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In vivo phosphoproteome

Supplementary Figures and Methods

FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ’s catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In part, this reflects the availability of methods for the identification of break sites, and characterization of factors recruited to DSBs at those sequences. However, DSBs also appear as intermediates during the processing of DNA adducts formed by compounds that do not directly cause breaks, and do not react at specific sequence sites. Consequently, for most of these agents, technologies that permit the analysis of binding interactions with response factors and repair proteins are unknown. For example, DNA interstrand crosslinks (ICLs) can provoke breaks following replication fork encounters. Although formed by drugs widely used as cancer chemotherapeutics, there has been no methodology for monitoring their interactions with replication proteins. Here, we describe our strategy for following the cellular response to fork collisions with these challenging adducts. We linked a steroid antigen to psoralen, which forms photoactivation dependent ICLs in nuclei of living cells. The ICLs were visualized by immunofluorescence against the antigen tag. The tag can also be a partner in the Proximity Ligation Assay (PLA) which reports the close association of two antigens. The PLA was exploited to distinguish proteins that were closely associated with the tagged ICLs from those that were not. It was possible to define replisome proteins that were retained after encounters with ICLs and identify others that were lost. This approach is applicable to any structure or DNA adduct that can be detected immunologically.

Purpose: Somatic inactivating mutations in ARID1A, a component of the SWI/SNF chromatin remodeling complex, are detected in various types of human malignancies. Loss of ARID1A compromises DNA damage repair. The induced DNA damage burden may increase reliance on PARP-dependent DNA repair of cancer cells to maintain genome integrity and render susceptibility to PARP inhibitor therapy. Experimental Design: Isogenic ARID1A−/− and wild-type cell lines were used for assessing DNA damage response, DNA compactness, and profiling global serine/threonine phosphoproteomic in vivo. A panel of inhibitors targeting DNA repair pathways was screened for a synergistic antitumor effect with irradiation in ARID1A−/− tumors. Results: ARID1A-deficient endometrial cells exhibit sustained levels in DNA damage response, a result further supported by in vivo phosphoproteomic analysis. Our results show that ARID1A is essential for establishing an open chromatin state upon DNA damage, a process required for recruitment of 53BP1 and RIF1, key mediators of non-homologous end-joining (NHEJ) machinery, to DNA lesions. The inability of ARID1A−/− cells to mount NHEJ repair results in a partial cytotoxic response to radiation. Small-molecule compound screens revealed that PARP inhibitors act synergistically with radiation to potentiate cytotoxicity in ARID1A−/− cells. Combination treatment with low-dose radiation and olaparib greatly improved antitumor efficacy, resulting in long-term remission in mice bearing ARID1A-deficient tumors. Conclusions: ARID1A-deficient cells acquire high sensitivity to PARP inhibition after exposure to exogenously induced DNA breaks such as ionizing radiation. Our findings suggest a novel biologically informed strategy for treating ARID1A-deficient malignancies.

Duplication of mammalian genomes requires replisomes to overcome numerous impediments during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of replication stress characterize mixed populations of challenged and unchallenged replication forks, averaged across S phase, and model a single species of “stressed” replisome. Here, in cells containing potent obstacles to replication, we find two different lesion proximal replisomes. One is bound by the DONSON protein and is more frequent in early S phase, in regions marked by euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. ChIP-seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions that replicate early and the skew of the latter towards regions that replicate late., Eukaryotic replisomes are multiprotein complexes. Here the authors reveal two distinct stressed replisomes, associated with DONSON and FANCM, displaying a bias in replication timing and chromatin domain.

Deficiency of FANCD2/FANCI-associated nuclease 1 (FAN1) in humans leads to karyomegalic interstitial nephritis (KIN), a rare hereditary kidney disease characterized by chronic renal fibrosis, tubular degeneration, and characteristic polyploid nuclei in multiple tissues. The mechanism of how FAN1 protects cells is largely unknown but is thought to involve FAN1's function in DNA interstrand cross-link (ICL) repair. Here, we describe a Fan1-deficient mouse and show that FAN1 is required for cellular and organismal resistance to ICLs. We show that the ubiquitin-binding zinc finger (UBZ) domain of FAN1, which is needed for interaction with FANCD2, is not required for the initial rapid recruitment of FAN1 to ICLs or for its role in DNA ICL resistance. Epistasis analyses reveal that FAN1 has cross-link repair activities that are independent of the Fanconi anemia proteins and that this activity is redundant with the 5′–3′ exonuclease SNM1A. Karyomegaly becomes prominent in kidneys and livers of Fan1-deficient mice with age, and mice develop liver dysfunction. Treatment of Fan1-deficient mice with ICL-inducing agents results in pronounced thymic and bone marrow hypocellularity and the disappearance of c-kit+ cells. Our results provide insight into the mechanism of FAN1 in ICL repair and demonstrate that the Fan1 mouse model effectively recapitulates the pathological features of human FAN1 deficiency.

Summary Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells’ ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process. EXD2 recruitment to stressed forks suppresses their degradation by restraining excessive fork regression. Accordingly, EXD2 deficiency leads to fork collapse, hypersensitivity to replication inhibitors, and genomic instability. Impeding fork regression by inactivation of SMARCAL1 or removal of RECQ1’s inhibition in EXD2−/− cells restores efficient fork restart and genome stability. Moreover, purified EXD2 efficiently processes substrates mimicking regressed forks. Thus, this work identifies a mechanism underpinned by EXD2’s nuclease activity, by which cells balance fork regression with fork restoration to maintain genome stability. Interestingly, from a clinical perspective, we discover that EXD2’s depletion is synthetic lethal with mutations in BRCA1/2, implying a non-redundant role in replication fork protection., Graphical Abstract, Highlights • EXD2 is required for cell survival in response to replicative stress • EXD2 protects replication forks from over resection by counteracting fork reversal • EXD2 is synthetic lethal with the deficiency in BRCA1/2 genes, Nieminuszczy et al. identify a key function of the EXD2 nuclease in DNA replication and alternative end-joining. EXD2 localizes to replication forks and promotes their stabilization by counteracting fork regression. Loss of EXD2 results in sensitivity to replicative inhibitors, degradation of regressed forks, and compromises survival of BRCA1/2-deficient tumors.

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Summary: Eukaryotic replisomes are driven by the mini chromosome maintenance (MCM [M]) helicase complex, an offset ring locked around the template for leading strand synthesis by CDC45 (C) and GINS (G) proteins. Although the CDC45 MCM GINS (CMG) structure implies that interstrand crosslinks (ICLs) are absolute blocks to replisomes, recent studies indicate that cells can restart DNA synthesis on the side of the ICL distal to the initial encounter. Here, we report that restart requires ATR and is promoted by FANCD2 and phosphorylated FANCM. Following introduction of genomic ICLs and dependent on ATR and FANCD2 but not on the Fanconi anemia core proteins or FAAP24, FANCM binds the replisome complex, with concomitant release of the GINS proteins. In situ analysis of replisomes proximal to ICLs confirms the ATR-dependent release of GINS proteins while CDC45 is retained on the remodeled replisome. The results demonstrate the plasticity of CMG composition in response to replication stress. : Replication of the mammalian genome is driven by the replisome complex, which unwinds DNA and must overcome many impediments. Zhang et al. find that the encounter of the CMG with a strong block triggers a change in replisome composition that is important for restart of replication past the obstruction. Keywords: replication traverse, interstrand crosslink, ICL, ATR, FANCM, FANCD2, CMG, GINS

Deletion of Hop2 in mice eliminates homologous chromosome synapsis and disrupts double-strand break (DSB) repair through homologous recombination. HOP2 in vitro shows two distinctive activities: when it is incorporated into a HOP2–MND1 complex it stimulates DMC1 and RAD51 recombination activities and the purified HOP2 alone is proficient in promoting strand invasion. We observed that a fraction of Mnd1−/− spermatocytes, which express HOP2 but apparently have inactive DMC1 and RAD51 due to lack of the HOP2–MND1 complex, exhibits a high level of chromosome synapsis and that most DSBs in these spermatocytes are repaired. This suggests that DSB repair catalyzed solely by HOP2 supports homologous chromosome pairing and synapsis. In addition, we show that in vitro HOP2 promotes the co-aggregation of ssDNA with duplex DNA, binds to ssDNA leading to unstacking of the bases, and promotes the formation of a three-strand synaptic intermediate. However, HOP2 shows distinctive mechanistic signatures as a recombinase. Namely, HOP2-mediated strand exchange does not require ATP and, in contrast to DMC1, joint molecules formed by HOP2 are more sensitive to mismatches and are efficiently dissociated by RAD54. We propose that HOP2 may act as a recombinase with specific functions in meiosis.

The replicative machinery encounters many impediments, some of which can be overcome by lesion bypass or replication restart pathways, leaving repair for a later time. However, interstrand crosslinks (ICLs), which preclude DNA unwinding, are considered absolute blocks to replication. Current models suggest that fork collisions, either from one or both sides of an ICL, initiate repair processes required for resumption of replication. To test these proposals, we developed a single molecule technique for visualizing encounters of replication forks with ICLs, as they occur in living cells. Surprisingly, the most frequent patterns were consistent with replication traverse of an ICL, without lesion repair. The traverse frequency was strongly reduced by inactivation of the translocase and DNA binding activities of the FANCM/MHF complex. The results indicate that translocase-based mechanisms enable DNA synthesis to continue past ICLs, and that these lesions are not always absolute blocks to replication.

An oncogenic role for CHD4, a NuRD component, is defined for initiating and supporting tumor suppressor gene (TSG) silencing in human colorectal cancer. CHD4 recruits repressive chromatin proteins to sites of DNA damage repair, including DNA methyltransferases where it imposes de novo DNA methylation. At TSGs, CHD4 retention helps maintain DNA hypermethylation-associated transcriptional silencing. CHD4 is recruited by the excision repair protein OGG1 for oxidative damage to interact with the damage-induced base 8-hydroxydeoxyguanosine (8-OHdG), while ZMYND8 recruits it to double-strand breaks. CHD4 knockdown activates silenced TSGs, revealing their role for blunting colorectal cancer cell proliferation, invasion, and metastases. High CHD4 and 8-OHdG levels plus low expression of TSGs strongly correlates with early disease recurrence and decreased overall survival.

Both in mice and humans, two major SPO11 isoforms are generated by alternative splicing: SPO11alpha (exon 2 skipped) and SPO11beta. Thus, the alternative splicing event must have emerged before the mouse and human lineages diverged and was maintained during 90 million years of evolution, arguing for an essential role for both isoforms. Here we demonstrate that developmental regulation of alternative splicing at the Spo11 locus governs the sequential expression of SPO11 isoforms in male meiotic prophase. Protein quantification in juvenile mice and in prophase mutants indicates that early spermatocytes synthesize primarily SPO11beta. Estimation of the number of SPO11 dimers (betabeta/alphabeta/alphaalpha) in mutants in which spermatocytes undergo a normal number of double strand breaks but arrest in midprophase due to inefficient repair argues for a role for SPO11beta-containing dimers in introducing the breaks in leptonema. Expression kinetics in males suggested a role for SPO11alpha in pachytene/diplotene spermatocytes. Nevertheless, we found that both alternative transcripts can be detected in oocytes throughout prophase I, arguing against a male-specific function for this isoform. Altogether, our data support a role for SPO11alpha in mid- to late prophase, presumably acting as a topoisomerase, that would be conserved in male and female meiocytes.

DNA instability disorders are associated with cancer, neurodegeneration, and premature aging. The genome integrity is most vulnerable during S phase during which the replication apparatus encounters many impediments. Among the most severe are interstrand crosslinks (ICLs) which are absolute blocks to helicases, and have always been regarded as absolute blocks to replication. Cells from patients with the genome instability disorder Fanconi Anemia (FA) are highly sensitive to crosslinking agents. We have developed a novel single molecule strategy, based on DNA fibers, to visualize collisions of replication forks with ICLs. Remarkably, we found that DNA synthesis can resume past an ICL, leaving behind still crosslinked parental strands, a process that requires only a few minutes. Traverse of ICLs requires the activity of the Fanconi Anemia translocase FANCM. Furthermore, while the FA “core” complex proteins, which monoubiquitinate the FANCD2 protein, were not required, FANCD2 was. These results indicate that...

Poly (ADP-ribose) polymerase (PARP) inhibitors represent one of the most exciting recent developments in cancer therapy. While substantial efficacy has been shown with clinically available PARP inhibitors (PARPis), to date, in treatment of hereditary deletions of BRCA1/2 in breast and ovarian cancers, the high promise of these drugs has not yet been realized in sporadic cancers. We present here strong preclinical data for a novel, mechanistically based, combinatorial approach to using DNA methyltransferase inhibitors (DNMTis), such as decitabine (DAC) and 5-Azacytidine (5-AZA), with PARP inhibitors (PARPis) as a treatment strategy for acute myelogenous leukemias (AML) and triple negative breast cancer (TNBC). We have previously demonstrated that low doses of 5-AZA and DAC alone show efficacy in AML and TNBC, and propose treatment with PARPis to enhance sensitivity of cancer cells to DNMTis. The mechanistic rationale for our approach is based upon: 1) data from our group and others showing DNMT1 and PARP1 associate in a complex, and this association increases with DNA damage; 2) the fact that 5-AZA and DAC trap DNMTs led us to hypothesize that these drugs might also increase PARP trapping at DNA damage sites; and 3) the cytotoxicity of the most potent PARPis (e.g. BMN 673) appears to correlate with the degree of trapping of PARP1 in chromatin. We first find that in cultured human AML and TNBC cells, the DNMTis (5 to 20 nM DAC or 100 to 200nM 5-AZA) and PARPis (1 to 10 nM BMN 673) alone trap PARP into chromatin, and this effect is enhanced when the drugs are combined. In addition, the PARPi-DNMTi combination treatment of TNBC cell line MDA-MB-231 resulted in significantly enhanced retention of PARP1 and DNMT1 at sites of double strand breaks (DSBs) induced by laser microirradiation. Concomitant with this, the combined doses resulted in significant increases in cytotoxic DSBs, observed 4-24 hours after DSB induction, when compared to single-drug treatments. Homologous recombination (HR) DSB repair activity also appears decreased, as measured by GFP reporter assays. In keeping with these findings, colony survival assays demonstrated that the combination treatment, compared to either drug alone, strongly inhibited colony formation of TNBC cell lines (N=4). Notably non-tumorigenic MCF10A cells showed no significant differences in colony numbers with single or combination drug treatments. Similar to TNBCs, AML cell lines (N=3) as well as primary AML cells (N=8) showed dramatic decreases in colonies in combination vs single agent drug treatments. In the most important translational implications of the preliminary studies, in in vivo therapy TNBC and AML models in immune-deficient mice, our low dose combinations of DNMTis and PARPis provide for potent anti-tumor responses. Mouse xenograft experiments using BRCA mutant TNBC cell line SUM149PT demonstrated that the combination treatment has a significant (p Citation Format: Nidal Muvarak, Khadiza Chowdhury, Carine Robert, Xia Limin, Eun Yong Choi, Yi Cai, Marina Bellani, Michael Seidman, Maria R. Baer, Rena Lapidus, Stephen B. Baylin, Feyruz V. Rassool. Combination of DNA methyltransferase and PARP inhibitors as a novel therapy strategy for multiple cancers: Key data in AML and triple negative breast cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA13.

Replication of the genome can be slowed or stalled by DNA interstrand crosslinks (ICLs), which are absolute blocks to helix unwinding. Current models propose that fork collisions, either from one (...

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPis) have shown efficacy in treatment of breast and ovarian cancers with hereditary deletions of BRCA1/2, but the high promise of these drugs has not yet been realized in sporadic cancers. We present here strong preclinical data for a novel, mechanistically based, combinatorial approach to using DNA methyltransferase inhibitors (DNMTi’s), such as decitabine (DAC) and 5-Azacytidine (5-AZA), with PARP inhibitors (PARPi’s) as a treatment strategy for acute myelogenous leukemias (AML) and estogen-, progesterone- and HER2-receptor negative, or triple negative breast cancer (TNBC). We have previously demonstrated that low doses of 5-AZA and DAC alone show efficacy in AML and TNBC, and propose treatment with PARPi's to enhance sensitivity of cancer cells to DNMTis. The mechanistic rationale for our approach is based upon: 1) data from our group and others showing DNMT1 and PARP1 associate in a complex, and this association increases with DNA damage; 2) the fact that 5-AZA and DAC trap DNMT's led us to hypothesize that these drugs might also increase PARP trapping at DNA damage sites; and 3) the cytotoxicity of the most potent PARPi's (e.g. Talazoparib) appears to correlate with the degree of trapping of PARP1 in chromatin. We find that in cultured human AML and TNBC cells, the DNMTi's (5 to 20 nM DAC or 100 to 200nM 5-AZA) and PARPi's (1 to 10 nM Talazoparib) alone trap PARP into chromatin, and this effect is enhanced when the drugs are combined. In addition, the PARPi-DNMTi combination treatment in TNBC MDA-MB-231 and AML MOLM-14 cell lines resulted in significantly increased DNA double strand breaks (DSBs) and enhanced retention of PARP1 and DNMT1 at laser microirradiation DNA damage sites. Compared with non-tumorigenic MCF10A cells, in TNBC cell lines (N = 4), the combined doses resulted in significant (p Citation Format: Feyruz V. Rassool, Nidal Muvarak, Khadiza Chowdury, Carine Robert, Limin Xia, Eun Yong Choi, Yi Cai, Marina Bellani, Ying Zou, Michael Seidman, Søren Bentzen, Maria Baer, Rena Lapidus, Stephen B. Baylin. Combination of DNA methyltransferase and PARP inhibitors as a novel therapy strategy for poor prognosis acute myeloid leukemia and triple-negative breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-205.

Transcriptional silencing of the sex chromosomes during male meiosis is regarded as a manifestation of a general mechanism active in both male and female germ cells, called meiotic silencing of unsynapsed chromatin (MSUC). MSUC is initiated by the recruitment of the tumor suppressor protein BRCA1 to the axes of unsynapsed chromosomes. We now show that Sycp3, a structural component of the chromosome axis, is required for localization of BRCA1 to unsynapsed pachytene chromosomes. Importantly, we find that oocytes carrying an excess of two to three pairs of asynapsed homologous chromosomes fail to recruit enough BRCA1 to the asynapsed axes to activate MSUC. Furthermore, loss of MSUC function only transiently rescues oocytes from elimination during early postnatal development. The fact that the BRCA1-dependent synapsis surveillance system cannot respond to higher degrees of asynapsis and is dispensable for removal of aberrant oocytes argues that MSUC has a limited input as a quality control mechanism in female germ cells.

SPO11 introduces double-strand breaks (DSBs) that trigger the phosphorylation of H2AX during meiotic prophase. In mice, SPO11 is strictly required for initiation of meiotic recombination and synapsis, yet SPO11 is still considered to be dispensable for sex-body formation in mouse spermatocytes. We provide conclusive evidence showing that functional SPO11, and consequently recombination and synapsis, are required for phosphorylation of H2AX in the X-Y chromatin and for sex-body formation in mouse spermatocytes. We investigated the role in meiosis of the three kinases [ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia- and Rad-3-related) and DNA-PKcs (DNA-dependent-protein-kinase catalytic subunit)] known to phosphorylate H2AX in mitotic cells. We found that DNA-PKcs can be ruled out as an essential kinase in this process, whereas ATM is strictly required for the chromatin-wide phosphorylation of H2AX occurring in leptotene spermatocytes in response to DSBs. Remarkably, we discovered that Spo11 heterozygosity can rescue the prophase-I-arrest characteristic of ATM-deficient spermatocytes. Characterization of the rescued Atm -/- Spo11 +/- mutant indicates that ATM is dispensable for sex-body formation and phosphorylation of H2AX in this subnuclear domain. The co-localization of ATR, phosphorylated H2AX and the sex chromatin observed in the Atm -/- Spo11 +/- mutant, along with ATR transcription kinetics during the first wave of spermatogenesis, confirm and expand recent findings indicating that ATR is the kinase involved in H2AX phosphorylation in the sex body.