Allele-specific ATAC-seq

One of the two X chromosomes in female somatic cells is transcriptionally silenced across cell generations, a classic paradigm of epigenetic regulation.  Although most genes are stably silenced, certain X-linked genes escape X-chromosome inactivation (XCI), providing a fundamental dosage difference between females and males.  A role for chromosome conformation has been proposed in XCI as the process is  accompanied by a massive structural reorganisation. However a detailed molecular understanding of the three-dimensional architecture of the Xi has been lacking.  Here we reveal an unusual three-dimensional configuration of the Xi, that provides novel insights into the relationship between gene expression and TAD organisation.  Using allele-specific Hi-C, RNA-Seq and ATAC-seq, as well as DNA FISH, we show that the Xi is spatially segregated into two ‘mega-domains’ separated by a 200kb boundary including the DXZ4 macrosatellite, and is globally devoid of typical autosomal structural features such as active/inactive compartments and topologically associating domains (TADs). However, we find that a few regions along the Xi display TAD signatures corresponding to regions containing escape genes, and display DNA accessibility at promoter-proximal TF binding sites.  Strikingly, when the DXZ4-containing boundary region is deleted prior to XCI, most facultative escapees are no longer expressed from the Xi, and the escape associated  chromatin domains are no longer observed. Deletion of the DXZ4-containing boundary region from the Xi also results in fusion of the two mega-domains.   We show that Xist A-repeat containing (gene silencing competent) RNA is sufficient to induce this spatial segregation of the X chromosome. Combined, our results point to a critical role for the DXZ4-boundary region, together with Xist RNA, in shaping the higher order structure of the Xi and in allowing facultative escape from XCI during differentiation. They also point to direct and uncover a close relationships between transcription and TAD formation organisation in the context of the Xi. ATAC-seq was performed in mESCs and mNPCs as well as deltaFT NPCs. Libraries(2 replicates per line) were sequenced on an Illumina NextSeq (2x75bp) and data was analyzed allele-specifically. ATAC-seq was also performed inmale mESCs containing a doxycycline-inducible Xist construct at the endogenous locus with or without the functional RepA region. Samples with Xist induction (+dox) and without (-dox) are included.