Project description:Insight into mechanisms controlling gene expression in the spermatogonial stem cell (SSC) will improve our understanding of the processes regulating spermatogenesis and aid in treating problems associated with male infertility. In this study we explored the global gene expression profiles of glial cell line-derived neurotrophic factor (GDNF) regulated transcription factors, Ets variant gene 5 (Etv5), B-cell CLL/lymphoma 6, member B (Bcl6b) and POU domain, class-3 transcription factor-1 (Pou3f1). We reasoned that these three factors may function as a core-set of transcription factors, regulating genes responsible for maintaining the SSC population. Using transient short-interfering RNA oligonucleotides (siRNA) to individually target Etv5, Bcl6b and Pou3f1 within mouse SSC cultures, we examined changes to the global gene expression profiles associated with these transcription factors. While there were only modest overlaps in the target genes regulated by the three factors, ETV5 was found to be a critical downstream regulator of GDNF signaling that mediated the expression of several known SSC self-renewal related genes including, Bcl6b and LIM homeobox 1 (Lhx1). Notably, ETV5 was identified as a regulator of Brachyury and CXC chemokine Receptor, type 4 (Cxcr4), and we show that ETV5 binding to the Brachyury gene promoter region is associated with an active state of transcription. Moreover, in vivo transplantation of SSCs following silencing of Brachyury significantly reduced the number of donor cell-derived colonies formed within recipient mouse testes. These results suggest Brachury is of biological importance, and functions as part of GDNF/ETV5 signaling to promote self-renewal of mouse SSCs cultured in vitro. Microarray gene expression analysis was conducted with Affymetrix Mouse 430 2.0 GeneChips (Affymetrix Inc.).Following with gene knockdown, total RNA from spermatogonial stem cells was converted to cDNA. There are total 16 samples (Four groups and four samples per group) Negative control (N), Bcl6b Knockdown (B), Etv5 knockdown (E), and Pou3f1 knockdown (O), respectively.
Project description:It is known that ubiquitination is important for T cell receptor (TCR) signaling during T cell activation but the breadth of ubiquitination events triggered during TCR signaling is not completely understood. This dataset utilizes di-glycine remnant profiling combined with mass spectrometry to identify a global landscape of ubiquitination events downstream of the TCR and to quantify changes ubiquitin abundance in response to TCR stimulation. Additionally, whole cell proteomics data were generated to measure protein abundances during TCR stimulation. Mouse primary T cells were isolated, proliferated and either remained resting or stimulated with CD3/CD28 to activate downstream signaling through the TCR and co-stimulatory pathways. Di-glycine remnant profiling and whole cell proteomics was performed on rested cells and cells that had undergone CD3/CD28 TCR stimulation for 4 hours. These data were analyzed to identify the ubiquitination events during TCR activation and to quantify the change in peptide-based ubiquitin abundance and total protein abundance over the course of the 4 hour TCR stimulation. Integration of di-glycine and whole cell proteomics was used to generate protein-specific predictions of whether ubiquitination events downstream of TCR signaling lead to a decrease in associated protein abundance. The analysis of these data suggests that T cell activation leads to an increase in ubiquitination that is not associated with proteasomal or lysosomal degradation.
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.