The Regulatory Mechanisms And Functions Exploration Of Mouse Endogenous Retrovirus RLTR4

In mammalian genomes,the activity of endogenous retroviruses（ERVs）is strictly regulated.Transcriptional repression of ERV is a key pathway to repress its activity and transposition,which is usually achieved through heterochromatin generated by coordinated epigenetic modifications.Mouse endogenous MLV（also known as RLTR4）has been widely studied as a classic type of ERV.The combination of the histone modifications at the RLTR4copies is unique,which is occupied by both H3K9me3 and H3K27me3.The recruiting mechanism of the PBS-dependent H3K9me3 modification has been studied in detail,but how H3K27me3 is established remains unclear.In our previous work,we confirmed that the PBS^Glnsequence of RLTR4 was necessary for the recruitment of Polycomb proteins which can catalyze H3K27me3 modification,but the relationship between these two histone modifications and also the potential interconnection between histone modifications and DNA methylation remains unclear.Studies have shown that the abnormal activation of RLTR4 in mice causes the accumulation of the virus proteins and make the mice more susceptible to the autoimmune disease systemic lupus erythematosus,while whether there is any physiological functions of RLTR4 needs to be further explored.Furthermore,KRAB-ZFP is a supper transcription factor family that specifically recognizes PBS sequences and recruits the epigenetic repressive complex to establish the repression of ERV.From an evolutionary point of view,the KRAB-ZFP family can rapidly evolve new members in response to the expansion of newly invasive ERVs in the genome,but the details of the rapid evolution of KRAB-ZFPs are still poorly understood.In this thesis,we studied the molecular basis of the rapid evolution of the KRAB-ZFP family by systematically analyzing the sequence characteristics of the KRAB-ZFP family.We analyzed H3K27me3 modification levels on different RLTR4 monomers in mouse embryonic stem cells（mESCs）.It was found that H3K27me3 was specifically enriched on some RLTR4 copies,and the viral copies with high H3K27me3 enrichment usually contained PBS^Gln,while the copies containing PBS^Pro were hardly marked by H3K27me3.Using inhibitors and RNA interference,we demonstrated that both H3K9me3 and H3K27me3 were functional for the repression of RLTR4 activity.Through the Ch IP-seq data analysis of related modifications,we focused on the relationship between various epigenetic modifications during RLTR4 inhibition.The results showed that the establishment of H3K27me3 on RLTR4 was not dependent on the H3K9me3 produced by SETDB1,but it was negatively correlated with the degree of DNA methylation to some extent,suggesting that H3K27me3 may serve as a compensatory mechanism for the reduction of DNA methylation on ERV to maintain epigenetic repression of RLTR4.In addition,we found that many RLTR4-LTR fragments（called Solo-LTRs）that did not contain PBS sequences were scattered in the mouse genome,and these Solo-LTRs were rich in active chromatin-associated histone modifications such as H3K4me3 and H3K27ac.Further studies have shown that the LTR of RLTR4 has promoter activity,and the U3 region of LTR is necessary for the promoter activity.The functional study of RLTR4 found that RLTR4 was usually in a suppressed state in mESCs,and continuous expression of exogenous RLTR4 fragments resulted in the abnormal morphology of mESCs and a sharp decrease in proliferation rate,and the expression of differentiation-related genes such as Gata4and Gata6 was significantly activated,and mESCs showed a state of differentiation disorder.RLTR4 is transiently activated during normal mouse embryonic development.In addition,KRAB-ZFP,as a key factor for inhibiting ERV,is distributed in clusters in the genome.KRAB-ZFPs in the same cluster have similar characteristics.Sequence analysis of more than 300KRAB-ZFPs in the mouse genome,we found that the Linker sequences between zinc fingers of KRAB-ZFP members in the same cluster are highly conserved,and human KRAB-ZFPs also exhibit similar characteristics.This finding suggests that these conserved Linker sequences may serve as the sequence basis for the rapid evolution of the KRAB-ZFP family.In summary,this thesis thoroughly explores the inhibitory mechanism and new functions of RLTR4.In terms of repression mechanism,the established mechanism of H3K27me3 and the relationship between various epigenetic repression mechanisms were analyzed in detail,and the internal reason for the rapid evolution of KRAB-ZFP was analyzed from the sequence characteristics.In terms of functional exploration,we found that the LTR of RLTR4 has promoter activity,and abnormal activation of RLTR4 can cause mESC differentiation disorder.These findings help to elucidate the diverse epigenetic inhibitory mechanisms of ERVs and unearth their physiological functions upon activation.