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Dissecting Function Of DNA Methylation In Mammals:from Genome-wide Mapping To Site-specific Editing

Posted on:2015-12-08Degree:DoctorType:Dissertation
Country:ChinaCandidate:J ZhangFull Text:PDF
GTID:1360330461460519Subject:Biology
Abstract/Summary:PDF Full Text Request
The reprogramming of parental methylomes is essential for embryonic development.In mammals,paternal 5-methylcytosines(5mCs)have been proved to be actively converted to oxidized bases such as 5-hydroxymethylcytosine(5hmC),5-formylcytosine(5fC)and 5-caboxycytosine(5caC).These paternal oxidized bases and maternal 5mCs are believed to be passively diluted by cell divisions.Herein,we generated allele-specific,and single-base resolution DNA methylomes from mouse gametes,early embryos and primordial germ cell(PGC),as well as single-base resolution maps of oxidized cytosine bases for two-cell embryos.Our data show that the PGCs E13.5 methylome is almost fully erased.More surprisingly,during early embryogenesis,5hmC and 5fC exist in both maternal and paternal genomes and find that 5mC or its oxidized derivatives,at the majority of demethylated CpGs are converted to unmodified cytosines independent of passive dilution from gametes to four-cell embryos.Therefore,we conclude that paternal methylome and at least a significant proportion of maternal methylome go through active demethylation during embryonic development.Genomic imprinting in mammals is important for embryonic development.Based on our single-base resolution oocyte DNA methylome,all the known imprinting control regions(ICRs)were classified into germ line(gICRs)and somatic ICRs.Unexpectedly,three gICRs undergo demethylation in early embryos.Allelic-specific expression show that most maternal gICRs expressed in early embryos from paternal copy.This study refines the current knowledge on DNA methylation reprogramming in mammals and provides a powerful resource for early developmental studies.Whole genome DNA methylation sequence have been generated from many different cell types and have yielded important biological insights,but such data are largely correlative.A crucial challenge in the field is determining the functional significance of specific methylation events,owing to the lack of method for removing such modifications at a specific site.Here we use nuclease-deficient Cas9(dCas9)as an artificial DNA-binding factor to specific binding a methylated CpG site,we demonstrated that DNA-binding factor and its physical binding is necessary and sufficient to create these low methylated sites.dCas9 can induce site specific DNA demethylation,offer a powerful tool to in vivo study the interaction between methylated DNA element and transcription factor.Moreover,some transcription factors are sensitive to DNA methylation,so we can use dCas9 to regulate gene expression by modulating the level of DNA methylation in the promoter region of targeting gene.Furthermore,we fused Kdm4d catalytic domain with dCas9,we use major satellites as a paradigm and proved that this new Cas9 deviant can mediate site specific histone H3K9me2/3 demethylation.Combining these two approaches,will make site-specific chromatin epigenetic modification come true.For DNA methylation and H3K9me2/3 both are repression marks,site specific target these modifications can used to reactive developmental silenced genes such as imprinted gene.These approaches expand the application of Cas9 for epigenetic modification.
Keywords/Search Tags:DNA methylation, DNA demethylation, Genomic imprinting, CRISPR/Cas9, Epigenetic editing
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