Identification of a novel imprinting mechanism at the X-linked imprinted locus, X-linked Lymphocyte Regulated 3/4 ( Xlr3/4 )

摘要

BackgroundGenomic imprinting is the epigenetic process by which asubset of genes is expressed from only one allele basedon the parent of origin. To date there are approximately150 confirmed autosomal imprinted loci in the mammaliangenome. The epigenetic hallmark of an autosomalimprinted locus is the presence of differential methylationat CpG islands otherwise known as a DMR[1].Imprint maintenance at autosomal loci is accomplishedthrough specific histone modifications[2]. In 2005,Raefski et.al. found the first X-linked imprinted locus,Xlr3/4 of which three paralogs, Xlr3b/4b/4c, were maternallyexpressed and paternally silenced in mouse neonatalbrain[3]. Here we use a series of techniques to uncoverthe imprinting mechanism at the Xlr locus using knownautosomal imprinting mechanisms as a model.Materials and methodsMice were generated that either carried the maternal (39,Xm) or paternal (39, Xp) X chromosome. To measure thedifference in both primary transcript and mRNA levels ofXlr3b/4b/4c between these samples quantitative real timePCR was performed. To search for differential methylation,MeDIP followed by hybridization to an X chromosometiling array was performed for a global view ofmethylation at the Xlr3/4 locus. For confirmation, targetedbisulfite sequencing was performed at CpG islands. ChromatinImmunoprecipitation (ChIP) followed by real timePCR was used to assess differential enrichment ofH3K4me3, H3K27me3, H3K9me2, and H3K36me3 atXlr3b. Furthermore, ChIP-Seq was performed to traceRNA Polymerase II (PolII) and H3K36me3 across theXlr3/4 locus.ResultsWe show that there is no differential methylation at theXlr3/4 locus between 39, Xm and 39XP samples. Potentialcandidate regions for differential methylation according toMeDIP assays were disproven by bisulfite sequencing ofCpG islands within those regions. Furthermore, all regionsassayed at Xlr3b were hypermethylated on both alleles.We show that H3K4me3 and H3K27me3 are not enrichedat either allele of Xlr3b while H3K9me2 is equallyenriched on both. We show that the primary transcript ofXlr3b only shows imprinted expression at the 3’ end of thegene and this is confirmed by PolII enrichment indicatinga stall on the paternal allele during transcriptional elongation.Lastly, H3K36me3 enrichment mirrors that of RNAPolII.ConclusionsWe show that there is no DMR to govern gene expressionat the Xlr3/4 locus and thus a novel mechanism must existto regulate the imprint. The current model for thismechanism involves PolII stalling during transcriptionalelongation along the paternal allele of Xlr3b. On the maternalallele PolII transcribes through the 3’end of the geneunimpeded. While the cause of the stall remains unknownit may involve H3K36me3 histone methyltransferases.