Research On Regulatory Mechanisms Of Genomic Imprinting Dynamics In Primates

In multicellular organisms,such as mammals,genomic imprinting is an allelic gene expression phenomenon and remarkable epigenetically regulated process.It plays critical roles in multiple biological processes including development,metabolism,diseases and psychiatric disorders.Despite the intensive studies on genomic imprinting in the last decades,our knowledge of genomic imprinting,especially in outbred animals such as primates,is still limited.DNA methylation has long been considered as the only primary imprinting mark in mammals until recently recent research demonstrated that maternal-biased H3K27me3 can also mediate dozens of paternal-biasedly expressed genes(PEGs)in mouse preimplantation embryos.However,whether this imprinting mechanism is conserved in primates remains to be resolved.Genomic imprinting plays an important role in regulating the growth and development of embryos,placenta and neonates.In general,paternally expressed imprinted genes play a positive role in regulating the development of embryos,while those expressed by maternal allele do the opposite.In addition,recent studies have shown a close relationship between genomic imprinting and development potential of somatic cell nuclear transfer(SCNT)animals.Abnormal H3K27me3-mediated noncanonical imprinting led to excessive placental growth in cloned mice.However,due to the lack of research on genomic imprinting in primates,it is not clear whether the lower developmental capacity of cloned monkeys is related to imprinting genes.Lack of appropriate research methods is the main reason for the slow progress of primate genomic imprinting research.Specifically,there is currently no effective method to distinguish between parental chromosomes or alleles in outbred animals,such as primates.Currently,de novo identification of genome-wide allelic DMRs in human somatic tissues requires integrating hundreds of methylomes from different individuals using single-nucleotide polymorphism(SNP)-based analyses.However,these methods are too expensive and impractical to be widely used in the studies of genomic imprinting in outbred animals.Unisexual embryos are important materials for studying genomic imprinting.Due to ethical restrictions,human unisexual embryos are difficult to obtain.Nonhuman primates are ideal animal models for studying human embryonic development and disease.Therefore,this study first comprehensively analyzed the transcriptome,DNA methylome and H3K27me3 histone modification of cynomolgus monkey uniparental embryos.These results demonstrate that,different from mouse,the major regulatory mechanism of maternal imprinting in early monkey embryos is DNA methylation rather than H3K27me3.In order to overcome the technical barrier,we further developed two accurate and efficient methods for identifying germline differentially methylated regions(g DMRs),namely tissue-associated,reads-based,SNP-free method for identifying imprint-DMRs(TARSII)and Cp G-island-associated,reads-based,SNPfree method for identifying imprint-DMRs(CARSII).These two methods do not rely on SNP analysis,and only need to use DNA methylation data from a few different adult tissues to identify g DMRs in somatic tissues(by TARSII analysis)or g DMRs in a specific tissue(by CARSII analysis).Using these methods,we revealed genomic imprinting differences in primates between early embryos and somatic tissues,as well as between embryonic and extraembryonic tissues.Interestingly,primate germline DMRs are different from PEG-associated DMRs in early embryos and are enriched in placenta.By comparing the g DMRs of wild-type placenta,cloned monkey placenta and somatic tissue,we found that the genomic imprinting pattern of cloned monkey placenta was closer to that of somatic tissue,and most of the original imprinting of wild-type placenta was lost.In addition,placenta-specific imprinted genes were also abnormally expressed in cloned monkey placentas.This genomic imprinting defect in cloned monkey embryos is likely to ultimately affect the development and birth of cloned monkey fetuses.In conclusion,in this study,we developed two SNP-free methods for identifying imprinted DMRs.By using them to investigate the dynamic of genomic imprinting in primate early embryonic and adult tissues,we finally reveal that somatic tissues of primates preserve much fewer mg DMRs compared with early embryos or placenta.Interestingly,such dememorization of mg DMRs is non-reversible and could lead to imprinting defects in placenta of developing embryos generated from somatic cell reprogramming.These results and conclusion provide important clues for primate nuclear transfer research,and also will increase the efficiency of primate cloning.