ZBTB16/PLZF regulates self-renewal and differentiation of spermatogonial stem cells through an extensive transcription factor-chromatin poising network [RNA-Seq]
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ABSTRACT: Spermatogonial stem cells (SSCs) balance self-renewal versus differentiation/spermatogenesis to ensure continuous sperm production. Here, we uncover multiple roles for the transcription factor ZBTB16/PLZF in juvenile mouse undifferentiated spermatogonia (uSPG). ZBTB16 activates genes in uSPG promoting self-renewal and cell cycle progression (Ccnd1) to maintain uSPG and transit-amplifying states. Remarkably, in uSPG, ZBTB16, SALL4, SOX3 all co-localize at over 12,000 promoters regulating uSPG and meiosis. These regions also feature broad H3K4me3 and H3K27ac marks, DNA hypomethylation, and often CTCF binding. Hi-C analyses reveal robust promoter-promoter physical interactions, revealing a transcription factor and higher-order active chromatin interaction network within uSPG that poises meiotic promoters for subsequent activation. Conversely, these factors do not occupy germline-specific promoters driving spermiogenesis, which instead lack promoter-promoter physical interactions and bear DNA hypermethylation. Therefore, ZBTB16 ensures uSPG cell cycle progression and colocalizes with SALL4, SOX3 and often CTCF to establish a novel chromatin poising network.Spermatogonial stem cells (SSCs) balance self-renewal and differentiation to ensure continuous sperm production in the testis. The transcription factor Zbtb16 (PLZF) supports undifferentiated SSC maintenance through partly unknown mechanisms. We combined genomics (RNA-seq and ChIP-seq) and genetic approaches to reveal multiple functions of Zbtb16 in juvenile mouse SSCs. Zbtb16-bound loci show a striking correlation with active promoters bearing H3K4me3 and the activator Sall4. Zbtb16 activates genes that support SSC self-renewal and cell cycle progression (e.g., Ccnd1) that help maintain undifferentiated SSC pools, including both self-renewing SSCs and transit-amplifying progenitors. Zbtb16 also attenuates certain genes, including meiotic genes and specific retrotransposons that confer genome instability. Notably, Zbtb16 genome localization and its impact on the transcriptome are dynamic, displaying mesenchymal gene targets in vivo, which are not maintained in cultured SSCs. Our data reveal dynamic roles for Zbtb16 in ensuring SSC identity, amplification, and maintenance in vivo.
Project description:Spermatogonial stem cells (SSCs) balance self-renewal versus differentiation/spermatogenesis to ensure continuous sperm production. Here, we uncover multiple roles for the transcription factor ZBTB16/PLZF in juvenile mouse undifferentiated spermatogonia (uSPG). ZBTB16 activates genes in uSPG promoting self-renewal and cell cycle progression to maintain uSPG and transit-amplifying states. Remarkably, in uSPG, ZBTB16, SALL4, SOX3 all co-localize at over 12,000 promoters regulating uSPG and meiosis. These regions feature broad H3K4me3 and H3K27ac, DNA hypomethylation, RNAPol2 and often CTCF. Hi-C analyses reveal robust 3D physical interactions among these co-bound promoters, revealing a transcription factor and higher-order active chromatin interaction network within uSPG that poises meiotic promoters for subsequent activation. Conversely, these factors do not occupy germline-specific promoters driving spermiogenesis, which instead lack promoter-promoter physical interactions and bear DNA hypermethylation, even when active. Overall, ZBTB16 promotes uSPG cell cycle progression and colocalizes with SALL4, SOX3, CTCF and RNAPol2 to establish a novel and extensive chromatin poising network.
Project description:Spermatogonial stem cells (SSCs) balance self-renewal versus differentiation/spermatogenesis to ensure continuous sperm production. Here, we uncover multiple roles for the transcription factor ZBTB16/PLZF in juvenile mouse undifferentiated spermatogonia (uSPG). ZBTB16 activates genes in uSPG promoting self-renewal and cell cycle progression (Ccnd1) to maintain uSPG and transit-amplifying states. Remarkably, in uSPG, ZBTB16, SALL4, SOX3 all co-localize at over 12,000 promoters regulating uSPG and meiosis. These regions also feature broad H3K4me3 and H3K27ac marks, DNA hypomethylation, and often CTCF binding. Hi-C analyses reveal robust promoter-promoter physical interactions, revealing a transcription factor and higher-order active chromatin interaction network within uSPG that poises meiotic promoters for subsequent activation. Conversely, these factors do not occupy germline-specific promoters driving spermiogenesis, which instead lack promoter-promoter physical interactions and bear DNA hypermethylation. Therefore, ZBTB16 ensures uSPG cell cycle progression and colocalizes with SALL4, SOX3 and often CTCF to establish a novel chromatin poising network.
Project description:Spermatogonial stem cells balance self-renewal and differentiation/spermatogenesis to ensure continuous sperm production. Here, we identify roles for the transcription factor ZBTB16/PLZF in juvenile mouse undifferentiated spermatogonia (uSPG) in promoting self-renewal and cell cycle progression to maintain uSPG and transit-amplifying states. Notably, ZBTB16, SALL4, SOX3 co-localize at over 12,000 promoters regulating uSPG and meiosis. These regions largely share broad H3K4me3 and H3K27ac, DNA hypomethylation, RNAPol2 and often CTCF. Hi-C analyses show robust 3D physical interactions among these co-bound promoters, suggesting the existence of a transcription factor and higher-order active chromatin interaction network within uSPG that poises meiotic promoters for subsequent activation. Conversely, these factors do not notably occupy germline-specific promoters driving spermiogenesis, which instead lack promoter-promoter physical interactions and bear DNA hypermethylation, even when active. Overall, ZBTB16 promotes uSPG cell cycle progression and colocalizes with SALL4, SOX3, CTCF and RNAPol2 to help establish an extensive and interactive chromatin poising network.
Project description:Maintenance and self-renewal of the spermatogonial stem cell (SSC) population is the cornerstone of male fertility. In this manuscript we have identified a key role for the nucleosome remodelling protein Chromodomain Helicase DNA binding protein 4 (CHD4) in regulating SSC function. Gene expression analyses revealed that CHD4 expression is largely restricted to spermatogonia in the mouse testis, and is particularly enriched in SSCs. Using spermatogonial transplantation techniques and RNAi mediated knockdown it was established that loss of Chd4 expression significantly impairs SSC regenerative capacity, resulting in a ~50% reduction in colonisation of recipient testes. A single cell RNA-seq comparison depicted reduced expression of ‘self-renewal’ genes such as Gfra1 and Pten following Chd4 knockdown, along with increased expression of signature progenitor genes, Neurog3 and Dazl. Co-immunoprecipitation analyses demonstrated that CHD4 regulates gene expression in spermatogonia not only though its traditional association with the remodelling complex NuRD, but also via interaction with the GDNF-responsive transcription factor SALL4. Cumulatively, the results of this study depict a previously unappreciated fundamental role for CHD4 in controlling fate decisions in the spermatogonial pool.
Project description:The spermatogonial stem cells (SSCs) niche is critical for SSC maintenance and the subsequent spermatogenesis. Numerous reproductive hazards impair the SSC niche, thereby result in aberrant SSC self-renewal and male infertility. However, promising agents targeting the impaired SSC niche to promote SSC self-renewal are still limited. Here, we screen out and assess the effects of Lovastatin on the self-renewal of mouse spermatogonial stem cells (mSSCs). Mechanistically, Lovastatin promotes the self-renewal of mSSCs and inhibits its inflammation and apoptosis through the regulation of isoprenoid intermediates. Likewise, other statins exhibit similar effects on SSC self-renewal. Remarkably, the treatment by Lovastatin could promote the self-renewal of mSSCs in the male gonadotoxicity model generated by busulfan injection. Noteworthy, we demonstrate that Lovastatin could significantly enhance the self-renewal of in vitro cultured primate SSCs. Collectively, our findings uncover that lovastatin could promote the self-renewal of both murine and primate SSCs and have implications for the treatment of certain male infertility using small compounds.
Project description:To better understand human spermatogonial stem cells (SSCs), we profiled their transciptome and epigenome, which revealed the mechanism how human SSCs regulates their self-renewal versus differentiation dermination, as well as how latent pluripotency is established in human SSCs. Remarkly, we discovered signaling pathways (e.g. LIF, BMP, WNT) that differentially regulated self-renewal vesus differentiation in SSCs. We also discovered that SSCs repress core pluripotent factors (Sox2, Pou5f1 and Nanog) yet activate ancillary factors (e.g. Klf4, Mbd3, Tcf3, Sall4) transcriptionally and epigenetically.
Project description:To better understand human spermatogonial stem cells (SSCs), we profiled their transciptome and epigenome, which revealed the mechanism how human SSCs regulates their self-renewal versus differentiation dermination, as well as how latent pluripotency is established in human SSCs. Remarkly, we discovered signaling pathways (e.g. LIF, BMP, WNT) that differentially regulated self-renewal vesus differentiation in SSCs. We also discovered that SSCs repress core pluripotent factors (Sox2, Pou5f1 and Nanog) yet activate ancillary factors (e.g. Klf4, Mbd3, Tcf3, Sall4) transcriptionally and epigenetically.
Project description:To better understand human spermatogonial stem cells (SSCs), we profiled their transciptome and epigenome, which revealed the mechanism how human SSCs regulates their self-renewal versus differentiation dermination, as well as how latent pluripotency is established in human SSCs. Remarkly, we discovered signaling pathways (e.g. LIF, BMP, WNT) that differentially regulated self-renewal vesus differentiation in SSCs. We also discovered that SSCs repress core pluripotent factors (Sox2, Pou5f1 and Nanog) yet activate ancillary factors (e.g. Klf4, Mbd3, Tcf3, Sall4) transcriptionally and epigenetically.
Project description:To better understand human spermatogonial stem cells (SSCs), we profiled their transciptome and epigenome, which revealed the mechanism how human SSCs regulates their self-renewal versus differentiation dermination, as well as how latent pluripotency is established in human SSCs. Remarkly, we discovered signaling pathways (e.g. LIF, BMP, WNT) that differentially regulated self-renewal vesus differentiation in SSCs. We also discovered that SSCs repress core pluripotent factors (Sox2, Pou5f1 and Nanog) yet activate ancillary factors (e.g. Klf4, Mbd3, Tcf3, Sall4) transcriptionally and epigenetically.
Project description:Spermatogonial stem cells (SSCs) are essential for the generation of sperm and have potential therapeutic value for male infertility, which afflicts >100 million men world-wide. To devise SSC therapy approaches, it is critical to first develop methods to culture human SSCs in vitro. Here, we report on a transcriptome approach to address this question. Using single-cell RNA-seq (scRNAseq), immunofluorescence, and germ-cell xenograft transplantation analyses, we identified a cell-surface protein, PLPPR3, that purifies human primitive undifferentiated spermatogonia (uSPG) enriched for SSCs. Comparative RNAseq analysis of PLPPR3+ cells (primitive uSPG) with KIT+ cells (enriched for differentiating [d] SPG) revealed that these two stages differentially express a remarkably large number of genes, including genes encoding key components in the TGF, GDNF, AKT, and JAK-STAT signaling pathways. Using scRNAseq analysis and conventional approaches, we tested the effect of manipulating these signaling pathways on cultured human SPG. This revealed that GDNF and BMP8B broadly support the culture of SPG, Activin A supports more advanced SPG, and one condition—AKT pathway inhibition—had the unique ability to selectively support primitive uSPG. These findings have implications for methods to culture and expand human SSCs for therapeutic uses in the future.