Function Of Histone H3 Methyltransferases EZH2 And GLP During The Early Development Of Mouse

Epigenetics,a major research field of functional genomics,describes cellular and physiological trait variations that do not follow the mendel’s laws of inheritance.Epigenetic changes are necessary for maintaining normal development and tissue-specific gene expression patterns.In mammalian developmental processes,there are two greatly important stages/cell types:early embryos and primordial germ cells(oocytes and sperm precursor cells).In early embryos,the genomes undergo a profound reprogramming.In recent years,researchers of epigenetics focused primarily on post-transcriptional modifications of histone,especially during the early embryonic development processes.The post-transcriptional modifications on N-terminus of core histones,including methylation,acetylation,ubiquitination,phosphorylation,and ADP-ribosylation,could affect chromosome remodelling,thereby regulating expression of nearby genes by recruiting transcriptional activators or repressors.In this study,we selected two well-understood modifications of histone,H3-Lysine 9 dimethylation(H3K9me2)and histone H3-Lysine 27 tri-methylation(H3K27me3),to investigate the potential role of EZH2 and GLP in controlling early embryo development.1.The expression level and function of EZH2 in mouse early embryosEnhancer of zeste homologue 2(EZH2)is essential for the development of pre-implantation mouse embryos.Loss of Ezh2 results in embryonic lethality in mouse.Ezh2-deficient embryos display impaired potential for outgrowth,represented by defects in establishing embryonic stem(ES)cells.Our current study investigated if Ezh2 controls the fate of embryos at an earlier stage by treating embryos with cycloheximide,the inhibitor of de novo protein,or microinjecting short interfering RNA(siRNA)at 2 hours post fertilization to restrict embryonic Ezh2 expression.Afterwards,we collected the oocytes and the fertilized zygotes for immunofluorescence staining and westernblot analysis,and the results showed that EZH2 were barely detected in oocytes but obviously observed in zygotes and embryos.In zygotes treated with Cycloheximide,the de novo EZH2 protein synthesis was inhibited,suggesting that EZH2 requires de novo synthesis duringfertilization stages.Furthermore,we found that loss of Ezh2 at the pronuclear stage causedsevere growth retardation and reduced blastocyst formation.What’s more,qPCR analysisshowed significantly decreased expression of the pluripotency-associated markers Oct4,Sox2 and Nanog were in embryos injected with Ezh2 siRNA.In addition,Ezh2 lossupregulated the expressions of genes involved in the differentiation of germ layers,including Gata6,Hoxbl and Hand1.Moreover,TUNEL assay demonstrated increasedapoptosis rate in the blastocyst embryos with Ezh2 knockdown.Finally,methylation ofhistone H3K27me2/3 was strongly reduced in Ezh2 knockdown embryos.In conclusion,Ezh2 is essential for early preimplantation embryo development.2.The expression level and function of GLP in mouse early embryosG9A-like protein(GLP)plays an important role in mouse early embryonicdevelopment.Glp-deficient embryos display severe growth retardation and defects that leadto lethality at approximately embryonic day 9.5.In this study,we investigated theexpression pattern of GLP in oocytes and early embryos.We microinjected specific siRNAof Glp into mouse zygotes at 2 hours post fertilization and observed the effect of Glp-defecton embryonic development in vitro.Our results showed that Glp knockdown inducedabnormal embryonic development and reduced blastocyst formation.Expressions of thepluripotency markers Oct4,Sox2,and Nanog were also significantly decreased inGlp-deficient embryos.In addition,the apoptotic indices,including Caspase 3/7 activitiesand expressions of Caspase 3/9,increased in Glp-deficient embryos.Moreover,themethylation levels of H3K9me2 decreased in Glp knockdown embryos.Therefore,ourresults suggested that GLP is involved in the modification of H3K9me2 and is essential forin vitro development of mouse embryo.This study was conducted to elucidate the function of histone methyltransferase EZH2and GLP in mouse early embryonic development.It was found that GLP and EZH2 wereessential for the maintenance of normal embryonic development.Deficient expression ofboth EZH2 and GLP lead to developmental defects and impaired pluripotency in embryos,which display altered methylation level of target histone.