PRC1 directs PRC2-H3K27me3 deposition to shield adult spermatogonial stem cells from differentiation.

Spermatogonial stem cells functionality reside in the slow-cycling and heterogeneous undifferentiated spermatogonia cell population. This pool of cells supports lifelong fertility in adult males by balancing self-renewal and differentiation to produce haploid gametes. However, the molecular mechanisms underpinning long-term stemness of undifferentiated spermatogonia during adulthood remain unclear. Here, we discover that an epigenetic regulator, Polycomb repressive complex 1 (PRC1), shields adult undifferentiated spermatogonia from differentiation, maintains slow cycling, and directs commitment to differentiation during steady-state spermatogenesis in adults. We show that PRC2-mediated H3K27me3 is an epigenetic hallmark of adult undifferentiated spermatogonia. Indeed, spermatogonial differentiation is accompanied by a global loss of H3K27me3. Disruption of PRC1 impairs global H3K27me3 deposition, leading to precocious spermatogonial differentiation. Therefore, PRC1 directs PRC2-H3K27me3 deposition to maintain the self-renewing state of undifferentiated spermatogonia. Importantly, in contrast to its role in other tissue stem cells, PRC1 negatively regulates the cell cycle to maintain slow cycling of undifferentiated spermatogonia. Our findings have implications for how epigenetic regulators can be tuned to regulate the stem cell potential, cell cycle and differentiation to ensure lifelong fertility in adult males.

Polycomb protein SCML2 mediates paternal epigenetic inheritance through sperm chromatin.

Sperm chromatin retains small amounts of histones, and chromatin states of sperm mirror gene expression programs of the next generation. However, it remains largely unknown how paternal epigenetic information is transmitted through sperm chromatin. Here, we present a novel mouse model of paternal epigenetic inheritance, in which deposition of Polycomb repressive complex 2 (PRC2) mediated-repressive H3K27me3 is attenuated in the paternal germline. By applying modified methods of assisted reproductive technology using testicular sperm, we rescued infertility of mice missing Polycomb protein SCML2, which regulates germline gene expression by establishing H3K27me3 on bivalent promoters with other active marks H3K4me2/3. We profiled epigenomic states (H3K27me3 and H3K4me3) of testicular sperm and epididymal sperm, demonstrating that the epididymal pattern of the sperm epigenome is already established in testicular sperm and that SCML2 is required for this process. In F1 males of X-linked Scml2-knockout mice, which have a wild-type genotype, gene expression is dysregulated in the male germline during spermiogenesis. These dysregulated genes are targets of SCML2-mediated H3K27me3 in F0 sperm. Further, dysregulation of gene expression was observed in the mutant-derived wild-type F1 preimplantation embryos. Together, we present functional evidence that the classic epigenetic regulator Polycomb mediates paternal epigenetic inheritance through sperm chromatin.

Polycomb directs timely activation of germline genes in spermatogenesis.

During spermatogenesis, a large number of germline genes essential for male fertility are coordinately activated. However, it remains unknown how timely activation of this group of germline genes is accomplished. Here we show that Polycomb-repressive complex 1 (PRC1) directs timely activation of germline genes during spermatogenesis. Inactivation of PRC1 in male germ cells results in the gradual loss of a stem cell population and severe differentiation defects, leading to male infertility. In the stem cell population, RNF2, the dominant catalytic subunit of PRC1, activates transcription of Sall4, which codes for a transcription factor essential for subsequent spermatogenic differentiation. Furthermore, RNF2 and SALL4 together occupy transcription start sites of germline genes in the stem cell population. Once differentiation commences, these germline genes are activated to enable the progression of spermatogenesis. Our study identifies a novel mechanism by which Polycomb directs the developmental process by activating a group of lineage-specific genes.

Use of Visual Information by Ant Species Occurring in Similar Urban Anthropogenic Environments.

Many insects, including ants, are known to respond visually to conspicuous objects. In this study, we compared orientation in an arena containing only a black target beacon as local information in six species of ants of widely varying degree of phylogenic relatedness, foraging strategy, and eye morphology (Aphaenogaster, Brachyponera, Camponotus, Formica, and two Lasius spp.), often found associated in similar urban anthropogenic habitats. Four species of ants displayed orientation toward the beacon, with two orienting toward it directly, while the other two approached it via convoluted paths. The two remaining species did not show any orientation with respect to the beacon. The results did not correlate with morphological parameters of the visual systems and could not be fully interpreted in terms of the species' ecology, although convoluted paths are linked to higher significance of chemical signals. Beacon aiming was shown to be an innate behavior in visually naive Formica workers, which, however, were less strongly attracted to the beacon than older foragers. Thus, despite sharing the same habitats and supposedly having similar neural circuits, even a very simple stimulus-related behavior in the absence of other information can differ widely in ants but is likely an ancestral trait retained especially in species with smaller eyes. The comparative analysis of nervous systems opens the possibility of determining general features of circuits responsible for innate and possibly learned attraction toward particular stimuli.

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.

Polycomb protein SCML2 facilitates H3K27me3 to establish bivalent domains in the male germline.

Repressive H3K27me3 and active H3K4me2/3 together form bivalent chromatin domains, molecular hallmarks of developmental potential. In the male germline, these domains are thought to persist into sperm to establish totipotency in the next generation. However, it remains unknown how H3K27me3 is established on specific targets in the male germline. Here, we demonstrate that a germline-specific Polycomb protein, SCML2, binds to H3K4me2/3-rich hypomethylated promoters in undifferentiated spermatogonia to facilitate H3K27me3. Thus, SCML2 establishes bivalent domains in the male germline of mice. SCML2 regulates two major classes of bivalent domains: Class I domains are established on developmental regulator genes that are silent throughout spermatogenesis, while class II domains are established on somatic genes silenced during late spermatogenesis. We propose that SCML2-dependent H3K27me3 in the male germline prepares the expression of developmental regulator and somatic genes in embryonic development.

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.

SCML2 promotes heterochromatin organization in late spermatogenesis.

Spermatogenesis involves the progressive reorganization of heterochromatin. However, the mechanisms that underlie the dynamic remodeling of heterochromatin remain unknown. Here, we identify SCML2, a germline-specific Polycomb protein, as a critical regulator of heterochromatin organization in spermatogenesis. We show that SCML2 accumulates on pericentromeric heterochromatin (PCH) in male germ cells, where it suppresses PRC1-mediated monoubiquitylation of histone H2A at Lysine 119 (H2AK119ub) and promotes deposition of PRC2-mediated H3K27me3 during meiosis. In postmeiotic spermatids, SCML2 is required for heterochromatin organization, and the loss of SCML2 leads to the formation of ectopic patches of facultative heterochromatin. Our data suggest that, in the absence of SCML2, the ectopic expression of somatic lamins drives this process. Furthermore, the centromere protein CENP-V is a specific marker of PCH in postmeiotic spermatids, and SCML2 is required for CENP-V localization on PCH. Given the essential functions of PRC1 and PRC2 for genome-wide gene expression in spermatogenesis, our data suggest that heterochromatin organization and spermatogenesis-specific gene expression are functionally linked. We propose that SCML2 coordinates the organization of heterochromatin and gene expression through the regulation of Polycomb complexes.

Dynamic reorganization of open chromatin underlies diverse transcriptomes during spermatogenesis.

During spermatogenesis, germ cells undergo massive cellular reconstruction and dynamic chromatin remodeling to facilitate highly diverse transcriptomes, which are required for the production of functional sperm. However, it remains unknown how germline chromatin is organized to promote the dynamic, complex transcriptomes of spermatogenesis. Here, using ATAC-seq, we establish the varied landscape of open chromatin during spermatogenesis. We identify the reorganization of accessible chromatin in intergenic and intronic regions during the mitosis-to-meiosis transition. During the transition, mitotic-type open chromatin is closed while the de novo formation of meiotic-type open chromatin takes place. Contrastingly, differentiation processes such as spermatogonial differentiation and the meiosis-to-postmeiosis transition involve chromatin closure without the de novo formation of accessible chromatin. In spermiogenesis, the germline-specific Polycomb protein SCML2 promotes the closure of open chromatin at autosomes for gene suppression. Paradoxically, we identify the massive de novo formation of accessible chromatin when the sex chromosomes undergo meiotic sex chromosome inactivation, and this is also mediated by SCML2. These results reveal meiotic sex chromosome inactivation as an active process for chromatin organization. Together, our results unravel the genome-wide, dynamic reorganization of open chromatin and reveal mechanisms that underlie diverse transcriptomes during spermatogenesis.

A rapidly evolved domain, the SCML2 DNA-binding repeats, contributes to chromatin binding of mouse SCML2†.

Genes involved in sexual reproduction diverge rapidly as a result of reproductive fitness. Here, we identify a novel protein domain in the germline-specific Polycomb protein SCML2 that is required for the establishment of unique gene expression programs after the mitosis-to-meiosis transition in spermatogenesis. We term this novel domain, which is comprised of rapidly evolved, DNA-binding repeat units of 28 amino acids, the SCML2 DNA-binding (SDB) repeats. These repeats are acquired in a specific subgroup of the rodent lineage, having been subjected to positive selection in the course of evolution. Mouse SCML2 has two DNA-binding domains: one is the SDB repeats and the other is an RNA-binding region, which is conserved in human SCML2. For the recruitment of SCML2 to target loci, the SDB repeats cooperate with the other functional domains of SCML2 to bind chromatin. The cooperative action of these domains enables SCML2 to sense DNA hypomethylation in an in vivo chromatin environment, thereby enabling SCML2 to bind to hypomethylated chromatin. We propose that the rapid evolution of SCML2 is due to reproductive adaptation, which has promoted species-specific gene expression programs in spermatogenesis.

Truncated UDP-glucuronosyltransferase (UGT) from a Crigler-Najjar syndrome type II patient colocalizes with intact UGT in the endoplasmic reticulum.

Mutations in the gene encoding bilirubin UDP-glucuronosyltransferase (UGT1A1) are known to cause Crigler-Najjar syndrome type II (CN-II). We previously encountered a patient with a nonsense mutation (Q331X) on one allele and with no other mutations in the promoter region or other exons, and proposed that CN-II is inherited as a dominant trait due to the formation of a heterologous subunit structure comprised of the altered UGT1A1 gene product (UGT1A1-p.Q331X) and the intact UGT1A1. Here, we investigated the molecular basis of CN-II in this case by expressing UGT1A1-p.Q331X in cells. UGT1A1-p.Q331X overexpressed in Escherichia coli or mammalian cells directly bound or associated with intact UGT1A1 in vitro or in vivo, respectively. Intact UGT1A1 was observed as a dimer using atomic force microscopy. Fluorescent-tagged UGT1A1-p.Q331X and intact UGT1A1 were colocalized in 293T cells, and fluorescence recovery after photobleaching analysis showed that UGT1A1-p.Q331X was retained in the endoplasmic reticulum (ER) without rapid degradation. These findings support the idea that UGT1A1-p.Q331X and UGT1A1 form a dimer and provide an increased mechanistic understanding of CN-II.

BRCT domain of DNA polymerase µ has DNA-binding activity and promotes the DNA polymerization activity.

DNA polymerase µ (pol µ) catalyzes nonhomologous end-joining in DNA double-stranded break repair. Pol µ consists of an amino-terminal BRCA1 carboxyl-terminal homology (BRCT) domain and a pol ß-like region, which contains the catalytic site. By DNA cellulose column chromatography, using full-length pol µ and five different deletion mutants, we found that the amino-terminal region has double-stranded DNA (dsDNA)-binding activity. Pol µ without BRCT domain reduces the DNA polymerization activity when compared to full-length pol µ. Observation by atomic force microscopy showed that full-length pol µ binds to the ends and middle part of dsDNA. Pol µ lacking the amino-terminal region or with a mutation within the BRCT domain bound only to DNA ends, whereas the amino-terminal region with the BRCT domain bound to both the ends and the middle part of dsDNA (mpdDNA). Terminal deoxynucleotidyltransferase, which, like pol µ, belongs to the X family DNA polymerases, also bound to mpdDNA through its amino-terminal region.

ATAC-Seq Analysis of Accessible Chromatin: From Experimental Steps to Data Analysis.

Accessible chromatin often represents gene regulatory elements, including promoters and enhancers, essential for gene expression. Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) is one of the most popular techniques to investigate chromatin accessibility across the genome. Here we describe, step by step, a series of optimized experimental methods and bioinformatics pipelines for ATAC-seq analysis. As an example, we present an analysis of murine spermatogenic cells: a method to isolate germ cells, a reaction step using Tn5 transposase to insert sequencing adapters into accessible DNA, a library preparation method for high-throughput sequencing, and bioinformatics analysis of sequencing data. Overall, we introduce a framework of ATAC-seq analysis that can be applied to any cell population to identify cell-type-specific gene regulatory elements and their cis-regulatory networks.

Bioinformatics Pipelines for Identification of Super-Enhancers and 3D Chromatin Contacts.

Precise regulation of gene expression is integral in development. Emerging studies have highlighted that super-enhancers (SEs), which are clusters of multiple enhancers, play critical roles in regulating cell type-specific gene expression via 3D chromatin, thereby defining the cellular identities of given cells. Here we provide optimized bioinformatics pipelines to identify SEs and 3D chromatin contacts. Our pipelines encompass the processing of chromatin immunoprecipitation sequencing (ChIP-seq) data to identify SEs and the processing of genome-wide chromosome conformation capture (Hi-C) data. We can then infer long-range chromatin contacts between SEs and other genomic regions. This integrative computational approach, which can be applied to CUT&RUN and CUT&Tag, alternative technologies to ChIP-seq, allows us to identify genomic locations of SEs and their 3D genome configuration, whereby multiple SEs act in concert. We show an analysis of mouse spermatogenesis as an example of this application.

KRAB-zinc-finger proteins regulate endogenous retroviruses to sculpt germline transcriptomes and genome evolution.

As transposable elements (TEs) coevolved with the host genome, the host genome exploited TEs as functional regulatory elements. What remains largely unknown are how the activity of TEs, namely, endogenous retroviruses (ERVs), are regulated and how TEs evolved in the germline. Here we show that KRAB domain-containing zinc-finger proteins (KZFPs), which are highly expressed in mitotically dividing spermatogonia, bind to suppressed ERVs that function following entry into meiosis as active enhancers. These features are observed for independently evolved KZFPs and ERVs in mice and humans, i.e., are evolutionarily conserved in mammals. Further, we show that meiotic sex chromosome inactivation (MSCI) antagonizes the coevolution of KZFPs and ERVs in mammals. Our study uncovers a mechanism by which KZFPs regulate ERVs to sculpt germline transcriptomes. We propose that epigenetic programming in the mammalian germline during the mitosis-to-meiosis transition facilitates coevolution of KZFPs and TEs on autosomes and is antagonized by MSCI.

CTCF-mediated 3D chromatin predetermines the gene expression program in the male germline.

Spermatogenesis is a unidirectional differentiation process that generates haploid sperm, but how the gene expression program that directs this process is established is largely unknown. Here we determine the high-resolution 3D chromatin architecture of male germ cells during spermatogenesis and show that CTCF-mediated 3D chromatin predetermines the gene expression program required for spermatogenesis. In undifferentiated spermatogonia, CTCF-mediated chromatin contacts on autosomes pre-establish meiosis-specific super-enhancers (SE). These meiotic SE recruit the master transcription factor A-MYB in meiotic spermatocytes, which strengthens their 3D contacts and instructs a burst of meiotic gene expression. We also find that at the mitosis-to-meiosis transition, the germline-specific Polycomb protein SCML2 resolves chromatin loops that are specific to mitotic spermatogonia. Moreover, SCML2 and A-MYB establish the unique 3D chromatin organization of sex chromosomes during meiotic sex chromosome inactivation. We propose that CTCF-mediated 3D chromatin organization enforces epigenetic priming that directs unidirectional differentiation, thereby determining the cellular identity of the male germline.

PRC1 suppresses a female gene regulatory network to ensure testicular differentiation.

Gonadal sex determination and differentiation are controlled by somatic support cells of testes (Sertoli cells) and ovaries (granulosa cells). In testes, the epigenetic mechanism that maintains chromatin states responsible for suppressing female sexual differentiation remains unclear. Here, we show that Polycomb repressive complex 1 (PRC1) suppresses a female gene regulatory network in postnatal Sertoli cells. We genetically disrupted PRC1 function in embryonic Sertoli cells after sex determination, and we found that PRC1-depleted postnatal Sertoli cells exhibited defective proliferation and cell death, leading to the degeneration of adult testes. In adult Sertoli cells, PRC1 suppressed specific genes required for granulosa cells, thereby inactivating the female gene regulatory network. Chromatin regions associated with female-specific genes were marked by Polycomb-mediated repressive modifications: PRC1-mediated H2AK119ub and PRC2-mediated H3K27me3. Taken together, this study identifies a critical Polycomb-based mechanism that suppresses ovarian differentiation and maintains Sertoli cell fate in adult testes.

TdIF2 is a nucleolar protein that promotes rRNA gene promoter activity.

Terminal deoxynucleotidyltransferase (TdT) interacting factor 2 (TdIF2) is an acidic protein that binds to TdT. TdIF2 binds to DNA and core histones and contains an acidic-amino acid-rich region in its C-terminus. It has therefore been suggested to function as a histone chaperone within the nucleus. TdIF2 localized within the nucleolus in HEK 293T cells, and its N-terminal (residues 1-234) and C-terminal (residues 606-756) regions were crucial for the nucleolar localization. A chromatin immunoprecipitation (ChIP) assay showed that TdIF2 associated with the promoter of human ribosomal RNA genes (hrDNAP), and an in vitro luciferase assay system showed that it promoted hrDNAP activity. Using the yeast two-hybrid system with TdIF2 as the bait, we isolated the cDNA encoding HIV Tat interactive protein 60 (Tip60), which has histone acetyltransferase (HAT) activity, as a TdIF2-binding protein. TdIF2 bound to Tip60 in vitro and in vivo, inhibited the Tip60 HAT activity in vitro and co-localized with Tip60 within the nucleolus. In addition, TdIF2 promotes upstream binding factor (UBF) acetylation in vivo. Thus, TdIF2 might promote hrDNAP activity by suppressing Tip60's HAT activity and promoting UBF acetylation.

PRC1-mediated epigenetic programming is required to generate the ovarian reserve.

The ovarian reserve defines the female reproductive lifespan, which in humans spans decades due to robust maintenance of meiotic arrest in oocytes residing in primordial follicles. Epigenetic reprogramming, including DNA demethylation, accompanies meiotic entry, but the chromatin changes that underpin the generation and preservation of ovarian reserves are poorly defined. We report that the Polycomb Repressive Complex 1 (PRC1) establishes repressive chromatin states in perinatal mouse oocytes that directly suppress the gene expression program of meiotic prophase-I and thereby enable the transition to dictyate arrest. PRC1 dysfuction causes depletion of the ovarian reserve and leads to premature ovarian failure. Our study demonstrates a fundamental role for PRC1-mediated gene silencing in female reproductive lifespan, and reveals a critical window of epigenetic programming required to establish ovarian reserve.

Loss of CAPS2/Cadps2 leads to exocrine pancreatic cell injury and intracellular accumulation of secretory granules in mice.

The type 2 Ca2+-dependent activator protein for secretion (CAPS2/CADPS2) regulates dense-core vesicle trafficking and exocytosis and is involved in the regulated release of catecholamines, peptidergic hormones, and neuromodulators. CAPS2 is expressed in the pancreatic exocrine acinar cells that produce and secrete digestive enzymes. However, the functional role of CAPS2 in vesicular trafficking and/or exocytosis of non-regulatory proteins in the exocrine pancreas remains to be determined. Here, we analyzed the morpho-pathological indicators of the pancreatic exocrine pathway in Cadps2-deficient mouse models using histochemistry, biochemistry, and electron microscopy. We used whole exosome sequencing to identify CADPS2 variants in patients with chronic pancreatitis (CP). Caps2/Cadps2-knockout (KO) mice exhibited morphophysiological abnormalities in the exocrine pancreas, including excessive accumulation of secretory granules (zymogen granules) and their amylase content in the cytoplasm, deterioration of the fine intracellular membrane structures (disorganized rough endoplasmic reticulum, dilated Golgi cisternae, and the appearance of empty vesicles and autophagic-like vacuoles), as well as exocrine pancreatic cell injury, including acinar cell atrophy, increased fibrosis, and inflammatory cell infiltration. Pancreas-specific Cadps2 conditional KO mice exhibited pathological abnormalities in the exocrine pancreas similar to the global Cadps2 KO mice, indicating that these phenotypes were caused either directly or indirectly by CAPS2 deficiency in the pancreas. Furthermore, we identified a rare variant in the exon3 coding region of CADPS2 in a non-alcoholic patient with CP and showed that Cadps2-dex3 mice lacking CAPS2 exon3 exhibited symptoms similar to those exhibited by the Cadps2 KO and cKO mice. These results suggest that CAPS2 is critical for the proper functioning of the pancreatic exocrine pathway, and its deficiency is associated with a risk of pancreatic acinar cell pathology.