isolation-murine-spermatogenic-cells-using-violet-excited-cell

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Azabu University

Tokyo University of Science

Tokyo University of science

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.

SCML2 promotes heterochromatin organization in late spermatogenesis.

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.

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

Germ cells manifest a unique gene expression program and regain totipotency in the zygote. Here, we perform Hi-C analysis to examine 3D chromatin organization in male germ cells during spermatogenesis. We show that the highly compartmentalized 3D chromatin organization characteristic of interphase nuclei is attenuated in meiotic prophase. Meiotic prophase is predominated by short-range intrachromosomal interactions that represent a condensed form akin to that of mitotic chromosomes. However, unlike mitotic chromosomes, meiotic chromosomes display weak genomic compartmentalization, weak topologically associating domains, and localized point interactions in prophase. In postmeiotic round spermatids, genomic compartmentalization increases and gives rise to the strong compartmentalization seen in mature sperm. The X chromosome lacks domain organization during meiotic sex-chromosome inactivation. We propose that male meiosis occurs amid global reprogramming of 3D chromatin organization and that strengthening of chromatin compartmentalization takes place in spermiogenesis to prepare the next generation of life.

Attenuated chromatin compartmentalization in meiosis and its maturation in sperm development.

Gene regulation in the germline ensures the production of high-quality gametes, long-term maintenance of the species and speciation. Male germline transcriptomes undergo dynamic changes after the mitosis-to-meiosis transition and have been subject to evolutionary divergence among mammals. However, the mechanisms underlying germline regulatory divergence remain undetermined. Here, we show that endogenous retroviruses (ERVs) influence species-specific germline transcriptomes. After the mitosis-to-meiosis transition in male mice, specific ERVs function as active enhancers to drive germline genes, including a mouse-specific gene set, and bear binding motifs for critical regulators of spermatogenesis, such as A-MYB. This raises the possibility that a genome-wide transposition of ERVs rewired germline gene expression in a species-specific manner. Of note, independently evolved ERVs are associated with the expression of human-specific germline genes, demonstrating the prevalence of ERV-driven mechanisms in mammals. Together, we propose that ERVs fine-tune species-specific transcriptomes in the mammalian germline.

Endogenous retroviruses drive species-specific germline transcriptomes in mammals.

Owing to bursts in the expression of thousands of germline-specific genes, the testis has the most diverse and complex transcriptome of all organs. By analyzing the male germline of mice, we demonstrate that the genome-wide reorganization of super-enhancers (SEs) drives bursts in germline gene expression after the mitosis-to-meiosis transition. SE reorganization is regulated by two molecular events: the establishment of meiosis-specific SEs via A-MYB (MYBL1), a key transcription factor for germline genes, and the resolution of SEs in mitotically proliferating cells via SCML2, a germline-specific Polycomb protein required for spermatogenesis-specific gene expression. Before entry into meiosis, meiotic SEs are preprogrammed in mitotic spermatogonia to ensure the unidirectional differentiation of spermatogenesis. We identify key regulatory factors for both mitotic and meiotic enhancers, revealing a molecular logic for the concurrent activation of mitotic enhancers and suppression of meiotic enhancers in the somatic and/or mitotic proliferation phases.

Article	Year
SCML2 promotes heterochromatin organization in late spermatogenesis. Journal of cell science\| PubMed ID: 30097555	2018
A rapidly evolved domain, the SCML2 DNA-binding repeats, contributes to chromatin binding of mouse SCML2†. Biology of reproduction\| PubMed ID: 30137219	2019
Attenuated chromatin compartmentalization in meiosis and its maturation in sperm development. Nature structural & molecular biology\| PubMed ID: 30778237	2019
Endogenous retroviruses drive species-specific germline transcriptomes in mammals. Nature structural & molecular biology\| PubMed ID: 32895553	2020
Super-enhancer switching drives a burst in gene expression at the mitosis-to-meiosis transition. Nature structural & molecular biology\| PubMed ID: 32895557	2020

So Maezawa

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