Landscape of Monoallelic DNA Accessibility and Gene Regulatory Networks during Reprogramming to Naive Pluripotency and X Chromosome Reactivation [smart-seq]
X chromosome reactivation (XCR) represents a paradigm to study epigenetic regulation and the reversal of chromatin silencing, although how it is linked to the pluripotency gene regulatory network, pluripotency transcription factors (TFs) and chromatin remodelling processes remains largely unexplained. Several pluripotency TFs have been linked to XCR through binding to the long non-coding RNA gene Xist, but whether other regulatory elements are involved is unclear. Here, we show that reprogramming to iPSCs induces gradual acquisition of chromatin accessibility at specific sites on the inactive X chromosome (Xi) which is subsequently propagated to other regulatory elements including enhancers and promoters. Regions that are genomically closer to day 0 accessible regions become biallelically accessible earlier, than the more distant ones. For distinct categories of chromatin regions that become biallelically accessible at different times, different sets of transcription factor (TF) motifs are enriched, including the pluripotency TFs KLF4 and cMYC. In addition, regions that become bialleliacally accessible earlier also show increased TF pluripotency  binding, supporting the link between pluripotency TFs in XCR. To further explore the relationship between pluripotency TFs and XCR, we construct the active Gene Regulatory Networks (GRNs) during iPSC reprogramming using scRNA-seq and computational analyses. We reveal gene targets of 311 TFs expressed across reprogramming to iPSCs. Using this single-cell regulatory atlas, we show that X-linked genes are preferentially targeted by several pluripotency TFs. Our results suggest a new model where XCR may be coordinated by direct targeting of regulatory elements on the X chromosome by pluripotency TFs, concomitant with step-wise acquisition of chromatin accessibility. Altogether, our results demonstrate how gradual acquisition of a new GRN during reprogramming of cellular identity is linked with dynamic induction of chromatin accessibility and overcomes stable chromatin silencing on the Xi. Overall design: single-cell RNA-seq using smart-seq protocol