Project description:ZFP541 is one of the candidates for a transcriptional regulator during mouse meiotic prophase. In order to address the role of ZFP541, transcriptomes of meiotic prophase-enriched population were compared between Zfp541 +/- and Zfp541 KO testes by RNA-seq. The meiotic prophase-enriched population was isolated from Zfp541 +/- and Zfp541 KO testes at postnatal day 18 (P18), on the Rec8-3FH-GFP KI background by fluorescent sorting of GFP positive cells.
Project description:ZFP541 is assumed to possess putative DNA-binding domains and associated with histone deacetylases, implying it plays a role in regulation of transcription via modulation of chromatin status. Therefore, we used ChIP-seq to search for targets of ZFP541 in meiotic prophase of mouse spermatocytes.
Project description:5'-hydroxymethylcytosine (5hmC), an important 5'-cytosine modification, is altered highly in order in male meiotic prophase. However, the regulatory mechanism of this dynamic change and the function of 5hmC in meiosis remain largely unknown. Using a knockout mouse model, we showed that UHRF1 regulated male meiosis. UHRF1 deficiency led to failure of meiosis and male infertility. Mechanistically, the deficiency of UHRF1 altered significantly the meiotic gene profile of spermatocytes. Uhrf1 knockout induced an increase of the global 5hmC level. The enrichment of hyper-5hmC at transcriptional start sites (TSSs) was highly associated with gene downregulation. In addition, the elevated level of the TET1 enzyme might have contributed to the higher 5hmC level in the Uhrf1 knockout spermatocytes. Finally, we reported Uhrf1, a key gene in male meiosis, repressed hyper-5hmC by downregulating TET1. Furthermore, UHRF1 facilitated RNA polymerase II (RNA-pol2) loading to promote gene transcription. Thus our study demonstrated a potential regulatory mechanism of 5hmC dynamic change and its involvement in epigenetic regulation in male meiosis.
Project description:MicroRNAs (miRNAs) are a class of endogenous, non-coding RNAs that mediate post-transcriptional gene silencing by inhibiting mRNA translation and promoting mRNA decay. DICER1, an RNAse III endonuclease encoded by Dicer1, is required for processing short 21-22 nucleotide miRNAs from longer double-stranded RNA precursors. Here, we investigate the loss of Dicer1 in mouse postnatal male germ cells to determine how disruptions in the miRNA biogenesis pathway may contribute to infertility. Reduced levels of Dicer1 transcripts and DICER1 were confirmed in germ cell knock-out (GCKO) testes by postnatal day 18 (P18). Compared to wild-type (WT) at 8 weeks, GCKO males had no change in body weight, yet showed significant reductions in testis mass and sperm number. Histology and fertility tests confirmed spermatogenic failure in GCKO males. Array analyses at P18 showed 96% of miRNA genes were down-regulated and 37% of protein-coding genes were differentially expressed in GCKO testes. Interestingly, we observed preferential overexpression of genes on the sex chromosomes in GCKO testes, with more than 80% of the genes overlapping those proposed to undergo meiotic sex chromosome inactivation (MSCI) in the germ cells. Compared to WT, GCKO mice showed higher percentages of cells at early meiotic stages (leptotene and zygotene) but lower percentages at later stages (pachytene, diplotene and metaphase I), providing evidence that deletion of Dicer1 leads to disruptions in meiotic progression. Furthermore, we observed fewer elongating spermatids with proper translational activation of transition protein 2 (Tnp2), protamine 1 and 2 (Prm1 and Prm2) in GCKO testes after step 12-14. Therefore, deleting Dicer1 in early postnatal germ cells causes misregulation of transcripts encoded by genes on the sex chromosomes, impairs meiotic progression and post-meiotic translational control and results in spermatogenic failure and infertility. Total RNA, including miRNAs, were purified from a total of six individual mouse samples. The tissue collected was obtained from wild-type (control; n=3) and Dicer1 germ cell knockout (mutant; n=3) testes on P18.
