Functinal Study Of Polycomb Group Protein PCGF6in Mouse Spermatogenesis And Long Non-coding RNA Expression Profiling Of Mouse Testis During Postnatal Development

In recent years, an increasing number of studies have demonstratedthat epigenetic mechanisms play important roles in development anddisease. Thus, epigenetic mechanism has gradually become an importantresearch aspect in the research field of molecular mechanisms onspermatogenesis.Polycomb group RING fingers (PCGFs) as core component of keyepigenetic regulatory complex PRC1extensively involves in varousindividual developmental processes via transcriptional repression ofrelated target genes mediated by PRC1. In this study we foucsd ourattention on the PCGF6due to its extremly high-level expression in testis.The expression analysis and functional characterization of PCGF6wereconducted. We found that the expression of both mRNA and protein levelof PCGF6in testes was low at early postnatal development (6dpp and12dpp), whereas it rose sharply in18dpp testes and it maintained at a higherlevel in adult testes. We further found that PCGF6was highly expressed in pachytene spermatocytes and elongated spermatids. Knockdown ofPCGF6promoted the proliferation of an immortalized spermatogeniccells GC-2spd by affecting cell cycle progression and could affect themRNA level of cell cycle regulatory genes in GC-2spd cells. We furtherdemonstrated that knockdown of PCGF6could reduced the proportion ofhaploid GC-2spd cells in whole cell populations after inducingdifferentiation at low temperature and mRNA expression level of markergenes for haploid male germ cell were declined in PCGF6knockdownGC-2spd cells. As expected we found that the! level of H2Aubiquitination was reduced in PCGF6knockdown GC-2spd cells,whereas the!level of H4acetylation and H3K4me3was increased. Withrespect to molecular mechanism, we identified a novel partner of PCGF6,HSPA2, a key gene required for normal meiosis in the male germ cell,could form complex with PCGF6but do not interact directly with eachother. Additionally, we revealed that PLZF and OVOL1, two keytranscription factors in spermatogenesis, could negtively and positively!modulate PCGF6promoter activity, respectively.Recent years, growing evidence suggests that long noncoding RNAs(lncRNAs) as a novel class of epigenetic regulator play important roles indevelopment and disease. However, the expression of lncRNAs and theirbiological functions in postnatal testis development remain unknownTo explore the global expression status of lncRNAs in! postnatal development of mouse testis we employed microarray technology toexamine lncRNA expression profiles of neonatal (6-day-old) and adult(8-week-old) mouse testes. We found that8265lncRNAs were expressedabove background levels during post-natal testis development, of which3025were differentially expressed. Candidate lncRNAs were identifiedfor further characterization by an integrated examination of genomiccontext, gene ontology (GO) enrichment of their associatedprotein-coding genes, promoter analysis for epigenetic modification, andevolutionary conservation of elements. Many lncRNAs overlapped orwere adjacent to key transcription factors and other genes involved inspermatogenesis, such as Ovol1, Ovol2, Lhx1, Sox3, Sox9, Plzf, c-Kit,Wt1, Sycp2, Prm1and Prm2. Most differentially expressed lncRNAsexhibited epigenetic modification marks similar to protein-coding genesand tend to be expressed in a tissue-specific manner. In addition, themajority of differentially expressed lncRNAs harbored evolutionaryconserved elements.Taken together, this work presents the first systematic study of roleof polycomb protein PCGF6in spermatogenesis and long non-codingRNA expression profiling of mouse testis during postnatal development.Studies of epigenetic regulatory mechanisms in mouse spermatogenesisfrom two perspectives have further enriched and improved theinvestigations into epigenetic regulatory mechanisms in mammalian spermatogenesis and also provide new clues and theoretic foundation forfurther exploration. Meanwhile, it may facilitate the uncovering ofmolecular mechanism on mammalian spermatogenesis and the molecularpathogenesis of male infertility and it also provide the basis for diagnosis,treatment and prevention of male infertility.