Project description:We report ATAC-seq analysis throughout spermatogonial development. We have assayed two biological replicates at 0, 18, 48, and 120 hours post WIN 18,446/RA treatment.
Project description:Total RNA from in vitro cultured murine undifferentiated and differentiating spermatogonia (by retinoic acid induction) were collected for RNA-seq
Project description:Chromatin modifications instruct genome function through spatiotemporal recruitment of regulatory factors to the genome. However, how these modifications define the proteome composition at distinct chromatin states remains to be fully characterized. Here, we made use of natural protein domains as modular building blocks to develop engineered chromatin readers (eCRs) selective for histone and DNA modifications. By stably expressing eCRs in mouse embryonic stem cells and measuring their subnuclear localisation, genomicdistribution and histone-PTM-binding preference, we first demonstrate their applicability as selective chromatin binders in living cells. Finally, we exploit the binding specificity of eCRs to establish ChromID, a new method for chromatin-dependent proteome identification based on proximity biotinylation. We use ChromID to reveal the proteome at distinct chromatin states in mouse stem cells, and by using a synthetic dual-modification reader, we furthermore uncover the protein composition at bivalent promoters marked by H3K4me3 and H3K27me3. These results highlight the applicability of ChromID as novel method to obtaina detailed view of the protein interaction network determined by the chemical language on chromatin.
Project description:Microarray analysis was performed in order to detail the gene expression profiles in murine b-2M-SPa-6+c-kit-undifferentiated and b-2M-SPa-6+c-kit+ differentiating spermatogonia. These data were used to compare human and mouse transcriptomes of undifferentiated spermatogonia.
Project description:To understand the functional role of CHD4, a member of the Nucleosome Remodeling and Deacetylase (NuRD) complex, in establishing chromatin states in the formation and maintenance of ovarian reserve and the maintenance of female and male germ cells, we eliminated CHD4 function in germ cells using Ddx4-Cre and Gdf9-iCre to generate CHD4 conditional knockout (Chd4 DcKO and GcKO) mice. We discover that CHD4 defines the chromatin state that ensures germ cell survival, thereby enabling the long-term maintenance of female and male germ cells. We then performed ATAC-seq in Chd4 Dctrl and Chd4 DcKO non-growing oocytes and undifferentiated spermatogonia.
Project description:Mammalian spermatogenesis is regulated by epigenetic mechanisms that maintain cell type-specific transcriptional programs. The epigenetic regulator BMI1, a PRC1 member, is required for maintaining undifferentiated spermatogonia, but the underlying mechanisms remain unclear. To address this issue, here we performed chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq).
Project description:Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) has been instrumental to our current view of chromatin structure and function. It allows genome-wide mapping of histone marks, which demarcate biologically relevant domains. However, ChIP-seq is an ensemble measurement reporting the average occupancy of individual marks in a cell population. Consequently, our understanding of the combinatorial nature of chromatin states relies almost exclusively on correlation between the genomic distributions of individual marks. Here, we report the development of Combinatorial-iChIP to determine the genome-wide co-occurrence of histone marks at single nucleosome resolution. By comparing to null model, we show that certain combinations of overlapping marks (H3K36me3 and H3K79me3) co-occur more frequently than expected by chance, while others (H3K4me3 and H3K36me3) do not, reflecting differences in the underlying chromatin pathways. We further use combinatorial-iChIP to illuminate aspects of the Set2-RPD3S pathway. This approach promises to improve our understanding of the combinatorial complexity of chromatin. Combinatorial iChIP in yeast.