Mechanistic Exploration On The Role Of AFF3 In X Chromosome Inactivation

X chromosome inactivation(XCI)and genomic imprinting are two classic epigenetic regulatory processes that cause mono-allelic gene expression.In female mammals,mono-allelic expression of the long non-coding RNA gene X-inactive specific transcript(XIST)is essential for initiation of X chromosome inactivation upon differentiation.The long non-coding RNA XIST is expressed exclusively from the inactive X chromosome,and cis-accumulated on the X chromosome to induce chromosome wide transcription silencing.The super elongation complex SEC contains the ELL family members ELL1/2/3,AF4/FMR2 family members AFF1/4,ENL,AF9 and the Pol II elongation factor P-TEFb.In addition,SEC-like complexes are also biochemically purified,AFF2 and AFF3 are the central factors in the formation of the SEC-like complexes super elongation complex-like 2(SEC-L2)and super elongation complex-like 3(SEC-L3),respectively.SEC,SEC-L2,and SEC-L3 demonstrate functional gene target specificity,SEC plays an important role in leukemia and HIV.SEC-L2 is associated with breast cancer and autism,and SEC-L3 affects the development of autoimmune diseases.Our previously studies have demonstrated that the central factor of SEC-L3,AFF3,which can regulate the imprinted gene expression in an allele-specific manner.AFF3 is enriched at gamete differentially methylated regions(DMRs)of the imprinted loci in a DNA methylation and H3K9 methylation-dependent mechanism;while,it can also bind to the enhancer by zinc finger protein ZFP281 to promote transcriptional elongation.However,it is unknown whether it can regulate the expression of XIST gene and further affect the X chromosome inactivation.Here we first analyzed our previously published RNA-Seq datasets,which showed that the expression of XIST is upregulated following sh RNA-mediated AFF3 knockdown.To further validate this phenomenon,we conducted real-time quantitative PCR analysis after knockdown of AFF3 using two independent sh RNA in IMR-90 female cells.The results confirmed that knockdown of AFF3 leads to de-repression of the inactive allele of XIST and a small percentage of cells display one more extra RNA clouds,as detected by RNA FISH.ENL and AF9,components of SEC-L3,are also required for XIST silencing.In addition,AFF3 can bind to the DMR downstream of the XIST promoter by performing chromatin immunoprecipitation(Ch IP-q PCR)and Ch IP-seq.We combined DNA methylation co-immunoprecipitation(Me DIP)and bisulfite-sequencing to reveal that binding of AFF3 to the XIST DMR relies on DNA methylation and also regulates DNA methylation level at DMR region.However,we found that the KAP1-H3K9 methylation machineries are not necessary in maintaining mono-allelic expression of XIST in these cells.Thus,our results suggest the differential mechanisms involving in XIST DMR and g DMR regulation,but both require AFF3 and DNA methylation.