Project description:Spermiogenesis defines the final phase of male germ cell differentiation. Multiple deubiquitinating enzymes have been linked to spermiogenesis, yet the impacts of deubiquitination on spermiogenesis remain poorly characterized. Here, we investigated the function of UAF1 in mouse spermiogenesis and male fertility. We selectively deleted Uaf1 in premeiotic germ cells using Stra8-Cre knock-in mouse strain (Uaf1 sKO), and found that Uaf1 is essential for spermiogenesis and male fertility. We found that UAF1 interacts and colocalizes with USP1 in testes. Conditional knockout of Uaf1 in testes results in disturbed expression and localization of USP1, suggesting that UAF1 regulates spermiogenesis through the function of the deubiquitinating enzyme USP1. We used tandem mass tag-based proteomics to identify differentially expressed proteins and potential underlying mechanisms, and found that conditional knockout Uaf1 in testes results in reduced levels of proteins essential for spermiogenesis. Thus, the UAF1/USP1 deubiquitinase complex is essential for normal spermiogenesis by regulating the levels of spermiogenesis-related proteins.
Project description:To identify RFX2-dependent genes during spermatogenesis, We chose 24-day-old wild-type and mutant testes samples. Spermiogenesis in mutants was normal before this time point while massive round spermatids detached from the tubules thereafter. RNAs were isolated from the total testicular cells.
Project description:We have performed a systems-level analysis of the RFX/Daf-19 family transcription factor, Rfx2. Using a combination of high-throughput sequencing of Rfx2-regulated transcripts and chromosomal binding sites, we provide a comprehensive accounting of the target genes by which Rfx2 controls ciliogenesis and cilia beating in vertebrates.
Project description:We have performed a systems-level analysis of the RFX/Daf-19 family transcription factor, Rfx2. Using a combination of high-throughput sequencing of Rfx2-regulated transcripts and chromosomal binding sites, we provide a comprehensive accounting of the target genes by which Rfx2 controls ciliogenesis and cilia beating in vertebrates. RNA-seq: two biological replicates for control and RFX2 knockdown by morpholino injection, ChIP-seq: RFX2-GFP pulldown with GFP antibody, GFP only expression used as control
Project description:Purpose: This study was carried out to determine the consequences of the Rfx2-/- genotype on spermatogenesis in the mouse Methods: DNA was extracted from decapsulated testes of 21 day old mice. ChIP sequencing was used to determine the binding sites of RFX2. Results: ChIP-Seq analysis identified ~880 binding sites of RFX2 near TSS. Conclusion: Spermatogenesis undergoes complete arrest just prior to the end of the round spermatid period of sperm development in mutant mice. Sequencing results showed that approximately 105 genes were downregulated 2 fold or more in the testes of mutant mice. Comparison of similar studies of targeted mutations in genes for other transcription factor demonstrate that Rfx2 has a large and nearly unique set of genes that depend on it directly or indirectly. A large number of downregulated genes are identified with cilia function.
Project description:Purpose: This study was carried out to determine the consequences of the Rfx2-/- genotype on spermatogenesis in the mouse Methods: DNA was extracted from decapsulated testes of 21 day old mice. ChIP sequencing was used to determine the binding sites of RFX2. Results: ChIP-Seq analysis identified ~880 binding sites of RFX2 near TSS. Conclusion: Spermatogenesis undergoes complete arrest just prior to the end of the round spermatid period of sperm development in mutant mice. Sequencing results showed that approximately 105 genes were downregulated 2 fold or more in the testes of mutant mice. Comparison of similar studies of targeted mutations in genes for other transcription factor demonstrate that Rfx2 has a large and nearly unique set of genes that depend on it directly or indirectly. A large number of downregulated genes are identified with cilia function. Testicular RFX2 binding sites were determined by deep sequencing using testes from 2 independentWT mice
Project description:The acetylation levels of histones and other proteins change during aging and have been linked to neurodegeneration. Here we show that deletion of the histone acetyltransferase (HAT) co-factor Trrap specifically impairs the function of the transcription factor Sp1, reduces its stability and causes a decrease in histone acetylation at Sp1 target genes. Modulation of Sp1 function by Trrap acts as a hub regulating multiple processes involved in neuron and neural stem cells function and maintenance including microtubule dynamics and the Wnt signaling pathway. Consistently, Trrap conditional mutants exhibit all hallmarks of neurodegeneration including dendrite retraction and axonal swellings, neuron death, astrogliosis, microglia activation, demyelination and decreased adult neurogenesis. Our results uncovered a novel functional network, essential to prevent neurodegeneration, and involving the specific regulation of Sp1 transcription factor and its downstream targets by Trrap-HAT.
Project description:Purpose: This study was carried out to determine the consequences of the Rfx2-/- genotype on spermatogenesis in the mouse Methods: RNA was extracted from decapsulated testes of 21 day old mixed background mice of either genotype. Deep sequencing was used to determine quantitative expression of the genomes from independent replicates of each genotype Results: RNA-Seq analysis identified some 105 genes that are down regulated at least 2-fold in Rfx2-/- testes, with ~50 being reduced at least 10-fold Conclusion: Spermatogenesis undergoes complete arrest just prior to the end of the round spermatid period of sperm development in mutant mice. Sequencing results showed that approximately 105 genes were downregulated 2 fold or more in the testes of mutant mice. Comparison of similar studies of targeted mutations in genes for other transcription factor demonstrate that Rfx2 has a large and nearly unique set of genes that depend on it directly or indirectly. A large number of downregulated genes are identified with cilia function. Testicular mRNA profiles were determined by deep sequencing using testes from 5 independent wild type and 6 independent Rfx2-/- mice