Project description:Sustained spermatogenesis in adult males and recovery of fertility following germ cell depletion are dependent on undifferentiated spermatogonia with self-renewal potential. We have previously demonstrated a critical role for the transcription factor Spalt-like 4 (SALL4) in spermatogonial differentiation. However, it remains unclear whether SALL4 has broader roles within the spermatogonial pool despite its ability to co-regulate genes with PLZF, a transcription factor required for undifferentiated cell maintenance. To identify genes regulated by SALL4 in the male germline, we established cultures of undifferentiated spermatogonia from a Sall4 inducible knockout mouse model. Cells were treated with vehicle (as control) or tamoxifen to induce gene deletion, then cells harvested and analysed by microarray to identify genes mis-expressed upon loss of SALL4.
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.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:Introgressed variants from other species can be an important source of genetic variation because they may arise rapidly, can include multiple mutations on a single haplotype, and have often been pretested by selection in the species of origin. Although introgressed alleles are generally deleterious, several studies have reported introgression as the source of adaptive alleles-including the rodenticide-resistant variant of Vkorc1 that introgressed from Mus spretus into European populations of Mus musculus domesticus. Here, we conducted bidirectional genome scans to characterize introgressed regions into one wild population of M. spretus from Spain and three wild populations of M. m. domesticus from France, Germany, and Iran. Despite the fact that these species show considerable intrinsic postzygotic reproductive isolation, introgression was observed in all individuals, including in the M. musculus reference genome (GRCm38). Mus spretus individuals had a greater proportion of introgression compared with M. m. domesticus, and within M. m. domesticus, the proportion of introgression decreased with geographic distance from the area of sympatry. Introgression was observed on all autosomes for both species, but not on the X-chromosome in M. m. domesticus, consistent with known X-linked hybrid sterility and inviability genes that have been mapped to the M. spretus X-chromosome. Tract lengths were generally short with a few outliers of up to 2.7 Mb. Interestingly, the longest introgressed tracts were in olfactory receptor regions, and introgressed tracts were significantly enriched for olfactory receptor genes in both species, suggesting that introgression may be a source of functional novelty even between species with high barriers to gene flow.
Project description:DDX5, or PLZF co-immunoprecipitation in lysates from cultured undifferentiated spermatogonia followed by identification of eluted proteins using mass spectrometry. IgG control IPs included.
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.