The Functions And Mechanisms Of ZFP281-releated Proteins In Mouse Embryonic Stem Cells

Genomic imprinting is an epigenetic regulation that affects gene expression in a parent-oforigin specific manner.Genes within one given imprinted locus are coordinately regulated by cisregulatory elements,epigenetic machineries and transcriptional factors to tightly control the allelespecific expression pattern.It is well accepted that imprinting control regions(ICRs)and enhancers are two major classes of cis-elements determining parent of origin-specific gene expression.Dysregulation of their interaction within the imprinted loci can lead to imprinted gene expression defects.Their misexpression have been found in numerous human congenital diseases,such as Angelman,Prader-Willi and Beckwith – Wiedemann Syndromes;and a number of cancers as well.Previously,we have demonstrated that the P-TEFb-containing super elongation complex(SEC)and super elongation complex-like(SEC-L2 and SEC-L3)can regulate different sets of genes with functional specificities in development and diseases.AFF3,the central component of SEC-L3,is enriched at both the intergenic-differentially methylated region(IG-DMR)and the Meg3 enhancer within the imprinted Dlk1-Dio3 locus to regulate the allelespecific gene expression in mouse embryonic stem(ES)cells.The localization of AFF3 to IG-DMR requires ZFP57.However,how AFF3 functions at the Meg3 enhancer in maintaining allele-specific gene expression and molecular signaling mechanism remains unclear.The Krüppel-like zinc finger transcription factor ZFP281 has previously been identified as a major regulator of stem cell pluripotency.We identified a consensus sequence of ZFP281 at the AFF3-occupied enhancer regions in mouse ES cells through a de novo motif search.Our genomewide analyses further identify ZFP281 as a critical factor generally associating with AFF3 at enhancers and functioning together with AFF3 in regulating the expression of a subset of genes.ZFP281 functionally interacts with AFF3 at the Meg3 enhancer,but not IG-DMR,to ensure proper expression of the Meg3 polycistron at the transcriptional elongation level.When Zfp281 is depleted by shRNA mediated RNAi,the recruitment of AFF3 to the Meg3 upstream enhancer is affected and the expression of the Meg3 polycistron is down-regulatedion.Thus,we have identified the molecular regulators involved in the recruitment of AFF3 to enhancers and provide mechanistic insight into the requirement of AFF3 at an enhancer for the expression of Meg3 polycistronic transcript within the imprinted Dlk1-Dio3 locus.Our results indicate that different zinc finger proteins,e.g.ZFP57 and ZFP281,can recruit AFF3 to different regulatory elements and differentially regulate the function of AFF3 in a context-dependent manner.