Project description:To investigate transcriptional changes in developing mouse testicular germ cells, cells were isolated via FACS or centrifugal elutriation Isolated cells were subjected to native ChIP-Seq with an antibody against H3K4me3
Project description:To investigate the complexity of RNA present in different stage germ cells from adult mice, we isolated cells via FACS We then performed paired end RNA-Seq to characterize the expression state of each cell type
Project description:Male germ cells establish a unique heterochromatin domain, the XY-body, early in meiosis. How this domain is maintained through the end of meiosis and into post-meiotic germ cell differentiation is poorly understood. ADAD2 is a late meiotic male germ cell specific RNA binding protein, loss of which leads to post-meiotic germ cell defects. Analysis of ribosome association in Adad2 mutants revealed defective translation of Mdc1, a key regulator of XY-body formation, late in meiosis. As a result, Adad2 mutants show normal establishment but failed maintenance of the XY-body. Observed XY-body defects are concurrent with abnormal autosomal heterochromatin and ultimately lead to severely perturbed post-meiotic germ cell heterochromatin and cell death. These findings highlight the requirement of ADAD2 for Mdc1 translation, the role of MDC1 in maintaining meiotic male germ cell heterochromatin, and the importance of late meiotic heterochromatin for normal post-meiotic germ cell differentiation.
Project description:Spermatogenesis is a complex process, dependent upon the successive activation and/or repression of thousands of gene products, and ends with the production of haploid male gametes. RNA sequencing of male germ cells in the rat identified thousands of novel testicular unannotated transcripts, named TUTs. Although such RNAs are usually annotated as long non-coding RNAs, it is possible that some of these TUTs code for protein. To test this possibility, we used a “Proteomics Informed by Transcriptomics” strategy, combining shotgun proteomics analyses and RNA sequencing data for enriched populations of rat testicular cells. Among 3559 TUTs and 506 lncRNAs found in meiotic and post-meiotic germ cells, 44 encoded at least one peptide. We show that these novel high-confidence protein-coding loci exhibit several genomic features intermediate between those of lncRNAs and mRNAs. We experimentally validated the testicular expression pattern of two of these novel protein-coding gene candidates, both highly conserved in mammals: one for a vesicle-associated membrane protein, we named VAMP-9, and the other for an enolase domain-containing protein. This study confirms the potential of PIT approaches for the discovery of protein coding transcripts initially thought to be untranslated, or unknown transcripts. Our results contribute to the understanding of spermatogenesis by characterizing two novel proteins, implicated by their strong expression in germ cells.