A novel cancer risk prediction score for the natural course of FA patients with biallelic BRCA2/FANCD1 mutations.

Biallelic germline mutations in BRCA2 occur in the Fanconi anemia (FA)-D1 subtype of the rare pediatric disorder, FA, characterized clinically by severe congenital abnormalities and a very high propensity to develop malignancies early in life. Clinical and genetic data from 96 FA-D1 patients with biallelic BRCA2 mutations were collected and used to develop a new cancer risk prediction score system based on the specific mutations in BRCA2. This score takes into account the location of frameshift/stop and missense mutations relative to exon 11 of BRCA2, which encodes the major sites for interaction with the RAD51 recombinase, and uses the MaxEnt and HBond splicing scores to analyze potential splice site perturbations. Among 75 FA-D1 patients with ascertained BRCA2 mutations, 66 patients developed 102 malignancies, ranging from one to three independent tumors per individual. The median age at the clinical presentation of peripheral embryonal tumors was 1.0, at the onset of hematologic malignancies 1.8 and at the manifestation of CNS tumors 2.7 years, respectively. Patients who received treatment lived longer than those without. Using our novel scoring system, we could distinguish three distinct cancer risk groups among FA-D1 patients: in the first, patients developed their initial malignancy at a median age of 1.3 years (n = 36, 95% CI = 0.9-1.8), in the second group at 2.3 years (n = 17, 95% CI = 1.4-4.4) and in the third group at 23.0 years (n = 22, 95% CI = 4.3-n/a). Therefore, this scoring system allows, for the first time, to predict the cancer manifestation of FA-D1 patients simply based on the type and position of the mutations in BRCA2.

Identification of new RAD51D-regulating microRNAs that also emerge as potent inhibitors of the Fanconi anemia/homologous recombination pathways.

The Fanconi anemia (FA) and homologous recombination (HR) pathways, which partially overlap and include RAD51 and its paralogs, are key for the repair of different types of DNA damage, such as DNA interstrand crosslinks. First, to broadly assess the impact of microRNA-mediated regulation, we examined microRNA expression profiles in five isogenic fibroblast cell pairs, either deficient in DNA repair due to germline mutations in FANCA, FANCB, FANCC, FANCI or BRIP1/FANCJ or proficient due to correction with retroviral vectors. In each pair, we observed lower abundance of specific microRNAs in the FA-deficient cells. From the list of microRNAs, we experimentally confirmed the effects of miR-141-3p and miR-369-3p targeting RAD51B and miR-15a-5p, miR-494-3p as well as miR-544a targeting RAD51D. However, by western blotting, only RAD51D protein was reduced by a mixture of its regulating microRNAs. Gene ontology analyses and identification of additional FA/HR factors as targets of miR-15a-5p, miR-494-3p and miR-544a strongly suggested the widespread influence of these microRNAs on HR. Interestingly, only miR-494-3p directly reduced RAD51 foci formation, while a mixture of miR-15a-5p, miR-494-3p and miR-544a strongly reduced HR activity in green fluorescent protein (GFP) repair assays. In summary, by successfully employing this novel loss- and gain-of-function strategy, we have identified new microRNAs strongly inhibiting HR in mammalian cells. Understanding and modulating such miRNA regulation of DNA repair genes/pathways might help to overcome the reduced repair capacity of FA patients with biallelic hypomorphic mutations or help to engineer synthetic lethality strategies for patients with mutations in cancer-associated FA/HR genes.

Head and Neck Cancer Susceptibility and Metabolism in Fanconi Anemia.

Fanconi anemia (FA) is a rare inherited, generally autosomal recessive syndrome, but it displays X-linked or dominant negative inheritance for certain genes. FA is characterized by a deficiency in DNA damage repair that results in bone marrow failure, and in an increased risk for various epithelial tumors, most commonly squamous cell carcinomas of the head and neck (HNSCC) and of the esophagus, anogenital tract and skin. Individuals with FA exhibit increased human papilloma virus (HPV) prevalence. Furthermore, a subset of anogenital squamous cell carcinomas (SCCs) in FA harbor HPV sequences and FA-deficient laboratory models reveal molecular crosstalk between HPV and FA proteins. However, a definitive role for HPV in HNSCC development in the FA patient population is unproven. Cellular metabolism plays an integral role in tissue homeostasis, and metabolic deregulation is a known hallmark of cancer progression that supports uncontrolled proliferation, tumor development and metastatic dissemination. The metabolic consequences of FA deficiency in keratinocytes and associated impact on the development of SCC in the FA population is poorly understood. Herein, we review the current literature on the metabolic consequences of FA deficiency and potential effects of resulting metabolic reprogramming on FA cancer phenotypes.

Understanding BRCA2 Function as a Tumor Suppressor Based on Domain-Specific Activities in DNA Damage Responses.

BRCA2 is an essential genome stability gene that has various functions in cells, including roles in homologous recombination, G2 checkpoint control, protection of stalled replication forks, and promotion of cellular resistance to numerous types of DNA damage. Heterozygous mutation of BRCA2 is associated with an increased risk of developing cancers of the breast, ovaries, pancreas, and other sites, thus BRCA2 acts as a classic tumor suppressor gene. However, understanding BRCA2 function as a tumor suppressor is severely limited by the fact that ~70% of the encoded protein has not been tested or assigned a function in the cellular DNA damage response. Remarkably, even the specific role(s) of many known domains in BRCA2 are not well characterized, predominantly because stable expression of the very large BRCA2 protein in cells, for experimental purposes, is challenging. Here, we review what is known about these domains and the assay systems that are available to study the cellular roles of BRCA2 domains in DNA damage responses. We also list criteria for better testing systems because, ultimately, functional assays for assessing the impact of germline and acquired mutations identified in genetic screens are important for guiding cancer prevention measures and for tailored cancer treatments.

The p.Ser64Leu and p.Pro104Leu missense variants of PALB2 identified in familial pancreatic cancer patients compromise the DNA damage response.

PALB2 has been identified as a breast and pancreatic cancer susceptibility gene. Utilizing a targeted sequencing approach, we discovered two novel germline missense PALB2 variants c.191C>T and c.311C>T, encoding p.Ser64Leu and p.Pro104Leu, respectively, in individuals in a pancreatic cancer registry. No missense PALB2 variants from familial pancreatic cancer patients, and few PALB2 variants overall, have been functionally characterized. Given the known role of PALB2, we tested the impact of p.Ser64Leu and p.Pro104Leu variants on DNA damage responses. Neither p.Ser64Leu nor p.Pro104Leu have clear effects on interactions with BRCA1 and KEAP1, which are mediated by adjacent motifs in PALB2. However, both variants are associated with defective recruitment of PALB2, and the RAD51 recombinase downstream, to DNA damage foci. Furthermore, p.Ser64Leu and p.Pro104Leu both largely compromise DNA double-strand break-initiated homologous recombination, and confer increased cellular sensitivity to ionizing radiation (IR) and the poly (ADP-ribose) polymerase (PARP) inhibitor Olaparib. Taken together, our results represent the first demonstration of functionally deleterious PALB2 missense variants associated with familial pancreatic cancer and of deleterious variants in the N-terminus outside of the coiled-coil domain. Furthermore, our results suggest the possibility of personalized treatments, using IR or PARP inhibitor, of pancreatic and other cancers that carry a deleterious PALB2 variant.

NF1 patient missense variants predict a role for ATM in modifying neurofibroma initiation.

In Neurofibromatosis type 1, NF1 gene mutations in Schwann cells (SC) drive benign plexiform neurofibroma (PNF), and no additional SC changes explain patient-to-patient variability in tumor number. Evidence from twin studies suggests that variable expressivity might be caused by unidentified modifier genes. Whole exome sequencing of SC and fibroblast DNA from the same resected PNFs confirmed biallelic SC NF1 mutations; non-NF1 somatic SC variants were variable and present at low read number. We identified frequent germline variants as possible neurofibroma modifier genes. Genes harboring variants were validated in two additional cohorts of NF1 patients and by variant burden test. Genes including CUBN, CELSR2, COL14A1, ATR and ATM also showed decreased gene expression in some neurofibromas. ATM-relevant DNA repair defects were also present in a subset of neurofibromas with ATM variants, and in some neurofibroma SC. Heterozygous ATM G2023R or homozygous S707P variants reduced ATM protein expression in heterologous cells. In mice, genetic Atm heterozygosity promoted Schwann cell precursor self-renewal and increased tumor formation in vivo, suggesting that ATM variants contribute to neurofibroma initiation. We identify germline variants, rare in the general population, overrepresented in NF1 patients with neurofibromas. ATM and other identified genes are candidate modifiers of PNF pathogenesis.

XRCC2 (X-ray repair cross complementing 2).

XRCC2 is one of five somatic RAD51 paralogs, all of which have Walker A and B ATPase motifs. Each of the paralogs, including XRCC2, has a function in DNA double-strand break repair by homologous recombination (HR). However, their individual roles are not as well understood as that of RAD51 itself. The XRCC2 protein forms a complex (BCDX2) with three other RAD51 paralogs, RAD51B, RAD51C and RAD51D. It is believed that the BCDX2 complex mediates HR downstream of BRCA2 but upstream of RAD51, as XRCC2 is involved in the assembly of RAD51 into DNA damage foci. XRCC2 can bind DNA and, along with RAD51D, can promote homologous pairing in vitro. Consistent with its role in HR, XRCC2-deficient cells have increased levels of spontaneous chromosome instability, and exhibit hypersensitivity to DNA interstrand crosslinking agents such as mitomycin C and cisplatin as well as ionizing radiation, alkylating agents and aldehydes. XRCC2 also functions in promoting DNA replication and chromosome segregation. Biallelic mutation of XRCC2 (FANCU) causes the FA-U subtype of FA, while heterozygosity for deleterious mutations in XRCC2 may be associated with an increased breast cancer risk. XRCC2 appears to function 'downstream' in the FA pathway, since it is not required for FANCD2 monoubiquitination, which is the central step in the FA pathway. Clinically, the only known FA-U patient in the world exhibits severe congenital abnormalities, but had not developed, by seven years of age, the bone marrow failure and cancer that are often seen in patients from other FA complementation groups.

CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites.

Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPR-Cas9 by homology-dependent repair (HDR) is limited by the competing error-prone non-homologous end-joining (NHEJ) DNA repair pathway. Here, we define a safer and efficient system that promotes HDR-based precise genome editing, while reducing NHEJ locally, only at CRISPR-Cas9-induced DSBs. We fused a dominant-negative mutant of 53BP1, DN1S, to Cas9 nucleases, and the resulting Cas9-DN1S fusion proteins significantly block NHEJ events specifically at Cas9 cut sites and improve HDR frequency; HDR frequency reached 86% in K562 cells. Cas9-DN1S protein maintains this effect in different human cell types, including leukocyte adhesion deficiency (LAD) patient-derived immortalized B lymphocytes, where nearly 70% of alleles were repaired by HDR and 7% by NHEJ. Our CRISPR-Cas9-DN1S system is clinically relevant to improve the efficiencies of precise gene correction/insertion, significantly reducing error-prone NHEJ events at the nuclease cleavage site, while avoiding the unwanted effects of global NHEJ inhibition.

SETD2 mutations confer chemoresistance in acute myeloid leukemia partly through altered cell cycle checkpoints.

SETD2, an epigenetic tumor suppressor, is frequently mutated in MLL-rearranged (MLLr) leukemia and relapsed acute leukemia (AL). To clarify the impact of SETD2 mutations on chemotherapy sensitivity in MLLr leukemia, two loss-of-function (LOF) Setd2-mutant alleles (Setd2F2478L/WT or Setd2Ex6-KO/WT) were generated and introduced, respectively, to the Mll-Af9 knock-in leukemia mouse model. Both alleles cooperated with Mll-Af9 to accelerate leukemia development that resulted in resistance to standard Cytarabine-based chemotherapy. Mechanistically, Setd2-mutant leukemic cells showed downregulated signaling related to cell cycle progression, S, and G2/M checkpoint regulation. Thus, after Cytarabine treatment, Setd2-mutant leukemic cells exit from the S phase and progress to the G2/M phase. Importantly, S and G2/M cell cycle checkpoint inhibition could resensitize the Mll-Af9/Setd2 double-mutant cells to standard chemotherapy by causing DNA replication collapse, mitotic catastrophe, and increased cell death. These findings demonstrate that LOF SETD2 mutations confer chemoresistance on AL to DNA-damaging treatment by S and G2/M checkpoint defects. The combination of S and G2/M checkpoint inhibition with chemotherapy can be explored as a promising therapeutic strategy by exploiting their unique vulnerability and resensitizing chemoresistant AL with SETD2 or SETD2-like epigenetic mutations.

Genetic Testing to Guide Risk-Stratified Screens for Breast Cancer.

Breast cancer screening modalities and guidelines continue to evolve and are increasingly based on risk factors, including genetic risk and a personal or family history of cancer. Here, we review genetic testing of high-penetrance hereditary breast and ovarian cancer genes, including BRCA1 and BRCA2, for the purpose of identifying high-risk individuals who would benefit from earlier screening and more sensitive methods such as magnetic resonance imaging. We also consider risk-based screening in the general population, including whether every woman should be genetically tested for high-risk genes and the potential use of polygenic risk scores. In addition to enabling early detection, the results of genetic screens of breast cancer susceptibility genes can be utilized to guide decision-making about when to elect prophylactic surgeries that reduce cancer risk and the choice of therapeutic options. Variants of uncertain significance, especially missense variants, are being identified during panel testing for hereditary breast and ovarian cancer. A finding of a variant of uncertain significance does not provide a basis for increased cancer surveillance or prophylactic procedures. Given that variant classification is often challenging, we also consider the role of multifactorial statistical analyses by large consortia and functional tests for this purpose.

Deficiency of the Fanconi anemia E2 ubiqitin conjugase UBE2T only partially abrogates Alu-mediated recombination in a new model of homology dependent recombination.

The primary function of the UBE2T ubiquitin conjugase is in the monoubiquitination of the FANCI-FANCD2 heterodimer, a central step in the Fanconi anemia (FA) pathway. Genetic inactivation of UBE2T is responsible for the phenotypes of FANCT patients; however, a FANCT patient carrying a maternal duplication and a paternal deletion in the UBE2T loci displayed normal peripheral blood counts and UBE2T protein levels in B-lymphoblast cell lines. To test whether reversion by recombination between UBE2T AluYa5 elements could have occurred in the patient's hematopoietic stem cells despite the defects in homologous recombination (HR) in FA cells, we constructed HeLa cell lines containing the UBE2T AluYa5 elements and neighboring intervening sequences flanked by fluorescent reporter genes. Introduction of a DNA double strand break in the model UBE2T locus in vivo promoted single strand annealing (SSA) between proximal Alu elements and deletion of the intervening color marker gene, recapitulating the reversion of the UBE2T duplication in the FA patient. To test whether UBE2T null cells retain HR activity, the UBE2T genes were knocked out in HeLa cells and U2OS cells. CRISPR/Cas9-mediated genetic knockout of UBE2T only partially reduced HR, demonstrating that UBE2T-independent pathways can compensate for the recombination defect in UBE2T/FANCT null cells.

The BRUCE-ATR Signaling Axis Is Required for Accurate DNA Replication and Suppression of Liver Cancer Development.

Replication fork stability during DNA replication is vital for maintenance of genomic stability and suppression of cancer development in mammals. ATR (ataxia-telangiectasia mutated [ATM] and RAD3-related) is a master regulatory kinase that activates the replication stress response to overcome replication barriers. Although many downstream effectors of ATR have been established, the upstream regulators of ATR and the effect of such regulation on liver cancer remain unclear. The ubiquitin conjugase BRUCE (BIR Repeat containing Ubiquitin-Conjugating Enzyme) is a guardian of chromosome integrity and activator of ATM signaling, which promotes DNA double-strand break repair through homologous recombination. Here we demonstrate the functions for BRUCE in ATR activation in vitro and liver tumor suppression in vivo. BRUCE is recruited to induced DNA damage sites. Depletion of BRUCE inhibited multiple ATR-dependent signaling events during replication stress, including activation of ATR itself, phosphorylation of its downstream targets CHK1 and RPA, and the mono-ubiquitination of FANCD2. Consequently, BRUCE deficiency resulted in stalled DNA replication forks and increased firing of new replication origins. The in vivo impact of BRUCE loss on liver tumorigenesis was determined using the hepatocellular carcinoma model induced by genotoxin diethylnitrosamine. Liver-specific knockout of murine Bruce impaired ATR activation and exacerbated inflammation, fibrosis and hepatocellular carcinoma, which exhibited a trabecular architecture, closely resembling human hepatocellular carcinoma (HCC). In humans, the clinical relevance of BRUCE down-regulation in liver disease was found in hepatitis, cirrhosis, and HCC specimens, and deleterious somatic mutations of the Bruce gene was found in human hepatocellular carcinoma in the Cancer Genome Atlas database. Conclusion: These findings establish a BRUCE-ATR signaling axis in accurate DNA replication and suppression of liver cancer in mice and humans and provides a clinically relevant HCC mouse model.

Setd2 regulates quiescence and differentiation of adult hematopoietic stem cells by restricting RNA polymerase II elongation.

SET domain containing 2 (Setd2), encoding a histone methyltransferase, is associated with many hematopoietic diseases when mutated. By generating a novel exon 6 conditional knockout mouse model, we describe an essential role of Setd2 in maintaining the adult hematopoietic stem cells. Loss of Setd2 results in leukopenia, anemia, and increased platelets accompanied by hypocellularity, erythroid dysplasia, and mild fibrosis in bone marrow. Setd2 knockout mice show significantly decreased hematopoietic stem and progenitor cells except for erythroid progenitors. Setd2 knockout hematopoietic stem cells fail to establish long-term bone marrow reconstitution after transplantation because of the loss of quiescence, increased apoptosis, and reduced multiple-lineage terminal differentiation potential. Bioinformatic analysis revealed that the hematopoietic stem cells exit from quiescence and commit to differentiation, which lead to hematopoietic stem cell exhaustion. Mechanistically, we attribute an important Setd2 function in murine adult hematopoietic stem cells to the inhibition of the Nsd1/2/3 transcriptional complex, which recruits super elongation complex and controls RNA polymerase II elongation on a subset of target genes, including Myc Our results reveal a critical role of Setd2 in regulating quiescence and differentiation of hematopoietic stem cells through restricting the NSDs/SEC mediated RNA polymerase II elongation.

PALB2 (partner and localizer of BRCA2).

PALB2 (Partner and Localizer of BRCA2) was first identified as a BRCA2-interacting protein. Subsequently, PALB2 has been recognized as a cog in the cellular machinery for DNA repair by homologous recombination (HR). PALB2 also mediates S and G2 DNA damage checkpoints, and has an apparent function in protecting transcriptionally active genes from genotoxic stress. PALB2 also interacts with, is localized by, and functions downstream of BRCA1. Further, PALB2 interacts with other essential effectors of HR, including RAD51 and RAD51C, as well as BRCA2. Consistent with its function in HR and its interaction with key HR proteins, PALB2-deficient cells are hypersensitive to ionizing radiation and DNA interstrand crosslinking agents such as mitomycin C and cisplatin. Mechanistically, PALB2 is required for HR by mediating the recruitment of BRCA2 and the RAD51 recombinase to sites of DNA damage. Similar to bi-allelic loss-of-function mutations of BRCA1, BRCA2, RAD51 and RAD51C, bi-allelic mutations in PALB2 cause Fanconi anemia (FA), a rare childhood disorder which is associated with progressive bone marrow failure, congenital anomalies, and a predisposition to leukemia and solid tumors. Due to their close functional relationship, bi-allelic mutations of PALB2 and BRCA2 cause particularly severe forms of FA, called FANCN and FANCD1, both characterized by severe congenital abnormalities and very early onset of various cancers. This includes acute leukemias, Wilms tumor, medulloblastoma and neuroblastomas. Also, heterozygous germ-line mutations of PALB2, like mutations in several other essential HR genes listed above, yield an increased susceptibility to breast and pancreatic cancer.

DNA repair-related functional assays for the classification of BRCA1 and BRCA2 variants: a critical review and needs assessment.

Mutation of BRCA1 and BRCA2 is the most common cause of inherited breast and ovarian cancer. Genetic screens to detect carriers of variants can aid in cancer prevention by identifying individuals with a greater cancer risk and can potentially be used to predict the responsiveness of tumours to therapy. Frequently, classification cannot be performed based on traditional approaches such as segregation analyses, including for many missense variants, which are therefore referred to as variants of uncertain significance (VUS). Functional assays provide an important alternative for classification of BRCA1 and BRCA2 VUS. As reviewed here, both of these tumour suppressors promote the maintenance of genome stability via homologous recombination. Thus, related assays may be particularly relevant to cancer risk. Progress in implementing functional assays to assess missense variants of BRCA1 and BRCA2 is considered here, along with current limitations and the path to more impactful assay systems. While functional assays have been developed to independently evaluate BRCA1 and BRCA2 VUS, high-throughput assays with sufficient sensitivity to characterise the large number of identified variants are lacking. Additionally, because of relatively low conservation of certain domains of BRCA1, and of BRCA2, between humans and rodents, heterologous expression in rodent cells may have limited reliability or capacity to assess variants present throughout either protein. Moving forward, it will be important to perform assays in human cell lines with relevance to particular tumour types, and to strengthen risk predictions based on multifactorial statistical analyses that also include available data on cosegregation and tumour pathology.

Manipulating DNA damage-response signaling for the treatment of immune-mediated diseases.

Antigen-activated lymphocytes undergo extraordinarily rapid cell division in the course of immune responses. We hypothesized that this unique aspect of lymphocyte biology leads to unusual genomic stress in recently antigen-activated lymphocytes and that targeted manipulation of DNA damage-response (DDR) signaling pathways would allow for selective therapeutic targeting of pathological T cells in disease contexts. Consistent with these hypotheses, we found that activated mouse and human T cells display a pronounced DDR in vitro and in vivo. Upon screening a variety of small-molecule compounds, we found that potentiation of p53 (via inhibition of MDM2) or impairment of cell cycle checkpoints (via inhibition of CHK1/2 or WEE1) led to the selective elimination of activated, pathological T cells in vivo. The combination of these strategies [which we termed "p53 potentiation with checkpoint abrogation" (PPCA)] displayed therapeutic benefits in preclinical disease models of hemophagocytic lymphohistiocytosis and multiple sclerosis, which are driven by foreign antigens or self-antigens, respectively. PPCA therapy targeted pathological T cells but did not compromise naive, regulatory, or quiescent memory T-cell pools, and had a modest nonimmune toxicity profile. Thus, PPCA is a therapeutic modality for selective, antigen-specific immune modulation with significant translational potential for diverse immune-mediated diseases.

Fancd2 in vivo interaction network reveals a non-canonical role in mitochondrial function.

Fancd2 is a component of the Fanconi anemia (FA) DNA repair pathway, which is frequently found defective in human cancers. The full repertoire of Fancd2 functions in normal development and tumorigenesis remains to be determined. Here we developed a Flag- and hemagglutinin-tagged Fancd2 knock-in mouse strain that allowed a high throughput mass spectrometry approach to search for Fancd2-binding proteins in different mouse organs. In addition to DNA repair partners, we observed that many Fancd2-interacting proteins are mitochondrion-specific. Fancd2 localizes in the mitochondrion and associates with the nucleoid complex components Atad3 and Tufm. The Atad3-Tufm complex is disrupted in Fancd2-/- mice and those deficient for the FA core component Fanca. Fancd2 mitochondrial localization requires Atad3. Collectively, these findings provide evidence for Fancd2 as a crucial regulator of mitochondrion biosynthesis, and of a molecular link between FA and mitochondrial homeostasis.

DEK is required for homologous recombination repair of DNA breaks.

DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition.

Coordination of the recruitment of the FANCD2 and PALB2 Fanconi anemia proteins by an ubiquitin signaling network.

Fanconi anemia (FA) is a chromosome instability syndrome and the 20 identified FA proteins are organized into two main arms which are thought to function at distinct steps in the repair of DNA interstrand crosslinks (ICLs). These two arms include the upstream FA pathway, which culminates in the monoubiquitination of FANCD2 and FANCI, and downstream breast cancer (BRCA)-associated proteins that interact in protein complexes. How, and whether, these two groups of FA proteins are integrated is unclear. Here, we show that FANCD2 and PALB2, as indicators of the upstream and downstream arms, respectively, colocalize independently of each other in response to DNA damage induced by mitomycin C (MMC). We also show that ubiquitin chains are induced by MMC and colocalize with both FANCD2 and PALB2. Our finding that the RNF8 E3 ligase has a role in recruiting FANCD2 and PALB2 also provides support for the hypothesis that the two branches of the FA-BRCA pathway are coordinated by ubiquitin signaling. Interestingly, we find that the RNF8 partner, MDC1, as well as the ubiquitin-binding protein, RAP80, specifically recruit PALB2, while a different ubiquitin-binding protein, FAAP20, functions only in the recruitment of FANCD2. Thus, FANCD2 and PALB2 are not recruited in a single linear pathway, rather we define how their localization is coordinated and integrated by a network of ubiquitin-related proteins. We propose that such regulation may enable upstream and downstream FA proteins to act at distinct steps in the repair of ICLs.

Elucidation of the Fanconi Anemia Protein Network in Meiosis and Its Function in the Regulation of Histone Modifications.

Precise epigenetic regulation of the sex chromosomes is vital for the male germline. Here, we analyze meiosis in eight mouse models deficient for various DNA damage response (DDR) factors, including Fanconi anemia (FA) proteins. We reveal a network of FA and DDR proteins in which FA core factors FANCA, FANCB, and FANCC are essential for FANCD2 foci formation, whereas BRCA1 (FANCS), MDC1, and RNF8 are required for BRCA2 (FANCD1) and SLX4 (FANCP) accumulation on the sex chromosomes during meiosis. In addition, FA proteins modulate distinct histone marks on the sex chromosomes: FA core proteins and FANCD2 regulate H3K9 methylation, while FANCD2 and RNF8 function together to regulate H3K4 methylation independently of FA core proteins. Our data suggest that RNF8 integrates the FA-BRCA pathway. Taken together, our study reveals distinct functions for FA proteins and illuminates the male sex chromosomes as a model to dissect the function of the FA-BRCA pathway.