scopus (150) Google Scholar; Figure 1A). The N-terminal region of BOD1L contains a region with sequence homology to the Shg1 component of the yeast COMPASS complex (Figure S1A) (PFAM: 05205; http://pfam.xfam.org/family/PF05205) (Marchler-Bauer et al., 2011Marchler-Bauer A. Lu S. Anderson J.B. Chitsaz F. Derbyshire M.K. DeWeese-Scott C. Fong J.H. Geer L.Y. Geer R.C. Gonzales N.R. et al.CDD: a Conserved Domain Database for the functional annotation of proteins.Nucleic Acids Res. 2011; 39: D225-D229Crossref PubMed Scopus (2346) Google Scholar, Roguev et al., 2001Roguev A. Schaft D. Shevchenko A. Pijnappel W.W. Wilm M. Aasland R. Stewart A.F. The Saccharomyces cerevisiae Set1 complex includes an Ash2 homologue and methylates histone 3 lysine 4.EMBO J. 2001; 20: 7137-7148Crossref PubMed Scopus (457) Google Scholar). We hypothesized that this “COMPASS-Shg1” domain may, by analogy, mediate the interaction of BOD1L with the SETD1A complex. To assess this, we generated glutathione S-transferase (GST)-tagged fragments of BOD1L spanning this domain or neighboring regions and analyzed their ability to interact with SETD1A. These experiments revealed that a fragment of BOD1L containing this COMPASS-Shg1 domain was necessary and sufficient to mediate interaction with SETD1A but not with SETD1B (Figure S1B). To analyze the functional consequences of this interaction, we depleted HeLa cells of BOD1L, SETD1A, or SETD1B, either alone or in combination (Figure 1B), and exposed them to mitomycin C (MMC), which induces DNA interstrand crosslinks (ICLs). Notably, depletion of SETD1A or BOD1L alone exquisitely hypersensitized cells to MMC (Figure 1C). Moreover, cells lacking SETD1A exhibited elevated chromosomal damage (Figures 1D, 1E, and S1C) and increased micronucleus formation after treatment with MMC (Figure 1F), indicating a critical role for this KMT in maintaining genome stability after replication damage. In all cases, loss of SETD1A alone or alongside BOD1L was comparable with BOD1L depletion, consistent with these two factors residing within the same protein complex. In contrast, depletion of SETD1B had no effect on cellular sensitivity to replication stress (Figure 1), in keeping with our interaction data, although we cannot completely exclude a role of this enzyme in regulating genome stability. Moreover, cells lacking SETD1A or BOD1L, but not SETD1B or BOD1 (a BOD1L paralog), were unable to suppress replication origin firing after MMC exposure (Figure S1D), a characteristic of BOD1L deficiency (Higgs et al., 2015Higgs M.R. Reynolds J.J. Winczura A. Blackford A.N. Borel V. Miller E.S. Zlatanou A. Nieminuszczy J. Ryan E.L. Davies N.J. et al.BOD1L is required to suppress deleterious resection of stressed replication forks.Mol. Cell. 2015; 59: 462-477Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Together with our previous data demonstrating that depletion of BOD1 had no effect on MMC cellular sensitivity (Higgs et al., 2015Higgs M.R. Reynolds J.J. Winczura A. Blackford A.N. Borel V. Miller E.S. Zlatanou A. Nieminuszczy J. Ryan E.L. Davies N.J. et al.BOD1L is required to suppress deleterious resection of stressed replication forks.Mol. Cell. 2015; 59: 462-477Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), these observations are consistent with a model in which BOD1L/SETD1A and BOD1/SETD1B form functionally distinct KMT complexes. Preventing aberrant replication fork resection is essential for genome integrity during replication stress. The RAD51 recombinase plays a crucial role in protecting stalled forks from such degradation (Schlacher et al., 2011Schlacher K. Christ N. Siaud N. Egashira A. Wu H. Jasin M. Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11.Cell. 2011; 145: 529-542Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar, Schlacher et al., 2012Schlacher K. Wu H. Jasin M. A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2.Cancer Cell. 2012; 22: 106-116Abstract Full Text Full Text PDF PubMed Scopus (609) Google Scholar, Zadorozhny et al., 2017Zadorozhny K. Sannino V. Beláň O. Mlčoušková J. Špírek M. Costanzo V. Krejčí L. Fanconi-anemia-associated mutations destabilize RAD51 filaments and impair replication fork protection.Cell Rep. 2017; 21: 333-340Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, Zellweger et al., 2015Zellweger R. Dalcher D. Mutreja K. Berti M. Schmid J.A. Herrador R. Vindigni A. Lopes M. Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells.J. Cell Biol. 2015; 208: 563-579Crossref PubMed Scopus (398) Google Scholar); indeed, our previous studies demonstrated that BOD1L suppresses degradation of stalled replication forks by stabilizing RAD51 at these sites (Higgs et al., 2015Higgs M.R. Reynolds J.J. Winczura A. Blackford A.N. Borel V. Miller E.S. Zlatanou A. Nieminuszczy J. Ryan E.L. Davies N.J. et al.BOD1L is required to suppress deleterious resection of stressed replication forks.Mol. Cell. 2015; 59: 462-477Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). To investigate whether SETD1A functioned in a similar fashion, we first analyzed DNA resection in cells depleted of BOD1L, SETD1A, or SETD1B, using phosphorylation of RPA2 on S4/S8 as a well-established marker of resected DNA. Interestingly, levels of MMC-induced RPA2-P-S4/8 were substantially elevated upon loss of BOD1L or SETD1A but not SETD1B (Figures 1G, 2A, and 2B), consistent with increased generation of single-stranded DNA (ssDNA). Moreover, similar to BOD1L, SETD1A was required to recruit or stabilize RAD51 at stalled forks upon exposure to either MMC or hydroxyurea (HU) (Figures 2C and 2D ). Co-depletion of SETD1A and BOD1L had no additional effect on RPA/RPA2-P-S4/8 or RAD51 focus formation, again suggesting that these factors act together (Figures 2B–2G). Next, using 5-ethynyl-2'-deoxyuridine (EdU)-Click coupled to proximity ligation (PLA) (Petruk et al., 2012Petruk S. Sedkov Y. Johnston D.M. Hodgson J.W. Black K.L. Kovermann S.K. Beck S. Canaani E. Brock H.W. Mazo A. TrxG and PcG proteins but not methylated histones remain associated with DNA through replication.Cell. 2012; 150: 922-933Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, Taglialatela et al., 2017Taglialatela A. Alvarez S. Leuzzi G. Sannino V. Ranjha L. Huang J.W. Madubata C. Anand R. Levy B. Rabadan R. et al.Restoration of replication fork stability in BRCA1- and BRCA2-deficient cells by inactivation of SNF2-family fork remodelers.Mol. Cell. 2017; 68: 414-430.e8Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), we analyzed whether the association of RAD51with nascent DNA was affected by loss of SETD1A. In agreement with our previous data, SETD1A depletion significantly decreased the levels of RAD51 on newly replicated DNA after HU exposure (Figure 2H). Given the importance of RAD51 in suppressing deleterious fork processing, we next assessed whether SETD1A was required to protect nascent DNA at replication forks. To this end, we used a well-characterized fork protection assay (Schlacher et al., 2011Schlacher K. Christ N. Siaud N. Egashira A. Wu H. Jasin M. Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11.Cell. 2011; 145: 529-542Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar) to monitor the stability of nascent DNA during prolonged replication arrest by HU. Loss of SETD1A, but not SETD1B, increased the degradation of HU-stalled forks, apparent as a decreased iododeoxyuridine (IdU):chlorodeoxyuridine (CldU) ratio (Figure 3A; Table S1), supporting our hypothesis that SETD1A and SETD1B are functionally distinct. Moreover, the fork degradation observed upon BOD1L loss was comparable with that arising from SETD1A depletion (Figure 3B; Table S1). Because fork remodeling enzymes such as SMARCAL1 catalyze fork reversal and, thus, provide a substrate for nucleolytic degradation in the absence of protective factors (Kolinjivadi et al., 2017Kolinjivadi A.M. Sannino V. De Antoni A. Zadorozhny K. Kilkenny M. Techer H. Baldi G. Shen R. Ciccia A. Pellegrini L. et al.Smarcal1-mediated fork reversal triggers Mre11-dependent degradation of nascent DNA in the absence of Brca2 and stable Rad51 nucleofilaments.Mol. Cell. 2017; 67: 867-881.e7Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), we investigated whether loss of SMARCAL1 suppressed fork resection observed upon SETD1A loss. Indeed, depletion of this annealing helicase reduced nascent strand degradation in cells lacking SETD1A (Figure 3C; Table S1). Together, this suggests that SETD1A-BOD1L prevent the resection of reversed replication forks. Recent studies have suggested that other members of the KMT2 family (KMT2C [MLL3] and KMT2D [MLL2]) promote nascent strand degradation in the absence of BRCA2 (Ray Chaudhuri et al., 2016Ray Chaudhuri A. Callen E. Ding X. Gogola E. Duarte A.A. Lee J.E. Wong N. Lafarga V. Calvo J.A. Panzarino N.J. et al.Replication fork stability confers chemoresistance in BRCA-deficient cells.Nature. 2016; 535: 382-387Crossref PubMed Scopus (489) Google Scholar). However, although we also observed that depletion of KMT2C/KMT2D rescued fork stability in the absence of BRCA2, this was not the case when these factors were co-depleted in combination with SETD1A (Figures 3D–3F; Table S1). Moreover, in stark contrast to cells lacking SETD1A, loss of KMT2C/D alone had no effect on fork stability after HU. These data reinforce the notion that different KMT2 enzymes have diverse functions during replication stress. Factors that regulate the stability of RAD51 filaments, such as PARI, BLM, FBH1, and the RAD51 paralogs, also play vital roles in maintaining replication fork stability (Mochizuki et al., 2017Mochizuki A.L. Katanaya A. Hayashi E. Hosokawa M. Moribe E. Motegi A. Ishiai M. Takata M. Kondoh G. Watanabe H. et al.PARI regulates stalled replication fork processing to maintain genome stability upon replication stress in mice.Mol. Cell. Biol. 2017; 37 (e00117-17)Crossref PubMed Scopus (9) Google Scholar, Somyajit et al., 2015Somyajit K. Saxena S. Babu S. Mishra A. Nagaraju G. Mammalian RAD51 paralogs protect nascent DNA at stalled forks and mediate replication restart.Nucleic Acids Res. 2015; 43: 9835-9855PubMed Google Scholar). Previous work has demonstrated that uncontrolled BLM/FBH1 activity can destabilize RAD51 nucleofilaments at stalled replication forks, leading to fork degradation (Higgs et al., 2015Higgs M.R. Reynolds J.J. Winczura A. Blackford A.N. Borel V. Miller E.S. Zlatanou A. Nieminuszczy J. Ryan E.L. Davies N.J. et al.BOD1L is required to suppress deleterious resection of stressed replication forks.Mol. Cell. 2015; 59: 462-477Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, Higgs and Stewart, 2016Higgs M.R. Stewart G.S. Protection or resection: BOD1L as a novel replication fork protection factor.Nucleus. 2016; 7: 34-40Crossref PubMed Scopus (10) Google Scholar, Leuzzi et al., 2016Leuzzi G. Marabitti V. Pichierri P. Franchitto A. WRNIP1 protects stalled forks from degradation and promotes fork restart after replication stress.EMBO J. 2016; 35: 1437-1451Crossref PubMed Scopus (52) Google Scholar). We therefore predicted that SETD1A may also counteract the activities of these two anti-recombinases to stabilize RAD51 at stalled forks. To investigate this, we co-depleted SETD1A and BLM (Figure S2A), exposed the cells to MMC, and monitored RPA S4/S8 phosphorylation and RAD51 focus formation. Strikingly, loss of BLM reduced MMC-induced RPA S4/S8 phosphorylation (Figure S2B) and restored HU- and MMC-induced RAD51 focus formation in the absence of SETD1A (Figures S2C and S2D). Moreover, depletion of either FBH1 or BLM abrogated the degradation of nascent DNA observed in cells lacking SETD1A (Figure S2E; Table S1). Because BOD1L, RAD51, and the FA pathway are crucial for suppressing fork resection by DNA2 (Higgs et al., 2015Higgs M.R. Reynolds J.J. Winczura A. Blackford A.N. Borel V. Miller E.S. Zlatanou A. Nieminuszczy J. Ryan E.L. Davies N.J. et al.BOD1L is required to suppress deleterious resection of stressed replication forks.Mol. Cell. 2015; 59: 462-477Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, Karanja et al., 2014Karanja K.K. Lee E.H. Hendrickson E.A. Campbell J.L. Preventing over-resection by DNA2 helicase/nuclease suppresses repair defects in Fanconi anemia cells.Cell Cycle. 2014; 13: 1540-1550Crossref PubMed Scopus (48) Google Scholar, Liu et al., 2016Liu W. Zhou M. Li Z. Li H. Polaczek P. Dai H. Wu Q. Liu C. Karanja K.K. Popuri V. et al.A selective small molecule DNA2 inhibitor for sensitization of human cancer cells to chemotherapy.EBioMedicine. 2016; 6: 73-86Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), we assessed whether the over-resection observed in the absence of SETD1A was dependent on DNA2 or whether other nucleases implicated in fork resection were also involved. In keeping with our previous observations in cells lacking BOD1L (Higgs et al., 2015Higgs M.R. Reynolds J.J. Winczura A. Blackford A.N. Borel V. Miller E.S. Zlatanou A. Nieminuszczy J. Ryan E.L. Davies N.J. et al.BOD1L is required to suppress deleterious resection of stressed replication forks.Mol. Cell. 2015; 59: 462-477Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), co-depletion of DNA2, but not EXO1 or MRE11, suppressed the degradation of HU-stalled forks in the absence of SETD1A (Figure S2F; Table S1). Therefore, SETD1A and BOD1L act together to stabilize RAD51 on nascent DNA by restraining the anti-recombinase functions of BLM/FBH1, protecting damaged replication forks from DNA2-dependent resection. Previous studies have demonstrated that the methyltransferase activity of SETD1A toward H3K4 is mediated by its C-terminal N-SET (COMPASS component N-Su(var)3-9, Enhancer-of-zeste, Trithorax domain) and SET catalytic domains, whereas interactions with WDR82, RNA, and RNA polymerase II (Pol II) occur via the N-terminal RRM domain (Lee and Skalnik, 2008Lee J.H. Skalnik D.G. Wdr82 is a C-terminal domain-binding protein that recruits the Setd1A Histone H3-Lys4 methyltransferase complex to transcription start sites of transcribed human genes.Mol. Cell. Biol. 2008; 28: 609-618Crossref PubMed Scopus (144) Google Scholar, Luciano et al., 2017Luciano P. Jeon J. El-Kaoutari A. Challal D. Bonnet A. Barucco M. Candelli T. Jourquin F. Lesage P. Kim J. et al.Binding to RNA regulates Set1 function.Cell Discov. 2017; 3: 17040Crossref PubMed Scopus (18) Google Scholar, Schlichter and Cairns, 2005Schlichter A. Cairns B.R. Histone trimethylation by Set1 is coordinated by the RRM, autoinhibitory, and catalytic domains.EMBO J. 2005; 24: 1222-1231Crossref PubMed Scopus (75) Google Scholar). To examine which of these domains is required for fork protection by SETD1A, we established U-2-OS cell lines in which endogenous SETD1A could be depleted with small interfering RNA (siRNA), and either full-length (FL) FLAG-tagged SETD1A or variants lacking the RRM (ΔRRM) or catalytic SET (ΔSET) domains could be inducibly expressed (Figures S3A and S3B). Strikingly, the genome instability (Figure 4A), defective RAD51 focus formation (Figures 4B and 4C), and increased fork degradation (Figure 4D) observed in the absence of endogenou" @default.
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• W2809592012 date "2018-07-01" @default.
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• W2809592012 title "Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2" @default.
• W2809592012 cites W1516566300 @default.
• W2809592012 cites W1531540594 @default.
• W2809592012 cites W1808263692 @default.
• W2809592012 cites W1838945435 @default.
• W2809592012 cites W1965343063 @default.
• W2809592012 cites W1970578083 @default.
• W2809592012 cites W1986586739 @default.
• W2809592012 cites W1987086578 @default.
• W2809592012 cites W1989388273 @default.
• W2809592012 cites W1997925338 @default.
• W2809592012 cites W2008461545 @default.
• W2809592012 cites W2012671266 @default.
• W2809592012 cites W2036124213 @default.
• W2809592012 cites W2037993016 @default.
• W2809592012 cites W2047343415 @default.
• W2809592012 cites W2048442759 @default.
• W2809592012 cites W2053964757 @default.
• W2809592012 cites W2054205113 @default.
• W2809592012 cites W2060740961 @default.
• W2809592012 cites W2074917365 @default.
• W2809592012 cites W2091230697 @default.
• W2809592012 cites W2092027270 @default.
• W2809592012 cites W2093958440 @default.
• W2809592012 cites W2096380982 @default.
• W2809592012 cites W2104584181 @default.
• W2809592012 cites W2110817748 @default.
• W2809592012 cites W2110951751 @default.
• W2809592012 cites W2114039383 @default.
• W2809592012 cites W2125490008 @default.
• W2809592012 cites W2140236604 @default.
• W2809592012 cites W2141897899 @default.
• W2809592012 cites W2148798204 @default.
• W2809592012 cites W2161711392 @default.
• W2809592012 cites W2164817558 @default.
• W2809592012 cites W2214304893 @default.
• W2809592012 cites W2296463804 @default.
• W2809592012 cites W2315977850 @default.
• W2809592012 cites W2408982653 @default.
• W2809592012 cites W2409124218 @default.
• W2809592012 cites W2476568892 @default.
• W2809592012 cites W2511945448 @default.
• W2809592012 cites W2526137059 @default.
• W2809592012 cites W2592850216 @default.
• W2809592012 cites W2607198330 @default.
• W2809592012 cites W2626471237 @default.
• W2809592012 cites W2737658553 @default.
• W2809592012 cites W2754855275 @default.
• W2809592012 cites W2763248055 @default.
• W2809592012 cites W2763383409 @default.
• W2809592012 cites W2765667278 @default.
• W2809592012 cites W2765875601 @default.
• W2809592012 cites W2766340225 @default.
• W2809592012 cites W2770177949 @default.
• W2809592012 cites W2771808827 @default.
• W2809592012 cites W2788063335 @default.
• W2809592012 cites W2793562500 @default.
• W2809592012 doi "https://doi.org/10.1016/j.molcel.2018.05.018" @default.
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