FANCI plays an essential role in spermatogenesis and regulates meiotic histone methylation

FANCI is an essential component of Fanconi anemia pathway, which is responsible for the repair of DNA interstrand cross-links (ICLs). As an evolutionarily related partner of FANCD2, FANCI functions together with FANCD2 downstream of FA core complex. Currently, growing evidences showed that the essential role of FA pathway in male fertility. However, the underlying mechanisms for FANCI in regulating spermatogenesis remain unclear. In the present study, we found that the male Fanci^−/− mice were sterile and exhibited abnormal spermatogenesis, including massive germ cell apoptosis in seminiferous tubules and dramatically decreased number of sperms in epididymis. Besides, FANCI deletion impaired maintenance of undifferentiated spermatogonia. Further investigation indicated that FANCI was essential for FANCD2 foci formation and regulated H3K4 and H3K9 methylation on meiotic sex chromosomes. These findings elucidate the role and mechanism of FANCI during spermatogenesis in mice and provide new insights into the etiology and molecular basis of nonobstructive azoospermia.Subject terms: Cell death, Spermatogenesis     

Differential Localization and Independent Acquisition of the H3K9me2 and H3K9me3 Chromatin Modifications in the Caenorhabditis elegans Adult Germ Line

Histone methylation is a prominent feature of eukaryotic chromatin that modulates multiple aspects of chromosome function. Methyl modification can occur on several different amino acid residues and in distinct mono-, di-, and tri-methyl states. However, the interplay among these distinct modification states is not well understood. Here we investigate the relationships between dimethyl and trimethyl modifications on lysine 9 of histone H3 (H3K9me2 and H3K9me3) in the adult Caenorhabditis elegans germ line. Simultaneous immunofluorescence reveals very different temporal/spatial localization patterns for H3K9me2 and H3K9me3. While H3K9me2 is enriched on unpaired sex chromosomes and undergoes dynamic changes as germ cells progress through meiotic prophase, we demonstrate here that H3K9me3 is not enriched on unpaired sex chromosomes and localizes to all chromosomes in all germ cells in adult hermaphrodites and until the primary spermatocyte stage in males. Moreover, high-copy transgene arrays carrying somatic-cell specific promoters are highly enriched for H3K9me3 (but not H3K9me2) and correlate with DAPI-faint chromatin domains. We further demonstrate that the H3K9me2 and H3K9me3 marks are acquired independently. MET-2, a member of the SETDB histone methyltransferase (HMTase) family, is required for all detectable germline H3K9me2 but is dispensable for H3K9me3 in adult germ cells. Conversely, we show that the HMTase MES-2, an E(z) homolog responsible for H3K27 methylation in adult germ cells, is required for much of the germline H3K9me3 but is dispensable for H3K9me2. Phenotypic analysis of met-2 mutants indicates that MET-2 is nonessential for fertility but inhibits ectopic germ cell proliferation and contributes to the fidelity of chromosome inheritance. Our demonstration of the differential localization and independent acquisition of H3K9me2 and H3K9me3 implies that the trimethyl modification of H3K9 is not built upon the dimethyl modification in this context. Further, these and other data support a model in which these two modifications function independently in adult C. elegans germ cells.     

H3K36me2 methyltransferase NSD2 orchestrates epigenetic reprogramming during spermatogenesis

Spermatogenesis is precisely controlled by sophisticated gene expression programs and is driven by epigenetic reprogramming, including histone modification alterations and histone-to-protamine transition. Nuclear receptor binding SET domain protein 2 (Nsd2) is the predominant histone methyltransferase catalyzing H3K36me2 and its role in male germ cell development remains elusive. Here, we report that NSD2 protein is abundant in spermatogenic cells. Conditional loss of Nsd2 in postnatal germ cells impaired fertility owing to apoptosis of spermatocytes and aberrant spermiogenesis. Nsd2 deficiency results in dysregulation of thousands of genes and remarkable reduction of both H3K36me2 and H3K36me3 in spermatogenic cells, with H3K36me2 occupancy correlating positively with expression of germline genes. Nsd2 deficiency leads to H4K16ac elevation in spermatogenic cells, probably through interaction between NSD2 and PSMA8, which regulates acetylated histone degradation. We further reveal that Nsd2 deficiency impairs EP300-induced H4K5/8ac, recognized by BRDT to mediate the eviction of histones. Accordingly, histones are largely retained in Nsd2-deficient spermatozoa. In addition, Nsd2 deficiency enhances expression of protamine genes, leading to increased protamine proteins in Nsd2-deficient spermatozoa. Our findings thus reveal a previously unappreciated role of the Nsd2-dependent chromatin remodeling during spermatogenesis and provide clues to the molecular mechanisms in epigenetic abnormalities impacting male reproductive health.     

Narrow H3K4me2 is required for chicken PGC formation

The development of primordial germ cells (PGCs) undergoes epigenetic modifications. The study of histone methylation in regulating PGCs is beneficial to understand the development and differentiation mechanism of germ stem cells. Notably, it provides a theoretical basis for directed induction and mass acquisition in vitro. However, little is known about the regulation of PGC formation by histone methylation. Here, we found the high enrichment of H3K4me2 in the blastoderm, genital ridges, and testis. Chromatin immunoprecipitation sequencing was performed and the results revealed that genomic H3K4me2 is dynamic in embryonic stem cells, PGCs, and spermatogonial stem cells. This trend was consistent with the H3K4me2 enrichment in the gene promoter region. Additionally, narrow region triggered PGC‐related genes (Bmp4, Wnt5a, and Tcf7l2) and signaling pathways (Wnt and transforming growth factor‐β). After knocking down histone methylase Mll2 in vitro and vivo, the level of H3K4me2 decreased, inhibiting Cvh and Blimp1 expression, then repressing the formation of PGCs. Taken together, our study revealed the whole genome map of H3K4me2 in the formation of PGCs, contributing to improve the epigenetic study in PGC formation and providing materials for bird gene editing and rescue of endangered birds.     

Epigenetic reprogramming in the porcine germ line

Background  

Epigenetic reprogramming is critical for genome regulation during germ line development. Genome-wide demethylation in mouse primordial germ cells (PGC) is a unique reprogramming event essential for erasing epigenetic memory and preventing the transmission of epimutations to the next generation. In addition to DNA demethylation, PGC are subject to a major reprogramming of histone marks, and many of these changes are concurrent with a cell cycle arrest in the G2 phase. There is limited information on how well conserved these events are in mammals. Here we report on the dynamic reprogramming of DNA methylation at CpGs of imprinted loci and DNA repeats, and the global changes in H3K27me3 and H3K9me2 in the developing germ line of the domestic pig.     

Dynamic patterns of histone H3 lysine 4 methyltransferases and demethylases during mouse preimplantation development

Extensive and dynamic chromatin remodeling occurs after fertilization, including DNA methylation and histone modifications. These changes underlie the transition from gametic to embryonic chromatin and are thought to facilitate early embryonic development. Histone H3 lysine 4 methylation (H3K4me) is an important epigenetic mechanism that associates with gene-specific activation and functions in development. However, dynamic regulation of H3K4me during early embryonic development remains unclear. Herein, the authors examined the dynamic changes of H3K4me and its key regulators (Ash1l, Ash2l, Kmt2a, Kmt2b, Kmt2c, Setd1a, Setd7, Kdm1a, Kdm1b, Kdm5a, Kdm5b, Kdm5c, and Kdm5d) in mouse oocytes and preimplantation embryos. An increase in levels of H3K4me2 and me3 was observed at the one- to two-cell stages (P?P?P?相似文献   

X chromosome activity in mouse XX primordial germ cells

In the early epiblast of female mice, one of the two X chromosomes is randomly inactivated by a Xist-dependent mechanism, involving the recruitment of Ezh2-Eed and the subsequent trimethylation of histone 3 on lysine 27 (H3K27me3). We demonstrate that this random inactivation process applies also to the primordial germ cell (PGC) precursors, located in the proximal region of the epiblast. PGC specification occurs at about embryonic day (E)7.5, in the extraembryonic mesoderm, after which the germ cells enter the endoderm of the invaginating hindgut. As they migrate towards the site of the future gonads, the XX PGCs gradually lose the H3K27me3 accumulation on the silent X chromosome. However, using a GFP transgene inserted into the X chromosome, we observed that the XX gonadal environment (independently of the gender) is important for the substantial reactivation of the inactive X chromosome between E11.5 and E13.5, but is not required for X-chromosome reactivation during the derivation of pluripotent embryonic germ cells. We describe in detail one of the key events during female PGC development, the epigenetic reprogramming of the X chromosome, and demonstrate the role of the XX somatic genital ridge in this process.     

H3K23me2 is a new heterochromatic mark in Caenorhabditis elegans

Genome-wide analyses in Caenorhabditis elegans show that post-translational modifications (PTMs) of histones are evolutionary conserved and distributed along functionally distinct genomic domains. However, a global profile of PTMs and their co-occurrence on the same histone tail has not been described in this organism. We used mass spectrometry based middle-down proteomics to analyze histone H3 N-terminal tails from C. elegans embryos for the presence, the relative abundance and the potential cross-talk of co-existing PTMs. This analysis highlighted that the lysine 23 of histone H3 (H3K23) is extensively modified by methylation and that tri-methylated H3K9 (H3K9me3) is exclusively detected on histone tails with di-methylated H3K23 (H3K23me2). Chromatin immunoprecipitation approaches revealed a positive correlation between H3K23me2 and repressive marks. By immunofluorescence analyses, H3K23me2 appears differentially regulated in germ and somatic cells, in part by the action of the histone demethylase JMJD-1.2. H3K23me2 is enriched in heterochromatic regions, localizing in H3K9me3 and heterochromatin protein like-1 (HPL-1)-positive foci. Biochemical analyses indicated that HPL-1 binds to H3K23me2 and interacts with a conserved CoREST repressive complex. Thus, our study suggests that H3K23me2 defines repressive domains and contributes to organizing the genome in distinct heterochromatic regions during embryogenesis.     

Structural and biochemical basis of interdependent FANCI‐FANCD2 ubiquitination

Di‐monoubiquitination of the FANCI‐FANCD2 (ID2) complex is a central and crucial step for the repair of DNA interstrand crosslinks via the Fanconi anaemia pathway. While FANCD2 ubiquitination precedes FANCI ubiquitination, FANCD2 is also deubiquitinated at a faster rate than FANCI, which can result in a FANCI‐ubiquitinated ID2 complex (I_UbD2). Here, we present a 4.1 Å cryo‐EM structure of I_UbD2 complex bound to double‐stranded DNA. We show that this complex, like ID2_Ub and I_UbD2_Ub, is also in the closed ID2 conformation and clamps on DNA. The target lysine of FANCD2 (K561) becomes fully exposed in the I_UbD2‐DNA structure and is thus primed for ubiquitination. Similarly, FANCI''s target lysine (K523) is also primed for ubiquitination in the ID2_Ub‐DNA complex. The I_UbD2‐DNA complex exhibits deubiquitination resistance, conferred by the presence of DNA and FANCD2. ID2_Ub‐DNA, on the other hand, can be efficiently deubiquitinated by USP1‐UAF1, unless further ubiquitination on FANCI occurs. Therefore, FANCI ubiquitination effectively maintains FANCD2 ubiquitination in two ways: it prevents excessive FANCD2 deubiquitination within an I_UbD2_Ub‐DNA complex, and it enables re‐ubiquitination of FANCD2 within a transient, closed‐on‐DNA, I_UbD2 complex.     

Mitotic Accumulation of Dimethylated Lysine 79 of Histone H3 Is Important for Maintaining Genome Integrity During Mitosis in Human Cells

The loss of genome stability is an early event that drives the development and progression of virtually all tumor types. Recent studies have revealed that certain histone post-translational modifications exhibit dynamic and global increases in abundance that coincide with mitosis and exhibit essential roles in maintaining genomic stability. Histone H2B ubiquitination at lysine 120 (H2Bub1) is regulated by RNF20, an E3 ubiquitin ligase that is altered in many tumor types. Through an evolutionarily conserved trans-histone pathway, H2Bub1 is an essential prerequisite for subsequent downstream dimethylation events at lysines 4 (H3K4me2) and 79 (H3K79me2) of histone H3. Although the role that RNF20 plays in tumorigenesis has garnered much attention, the downstream components of the trans-histone pathway, H3K4me2 and H3K79me2, and their potential contributions to genome stability remain largely overlooked. In this study, we employ single-cell imaging and biochemical approaches to investigate the spatial and temporal patterning of RNF20, H2Bub1, H3K4me2, and H3K79me2 throughout the cell cycle, with a particular focus on mitosis. We show that H2Bub1, H3K4me2, and H3K79me2 exhibit distinct temporal progression patterns throughout the cell cycle. Most notably, we demonstrate that H3K79me2 is a highly dynamic histone post-translational modification that reaches maximal abundance during mitosis in an H2Bub1-independent manner. Using RNAi and chemical genetic approaches, we identify DOT1L as a histone methyltransferase required for the mitotic-associated increases in H3K79me2. We also demonstrate that the loss of mitotic H3K79me2 levels correlates with increases in chromosome numbers and increases in mitotic defects. Collectively, these data suggest that H3K79me2 dynamics during mitosis are normally required to maintain genome stability and further implicate the loss of H3K79me2 during mitosis as a pathogenic event that contributes to the development and progression of tumors.     

Predominant expression of H3K9 methyltransferases in prehypertrophic and hypertrophic chondrocytes during mouse growth plate cartilage development

Histone lysine methylation (HKM) is an epigenetic change that establishes cell-specific gene expression and determines cell fates. In this study, we investigated the expression patterns of histone H3 lysine 9 methyltransferases (H3K9MTases) G9a (euchromatic histone lysine N-methyltransferase 2, Ehmt2), GLP (euchromatic histone lysine N-methyltransferase 1, Ehmt1), SETDB1 (SET domain, bifurcated 1), PRDM2 (PR domain containing 2), SUV39H1 (suppressor of variegation 3–9 homolog 1), and SUV39H2, as well as the distribution of 3 types of HKM at histone H3 lysine 9: mono- (H3K9me1), di- (H3K9me2), or tri-methylation (H3K9me3), during mouse growth plate development. In the forelimb cartilage primordial at embryonic day 12.5 (E12.5), none of the H3K9MTases were detected and H3K9me1, H3K9me2, and H3K9me3 were scarcely detected. At E14.5, the H3K9MTases were expressed at low levels in proliferating chondrocytes and at high levels in prehypertrophic and hypertrophic chondrocytes. Among the H3K9 methylations, H3K9me1 and H3K9me3 were markedly noted in these chondrocytes. At E16.5, G9, GLP, SETDB1, PRDM2, SUV39H1, and SUV39H2, as well as H3K9me1, H3K9me2, and H3K9me3, were detected in prehypertrophic and hypertrophic chondrocytes in the growth plate. Western blotting and real-time quantitative polymerase chain reaction analysis revealed the distributions of G9 and GLP proteins and the expression of all the H3K9MTase mRNAs in prehypertrophic and hypertrophic chondrocytes. These data suggest that H3K9 methyltransferases are predominantly expressed in prehypertrophic and hypertrophic chondrocytes, and that they could be involved in the regulation of gene expression and progression of chondrocyte differentiation by affecting the methylation state of histone H3 lysine 9 in the mouse growth plate.