Project description:Spermatogenic regeneration is key for male fertility and relies on activities of an undifferentiated spermatogonial population. Here, a high-throughput approach with primary cultures of mouse spermatogonia was devised to rapidly predict alterations in functional capacity. Combining the platform with a large-scale RNAi screen of transcription factors, we generated a repository of new information from which pathway analysis was able to predict candidate molecular networks regulating regenerative functions. Extending from this database, the SRCAP-CREBBP/EP300 (Snf2-related CREBBP activator protein-CREB binding protein/E1A binding protein P300) complex was found to mediate differential levels of histone acetylation between stem cell and progenitor spermatogonia to influence expression of key self-renewal genes including the previously undescribed testis-specific transcription factor ZSCAN2 (zinc finger and SCAN domain containing 2). Single cell RNA sequencing analysis revealed that ZSCAN2 deficiency alters key cellular processes in undifferentiated spermatogonia such as translation, chromatin modification, and ubiquitination. In Zscan2 knockout mice, while spermatogenesis was moderately impacted during steady state, regeneration after cytotoxic insult was significantly impaired. Altogether, these findings have validated the utility of our high-throughput screening approach and have generated a transcription factor database that can be utilized for uncovering novel mechanisms governing spermatogonial functions.
Project description:To understand the role of the transcription factor ZSCAN2 in regulating spermatogonial stem cell function, we performed single cell RNA-sequencing on testis cells enriched for spermatogonia, comparing control populations with populations from a CRISPR-generated Zscan2-knockout mouse line. Transcriptome profiling revealed that ZSCAN2 deficiency alters key cellular processes in undifferentiated spermatogonia such as translation, chromatin modification, and ubiquitination. These findings are cohesive with the decline in regenerative capacity that is experienced by spermatogonial stem cells in the Zscan2-knockout mouse testis following exposure to clastogens.
Project description:Docetaxel chemotherapy in metastatic prostate cancer offers only a modest survival benefit due to emerging resistance. To identify candidate therapeutic gene targets, we applied a murine prostate cancer orthograft model that recapitulates clinical invasive prostate cancer in a genome-wide CRISPR/Cas9 screen under docetaxel treatment pressure.
Project description:Purpose: Many young adults are in a state of stress due to social and psychological pressures, which may result in male reproductive dysfunction. To provide new insight into this phenomenon, we investigated the relationship between pathological changes in rat spermatogenic cells and the expression of genes specific to spermatogenic cell types under different stress conditions. Methods: After establishing rat stress models of different time durations, we observed pathological changes in testicular tissues through haematoxylin and eosin staining, and analysed gene expression in spermatogenic cells by RNA-seq, bioinformatic analysis, and reverse transcription qPCR (RT-qPCR).Three testicular samples were taken from each group to construct 12 cDNA libraries. For each sample, 3 μg RNA was used as the starting material. Ribosomal RNA was removed using the Epicentre Ribo-Zero™ Gold kit (Rat) (Epicentre, an Illumina company, Madison, WI, USA). Results:Compared with the control group, there were 1,194 DEGs in the 3-day RS+IS group , including 455 upregulated genes and 739 downregulated genes , 1,774 DEGs in the 14-day RS+IS group including 1,124 upregulated genes and 650 downregulated genes, and 2,267 DEGs in the 21-day RS+IS group including 1,366 upregulated genes and 901 downregulated genes. Mean-while, some same expression patterns were observed in three phenotypes.After comparison with single-cell sequencing data, 349 DEGs of spermatogenic cells at different developmental stages were found. Conclusions: Our study suggest that chronic psychosomatic stress can affect the spermatogenic transcriptome of the testes, leading to a significant change in the expression of the spermatogenic genes. At the same time, histopathological and immunohistochemical results showed that chronic stress may lead to pathological changes in spermatogenic cells and to a significant decrease in key regulatory proteins
Project description:This study aims to identify novel candidate variants from human Y-chromosomal genes DAZ, BPY2 and CDY1 by resequencing the coding regions of these genes from male patients with spermatogenic impairment.
The coding regions of the genes have been amplified by standard PCR, amplicon lengths range from 244 to 486 bp. A total of 61 amplicons were amplified for each of the 96 patients, totalling to approx. 25 kb per sample. Amplicons were quantified by gel electrophoresis and pooled in approx. equimolar concentrations per patient. For each of the 96 submitted samples, approx. 1 microgram of amplified DNA pool is provided in a total volume of 120 microlitres.
The samples need to be indexed and libraries prepared for a PE250bp Illumina MiSeq run.
This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Project description:Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disease. Approximately 20 percent of familial ALS cases are caused by mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Rodents expressing mutant SOD1 transgenes develop progressive, fatal motor neuron disease and disease onset and progression is dependent on the level of SOD1. We investigated the possibility that a reduction in SOD1 protein may be of therapeutic benefit in ALS and screened 30,000 compounds for inhibition of SOD1 transcription. The most effective inhibitor identified was N-{4-[4-(4-methylbenzoyl)-1-piperazinyl]phenyl}-2-thiophenecarboxamide (Compound ID 7687685), which in PC12 cells showed an EC50 of 10.6 microM for inhibition of SOD1 expression and an LD50 more than 30 microM. This compound was subsequently shown to reduce endogenous SOD1 levels in HeLa cells and to exhibit a modest reduction of SOD1 protein levels in mouse spinal cord tissue. These data suggest that the efficacy of compound 7687685 as an inhibitor of SOD1 gene expression is not likely to be clinically useful, although the strategy reported could be applied broadly to screening for small molecule inhibitors of gene expression.
Project description:We report genome-wide distribution of Linker histone variant H1t in mouse spermatogenic cells. We found that H1t was mainly located at around transcriptional start site of genic area, positively correlated with the gene expression. This study using ChIP-seq analysis provides genomic distribution of H1t in mouse spermatogenic cells.
Project description:Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are zoonotic pathogens that can cause severe respiratory disease in humans. Identification of the host factors that are necessary for viral infection and virus-induced cell death is critical to our understanding of the viral life cycle and can potentially aid the development of new treatment options. Here, we report CRISPR screen results of both SARS-CoV and MERS-CoV infections in derivatives of the human hepatoma cell line Huh7. Our screens identified the known entry receptors ACE2 for SARS-CoV and DPP4 for MERS-CoV. Additionally, the SARS-CoV screen uncovered several components of the NF-κB signaling pathway (CARD10, BCL10, MALT1, MAP3K7, IKBKG), while the MERS-CoV screen revealed the polypyrimidine tract-binding protein PTBP1, the ER scramblase TMEM41B, furin protease and several transcriptional and chromatin regulators as candidate factors for viral replication and/or virus-induced cell death. Together, we present several known and unknown coronavirus host factors that are of interest for further investigation.