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.

Chromosome-wide inactivation is an epigenetic signature of sex chromosomes. The mechanism by which the chromosome-wide domain is recognized and gene silencing is induced remains unclear. Here we identify an essential mechanism underlying the recognition of the chromosome-wide domain in the male germline. We show that mediator of DNA damage checkpoint 1 (MDC1), a binding partner of phosphorylated histone H2AX (gH2AX), defines the chromosome-wide domain, initiates meiotic sex chromosome inactivation (MSCI), and leads to XY body formation. Importantly, MSCI consists of two genetically separable steps. The first step is the MDC1-independent recognition of the unsynapsed axis by DNA damage response (DDR) factors such as ataxia telangiectasia and Rad3-related (ATR), TOPBP1, and gH2AX. The second step is the MDC1-dependent chromosome-wide spreading of DDR factors to the entire chromatin. Furthermore, we demonstrate that, in somatic cells, MDC1-dependent amplification of the gH2AX signal occurs following replicative stress and is associated with transcriptional silencing. We propose that a common DDR pathway underlies both MSCI and the response of somatic cells to replicative stress. These results establish that the DDR pathway centered on MDC1 triggers epigenetic silencing of sex chromosomes in germ cells.

SUMMARY Gametogenesis is dependent on the expression of germline-specific genes. However, it remains unknown how the germline epigenome is distinctly established from that of somatic lineages. Here we show that genes commonly expressed in somatic lineages and spermatogenesis-progenitor cells undergo repression in a genome-wide manner in late stages of the male germline and identify underlying mechanisms. SCML2, a germline-specific subunit of a Polycomb repressive complex 1 (PRC1), establishes the unique epigenome of the male germline through two distinct antithetical mechanisms. SCML2 works with PRC1 and promotes RNF2-dependent ubiquitination of H2A, thereby marking somatic/progenitor genes on autosomes for repression. Paradoxically, SCML2 also prevents RNF2-dependent ubiquitination of H2A on sex chromosomes during meiosis, thereby enabling unique epigenetic programming of sex chromosomes for male reproduction. Our results reveal divergent mechanisms involving a shared regulator by which the male germline epigenome is distinguished from that of the soma and progenitor cells.

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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.

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 (X P ). The mechanisms and events underlying X P imprinting remain unclear. Here, we find that the imprinted X P can be functionally divided into two domains, one comprising traditional coding genes (genic) and the other comprising intergenic repetitive elements. X P 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-toblastocyst 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 X P and X M . 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.Genomic imprinting refers to a parent-of-origin effect on gene expression in the developing embryo (3, 57). The existence of imprinting in the mammal means that male and female gametes contribute significantly different information to the zygote. One important difference is illustrated by X chromosome inactivation (XCI), the mechanism of dosage compensation in the mammal that results in the silencing of one X chromosome in the female embryo (2,33,34,49,64). While the eutherian form of XCI occurs randomly in the soma, the marsupial form is imprinted to occur exclusively on the paternal X (X P ) (54). Imprinted XCI also occurs in some eutherians but is restricted to the preimplantation embryo and the extraembryonic tissues (25,37,47,60). Imprinted XCI precedes random XCI in the early mouse embryo and continues through the placental lineages. In the epiblast (embryo proper), transient X reactivation is followed by random XCI, which accounts for the mosaic pattern of inactivation seen in all somatic tissues of the eutherian.The mechanisms and developmental timing of imprinted XCI remain unclear and are much debated. In principle, the maternal or paternal germ line (or both) may differentially mark the X chromosomes, with the maternal imprint protecting the maternal X (X M ) from inactivation and/or the paternal mark predestining X P for inactivation. The search for parentspecific regulators frequently has focused on the X inactivation center (Xic) (7), an X-linked region harboring several noncoding regulators for random XCI. Xist pr...

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.

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.