Studies On The Effects And Mechanisms Of MARF1 In The Control Of Oocyte Epigenetic Maturation In Mice

Oocyte maturation is a key step toward making a high-quality egg competent.In addition to the well known processes of meiotic and cytoplasmic maturation,oocytes also undergo extensive genomic modifications in their growth phase that regulate gene expression during oocyte development and post fertilization.These specialized epigenetic changes include the global DNA de novo methylation,histone modifications,and the large scale chromatin remodeling,which are collectively referred to as epigenetic maturation.The hallmarks of oocyte epigenetic maturation are the transition of the nuclear apparatus from the non-surrounded nucleolus(NSN)in the growing oocyte(GO)into the surrounded nucleolus(SN)in the fully-grown oocyte(FGO)and the establishment of the oocyte-specific histone marks,which are coincident with the global silencing of transcription in FGOs.Although these phenomena of epigenetic maturation have long been known,the underlying cellular and molecular mechanisms still remain largely elusive.In this thesis,effects of meiosis arrest female 1(MARF1)in the control of oocyte eppigenetic maturation and the potential mechanisms underlying the control by MARF1 were investigated based on the prior findings that MARF1,a master regulator of key oogenetic processes in mice,regulates the mRNA expression of several factors participating in chromatin remodeling,histone modification and transcription control in oocytes.Chromatin staining showed that,unlike the wild type FGOs that largely formed the normal appearing SNs in their GVs,immature germinal vesicle(GV)-stage FGOs isolated from Marf1 homozygous gene trap mutant mice(Marf1Gt(AS0671)Wtsi,hereafter referred to as Marfl-KO)displayed a skewed configuration of chromatin that is more similar to the NSNs in GOs of WT-mice.These Marf1-KO FGOs did not undergo global transcriptional silencing as compared to the WT-control FGOs,and showed high activity of transcription as revealed by transcriptional run-on assay and analysis of the phosphorylation of the carboxyl-terminal domain(CTD)of RNA polymerase ⅡA(POLR2A).The same abnormalities in chromatin structure and transcriptional activity were seen in ovulated Marf1-KO GV-stage oocytes.Associated with the abnormal chromatin configuration,immunofluorescence(IF)staining revealed that Marf1Gt/Gt FGOs have abnormal distribution of heterochromatin and pericentric chromatin markers,H3K9Me3 and H4K20Me3,as well as heterochromatin protein HP1,histone H2A variant H2AFY(macro-H2A),and proteins for chromatin remodeling(i.e.,ATRX and DAXX)in their GV.These data indicate that MARF1 controls chromatin remodeling and transcriptional silencing in oocytes.Treating Marf1-KO FGOs with both inhibitors for RNA polymerase Ⅰ(CX-5461)and Ⅱ(alpha-Amanitin)turned off the elevated transcriptional activity,and transformed the configuration of chromatin from NSN into SN in the GV,thus suggesting that these two abnormal phenotypes are mechanistically interconnected.In addition,IF and Western Blot analyses revealed that the expression of histone marks normally associated with active transcription,i.e.,H3K4me3,H3K36me3,H4K8ac and H4K12Ac,and repressive transcription,i.e.,H3K27me3 and H3R8me2s,respectively,are all altered in Marf1-KO FGOs.These data thus indicate that MARF1 controls histone modification in oocytes.By carefully mining the dataset on transcriptomic comparison of Marfl-KO and WT-oocytes,we found that genes involved in histone modification,transcriptional regulation,and heterochromatin formation were significantly up-regulated in Marf1-KO oocytes.However,treating Marfl-KO FGOs with inhibitors for RNA polymerase I(CX-5461)and/or Ⅱ(alpha-Amanitin)could not reverse the upregulated mRNA levels of these genes,thus indicating that this upregulation is not due to elevation of transcriptional activity.The defective eppigenetic phenotypes were partially relieved by microinjection of siRNAs to knock down the expression levels of two of the upregulated genes in Marfl-KO FGOs.Together,studies of this thesis revealed that MARF1 controls oocyte epigenetic maturation by modulating the expression of key factors responsible for histone modification,transcriptional regulation,and heterochromatin remodeling in oocytes.