ATF7IP2/MCAF2 directs H3K9 methylation and meiotic gene regulation in the male germline.

H3K9 trimethylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that in male meiosis, ATF7IP2 amasses on autosomal and X-pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X-pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the protein's essential roles in the maintenance of MSCI, suppression of retrotransposons, and global up-regulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.

ATF7IP2/MCAF2 directs H3K9 methylation and meiotic gene regulation in the male germline.

H3K9 tri-methylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that, in male meiosis, ATF7IP2 amasses on autosomal and X pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the protein's essential roles in the maintenance of MSCI, suppression of retrotransposons, and global upregulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.

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.

Active DNA damage response signaling initiates and maintains meiotic sex chromosome inactivation.

Meiotic sex chromosome inactivation (MSCI) is an essential process in the male germline. While genetic experiments have established that the DNA damage response (DDR) pathway directs MSCI, due to limitations to the experimental systems available, mechanisms underlying MSCI remain largely unknown. Here we establish a system to study MSCI ex vivo, based on a short-term culture method, and demonstrate that active DDR signaling is required both to initiate and maintain MSCI via a dynamic and reversible process. DDR-directed MSCI follows two layers of modifications: active DDR-dependent reversible processes and irreversible histone post-translational modifications. Further, the DDR initiates MSCI independent of the downstream repressive histone mark H3K9 trimethylation (H3K9me3), thereby demonstrating that active DDR signaling is the primary mechanism of silencing in MSCI. By unveiling the dynamic nature of MSCI, and its governance by active DDR signals, our study highlights the sex chromosomes as an active signaling hub in meiosis.

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.

Meiotic sex chromosome inactivation and the XY body: a phase separation hypothesis.

In mammalian male meiosis, the heterologous X and Y chromosomes remain unsynapsed and, as a result, are subject to meiotic sex chromosome inactivation (MSCI). MSCI is required for the successful completion of spermatogenesis. Following the initiation of MSCI, the X and Y chromosomes undergo various epigenetic modifications and are transformed into a nuclear body termed the XY body. Here, we review the mechanisms underlying the initiation of two essential, sequential processes in meiotic prophase I: MSCI and XY-body formation. The initiation of MSCI is directed by the action of DNA damage response (DDR) pathways; downstream of the DDR, unique epigenetic states are established, leading to the formation of the XY body. Accumulating evidence suggests that MSCI and subsequent XY-body formation may be driven by phase separation, a physical process that governs the formation of membraneless organelles and other biomolecular condensates. Thus, here we gather literature-based evidence to explore a phase separation hypothesis for the initiation of MSCI and the formation of the XY body.

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.

RNF8 is not required for histone-to-protamine exchange in spermiogenesis†.

While an E3 ubiquitin ligase, RNF8, was initially reported to be required for histone-to-protamine exchange in spermiogenesis, we subsequently demonstrated that RNF8 is not involved in this process. Nevertheless, reflecting a lingering misunderstanding in the field, a growing number of studies have continued to postulate a requirement for RNF8 in the histone-to-protamine exchange. For example, a recent study claimed that a mouse PIWI protein, MIWI, controls RNF8-mediated histone-to-protamine exchange. Here, confirming our earlier conclusions, we show that RNF8 is required neither for the establishment of histone H4K16 acetylation, which is an initial step in histone removal during spermiogenesis, nor for the incorporation of two protamine proteins, PRM1 and PRM2. Thus, whereas RNF8 mediates ubiquitination of H2A on the sex chromosomes in meiosis, during the prior stage of spermatogenesis, our genetic evidence underscores that RNF8 is not involved in histone-to-protamine exchange.

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.

The Initiation of Meiotic Sex Chromosome Inactivation Sequesters DNA Damage Signaling from Autosomes in Mouse Spermatogenesis.

Meiotic sex chromosome inactivation (MSCI) is an essential event in the mammalian male germline. MSCI is directed by a DNA damage response (DDR) pathway centered on the phosphorylation of histone variant H2AX at serine 139 (termed γH2AX). The failure to initiate MSCI is linked to complete meiotic arrest and elimination of germ cells; however, the mechanisms underlying this arrest and elimination remain unknown. To address this question, we established a new separation-of-function mouse model for H2ax that shows specific and complete defects in MSCI. The genetic change is a point mutation in which another H2AX amino acid residue important in the DDR, tyrosine 142 (Y142), is converted to alanine (H2ax-Y142A). In H2ax-Y142A meiosis, the establishment of DDR signals on the chromosome-wide domain of the sex chromosomes is impaired. The initiation of MSCI is required for stage progression, which enables crossover formation, suggesting that the establishment of MSCI permits the timely progression of male meiosis. Our results suggest that normal meiotic progression requires the removal of ATR-mediated DDR signaling from autosomes. We propose a novel biological function for MSCI: the initiation of MSCI sequesters DDR factors from autosomes to the sex chromosomes at the onset of the pachytene stage, and the subsequent formation of an isolated XY nuclear compartment-the XY body-sequesters DDR factors to permit meiotic progression from the mid-pachytene stage onward. VIDEO ABSTRACT.

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.

RNF8 and SCML2 cooperate to regulate ubiquitination and H3K27 acetylation for escape gene activation on the sex chromosomes.

The sex chromosomes are enriched with germline genes that are activated during the late stages of spermatogenesis. Due to meiotic sex chromosome inactivation (MSCI), these sex chromosome-linked genes must escape silencing for activation in spermatids, thereby ensuring their functions for male reproduction. RNF8, a DNA damage response protein, and SCML2, a germline-specific Polycomb protein, are two major, known regulators of this process. Here, we show that RNF8 and SCML2 cooperate to regulate ubiquitination during meiosis, an early step to establish active histone modifications for subsequent gene activation. Double mutants of Rnf8 and Scml2 revealed that RNF8-dependent monoubiquitination of histone H2A at Lysine 119 (H2AK119ub) is deubiquitinated by SCML2, demonstrating interplay between RNF8 and SCML2 in ubiquitin regulation. Additionally, we identify distinct functions of RNF8 and SCML2 in the regulation of ubiquitination: SCML2 deubiquitinates RNF8-independent H2AK119ub but does not deubiquitinate RNF8-dependent polyubiquitination. RNF8-dependent polyubiquitination is required for the establishment of H3K27 acetylation, a marker of active enhancers, while persistent H2AK119ub inhibits establishment of H3K27 acetylation. Following the deposition of H3K27 acetylation, H3K4 dimethylation is established as an active mark on poised promoters. Together, we propose a model whereby regulation of ubiquitin leads to the organization of poised enhancers and promoters during meiosis, which induce subsequent gene activation from the otherwise silent sex chromosomes in postmeiotic spermatids.