Satoshi H. Namekawa

Postmeiotic Sex Chromatin in the Male Germline of Mice

In mammals, the X and Y chromosomes are subject to meiotic sex chromosome inactivation (MSCI) during prophase I in the male germline, but their status thereafter is currently unclear. An abundance of X-linked spermatogenesis genes has spawned the view that the X must be active . On the other hand, the idea that the imprinted paternal X of the early embryo may be preinactivated by MSCI suggests that silencing may persist longer . To clarify this issue, we establish a comprehensive X-expression profile during mouse spermatogenesis. Here, we discover that the X and Y occupy a novel compartment in the postmeiotic spermatid and adopt a non-Rabl configuration. We demonstrate that this postmeiotic sex chromatin (PMSC) persists throughout spermiogenesis into mature sperm and exhibits epigenetic similarity to the XY body. In the spermatid, 87% of X-linked genes remain suppressed postmeiotically, while autosomes are largely active. We conclude that chromosome-wide X silencing continues from meiosis to the end of spermiogenesis, and we discuss implications for proposed mechanisms of imprinted X-inactivation.

THE RNASE III ENZYME DROSHA IS ESSENTIAL FOR MICRORNA PRODUCTION AND SPERMATOGENESIS

Background: miRNA biogenesis requires two RNase III enzymes, DROSHA and DICER. Results: Lack of DROSHA in the male germ line leads to deficiency in miRNA production and male infertility. Conclusion: DROSHA and DICER have both common and unique functions in male germ cell development. Significance: This study reveals an essential role of DROSHA, DICER, and DROSHA-/DICER-dependent small noncoding RNAs spermatogenesis. DROSHA is a nuclear RNase III enzyme responsible for cleaving primary microRNAs (miRNAs) into precursor miRNAs and thus is essential for the biogenesis of canonical miRNAs. DICER is a cytoplasmic RNase III enzyme that not only cleaves precursor miRNAs to produce mature miRNAs but also dissects naturally formed/synthetic double-stranded RNAs to generate small interfering RNAs (siRNAs). To investigate the role of canonical miRNA and/or endogenous siRNA production in spermatogenesis, we generated Drosha or Dicer conditional knock-out (cKO) mouse lines by inactivating Drosha or Dicer exclusively in spermatogenic cells in postnatal testes using the Cre-loxp strategy. Both Drosha and Dicer cKO males were infertile due to disrupted spermatogenesis characterized by depletion of spermatocytes and spermatids leading to oligoteratozoospermia or azoospermia. The developmental course of spermatogenic disruptions was similar at morphological levels between Drosha and Dicer cKO males, but Drosha cKO testes appeared to be more severe in spermatogenic disruptions than Dicer cKO testes. Microarray analyses revealed transcriptomic differences between Drosha- and Dicer-null pachytene spermatocytes or round spermatids. Although levels of sex-linked mRNAs were mildly elevated, meiotic sex chromosome inactivation appeared to have occurred normally. Our data demonstrate that unlike DICER, which is required for the biogenesis of several small RNA species, DROSHA is essential mainly for the canonical miRNA production, and DROSHA-mediated miRNA production is essential for normal spermatogenesis and male fertility.

Two-Step Imprinted X Inactivation: Repeat versus Genic Silencing in the Mouse

ABSTRACT Mammals compensate for unequal X-linked gene dosages between the sexes by inactivating one X chromosome in the female. In marsupials and in the early mouse embryo, X chromosome inactivation (XCI) is imprinted to occur selectively on the paternal X chromosome (XP). The mechanisms and events underlying XP imprinting remain unclear. Here, we find that the imprinted XP can be functionally divided into two domains, one comprising traditional coding genes (genic) and the other comprising intergenic repetitive elements. XP repetitive element silencing occurs by the two-cell stage, does not require Xist, and occurs several divisions prior to genic silencing. In contrast, genic silencing initiates at the morula-to-blastocyst stage and absolutely requires Xist. Genes translocate into the presilenced repeat region as they are inactivated, whereas active genes remain outside. Thus, during the gamete-embryo transition, imprinted XCI occurs in two steps, with repeat silencing preceding genic inactivation. Nucleolar association may underlie the epigenetic asymmetry of XP and XM. We hypothesize that transgenerational information (the imprint) is carried by repeats from the paternal germ line or that, alternatively, repetitive elements are silenced at the two-cell stage in a parent-of-origin-specific manner. Our model incorporates aspects of the so-called classical, de novo, and preinactivation hypotheses and suggests that Xist RNA functions relatively late during preimplantation mouse development.

Tsx Produces a Long Noncoding RNA and Has General Functions in the Germline, Stem Cells, and Brain

The Tsx gene resides at the X-inactivation center and is thought to encode a protein expressed in testis, but its function has remained mysterious. Given its proximity to noncoding genes that regulate X-inactivation, here we characterize Tsx and determine its function in mice. We find that Tsx is actually noncoding and the long transcript is expressed robustly in meiotic germ cells, embryonic stem cells, and brain. Targeted deletion of Tsx generates viable offspring and X-inactivation is only mildly affected in embryonic stem cells. However, mutant embryonic stem cells are severely growth-retarded, differentiate poorly, and show elevated cell death. Furthermore, male mice have smaller testes resulting from pachytene-specific apoptosis and a maternal-specific effect results in slightly smaller litters. Intriguingly, male mice lacking Tsx are less fearful and have measurably enhanced hippocampal short-term memory. Combined, our study indicates that Tsx performs general functions in multiple cell types and links the noncoding locus to stem and germ cell development, learning, and behavior in mammals.

Sex chromosome silencing in the marsupial male germ line.

In marsupials, dosage compensation involves silencing of the fathers X-chromosome. Because no XIST orthologue has been found, how imprinted X-inactivation occurs is unknown. In eutherians, the X is subject to meiotic sex chromosome inactivation (MSCI) in the paternal germ line and persists thereafter as postmeiotic sex chromatin (PMSC). One hypothesis proposes that the paternal X is inherited by the eutherian zygote as a preinactive X and raises the possibility of a similar process in the marsupial germ line. Here we demonstrate that MSCI and PMSC occur in the opossum. Surprisingly, silencing occurs before X–Y association. After MSCI, the X and Y fuse through a dense plate without obvious synapsis. Significantly, sex chromosome silencing continues after meiosis, with the opossum PMSC sharing features of eutherian PMSC. These results reveal a common gametogenic program in two diverse clades of mammals and support the idea that male germ-line silencing may have provided an ancestral form of mammalian dosage compensation.

RNF8 REGULATES ACTIVE EPIGENETIC MODIFICATIONS AND ESCAPE GENE ACTIVATION FROM INACTIVE SEX CHROMOSOMES IN POST-MEIOTIC SPERMATIDS

Sex chromosomes are uniquely subject to chromosome-wide silencing during male meiosis, and silencing persists into post-meiotic spermatids. Against this background, a select set of sex chromosome-linked genes escapes silencing and is activated in post-meiotic spermatids. Here, we identify a novel mechanism that regulates escape gene activation in an environment of chromosome-wide silencing in murine germ cells. We show that RNF8-dependent ubiquitination of histone H2A during meiosis establishes active epigenetic modifications, including dimethylation of H3K4 on the sex chromosomes. RNF8-dependent active epigenetic memory, defined by dimethylation of H3K4, persists throughout meiotic division. Various active epigenetic modifications are subsequently established on the sex chromosomes in post-meiotic spermatids. These RNF8-dependent modifications include trimethylation of H3K4, histone lysine crotonylation (Kcr), and incorporation of the histone variant H2AFZ. RNF8-dependent epigenetic programming regulates escape gene activation from inactive sex chromosomes in post-meiotic spermatids. Kcr accumulates at transcriptional start sites of sex-linked genes activated in an RNF8-dependent manner, and a chromatin conformational change is associated with RNF8-dependent epigenetic programming. Furthermore, we demonstrate that this RNF8-dependent pathway is distinct from that which recognizes DNA double-strand breaks. Our results establish a novel connection between a DNA damage response factor (RNF8) and epigenetic programming, specifically in establishing active epigenetic modifications and gene activation.

X-inactivation and X-reactivation: epigenetic hallmarks of mammalian reproduction and pluripotent stem cells

X-chromosome inactivation is an epigenetic hallmark of mammalian development. Chromosome-wide regulation of the X-chromosome is essential in embryonic and germ cell development. In the male germline, the X-chromosome goes through meiotic sex chromosome inactivation, and the chromosome-wide silencing is maintained from meiosis into spermatids before the transmission to female embryos. In early female mouse embryos, X-inactivation is imprinted to occur on the paternal X-chromosome, representing the epigenetic programs acquired in both parental germlines. Recent advances revealed that the inactive X-chromosome in both females and males can be dissected into two elements: repeat elements versus unique coding genes. The inactive paternal X in female preimplantation embryos is reactivated in the inner cell mass of blastocysts in order to subsequently allow the random form of X-inactivation in the female embryo, by which both Xs have an equal chance of being inactivated. X-chromosome reactivation is regulated by pluripotency factors and also occurs in early female germ cells and in pluripotent stem cells, where X-reactivation is a stringent marker of naive ground state pluripotency. Here we summarize recent progress in the study of X-inactivation and X-reactivation during mammalian reproduction and development as well as in pluripotent stem cells.

A Mammal-Specific Doublesex Homolog Associates with Male Sex Chromatin and Is Required for Male Meiosis

Gametogenesis is a sexually dimorphic process requiring profound differences in germ cell differentiation between the sexes. In mammals, the presence of heteromorphic sex chromosomes in males creates additional sex-specific challenges, including incomplete X and Y pairing during meiotic prophase. This triggers formation of a heterochromatin domain, the XY body. The XY body disassembles after prophase, but specialized sex chromatin persists, with further modification, through meiosis. Here, we investigate the function of DMRT7, a mammal-specific protein related to the invertebrate sexual regulators Doublesex and MAB-3. We find that DMRT7 preferentially localizes to the XY body in the pachytene stage of meiotic prophase and is required for male meiosis. In Dmrt7 mutants, meiotic pairing and recombination appear normal, and a transcriptionally silenced XY body with appropriate chromatin marks is formed, but most germ cells undergo apoptosis during pachynema. A minority of mutant cells can progress to diplonema, but many of these escaping cells have abnormal sex chromatin lacking histone H3K9 di- and trimethylation and heterochromatin protein 1β accumulation, modifications that normally occur between pachynema and diplonema. Based on the localization of DMRT7 to the XY body and the sex chromatin defects observed in Dmrt7 mutants, we conclude that DMRT7 plays a role in the sex chromatin transformation that occurs between pachynema and diplonema. We suggest that DMRT7 may help control the transition from meiotic sex chromosome inactivation to postmeiotic sex chromatin in males. In addition, because it is found in all branches of mammals, but not in other vertebrates, Dmrt7 may shed light on evolution of meiosis and of sex chromatin.

Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos

Dynamic reprogramming of the genome takes place during the gamete-to-embryo transition. This transition defines a period of continuous and global change but has been difficult to study because of extremely limited material and varying degrees of chromatin compaction. Improved methods of detecting chromatin and gene expression changes in the germ line and in the preimplantation embryo would greatly enhance the understanding of this crucial developmental transition. Here we describe a protocol developed and used by us that improves the sensitivity of existing fluorescence in situ hybridization (FISH) methods; the protocol described here has enabled us to visualize single-copy DNA targets and corresponding nascent RNA transcripts in preimplantation embryos and during spermatogenesis. Major improvements over alternative methods involve fixation and permeabilization steps. Chromatin epitopes can be visualized simultaneously by combining FISH with immunofluorescence; multicopy and repetitive element expression can also be reliably measured. This procedure (sample preparation and staining) requires 1–1.5 d to complete and will facilitate detailed examination of spatial relationships between chromatin epitopes, DNA and RNA during the dynamic transition from gamete to embryo.

Sex chromosome inactivation in germ cells: emerging roles of DNA damage response pathways

Sex chromosome inactivation in male germ cells is a paradigm of epigenetic programming during sexual reproduction. Recent progress has revealed the underlying mechanisms of sex chromosome inactivation in male meiosis. The trigger of chromosome-wide silencing is activation of the DNA damage response (DDR) pathway, which is centered on the mediator of DNA damage checkpoint 1 (MDC1), a binding partner of phosphorylated histone H2AX (γH2AX). This DDR pathway shares features with the somatic DDR pathway recognizing DNA replication stress in the S phase. Additionally, it is likely to be distinct from the DDR pathway that recognizes meiosis-specific double-strand breaks. This review article extensively discusses the underlying mechanism of sex chromosome inactivation.