GSE126229
Dynamic Erasure of Random X-Chromosome Inactivation during iPSC Reprogramming
Induction and reversal of chromatin silencing is critical for successful development, tissue homeostasis and the derivation of induced pluripotent stem cells (iPSCs). X-chromosome inactivation (XCI) and reactivation (XCR) in female cells represent chromosome-wide transitions between active and inactive chromatin states. While XCI has long been studied and provided important insights into gene regulation, the dynamics and mechanisms underlying the reversal of stable chromatin silencing of X-linked genes are much less understood. In this work, we use allele-resolution transcriptomic approaches to study XCR during mouse iPSC reprogramming in order to elucidate the timing and mechanisms of chromosome-wide reversal of gene silencing. Our findings reveal a sequential hierarchy of gene reactivation from the inactive X-chromosome (Xi). We show that gene reactivation initiates before the activation of late pluripotency genes and complete silencing of the long non-coding RNA (lncRNA) Xist, and is completed late in reprogramming. We reveal that the gene-specific timing of reactivation correlates with the genomic distance to genes that escape inactivation and with differential targeting by pluripotency transcription factors (TFs). We also show that histone deacetylases restrict XCR in reprogramming intermediates and that the severe hypoacetylation state of the Xi persists until late reprogramming stages. Therefore, randomly inactivated X-linked genes possess different degrees of epigenetic memory, the reversal of which may involve the combined action of chromatin regulators, pluripotency transcription factors and chromatin topology. Taken together, our study provides the chromosome-wide dynamics of gene reactivation from the randomly inactivated X-chromosome and a framework for understanding how transcription factors induce dynamic reversal of stable epigenetic memory by overcoming repressive chromatin barriers. Overall design: Transcriptome profiles of female Mus Musculus/Mus Castaneus mouse embryonic fibroblasts induced to reprogramming by overexpression of dox inducible Oct4, Sox2, Klf4, cMyc cassette were generated by next generation sequencing using NextSeq 500 platform. In total  7 samples were analyzed including: cells collected at Day 2 after dox treatment; Day 8 SSEA1+, Day 10 SSEA1+, Day 13 SSEA1+, Day 15 SSEA1+, iPSCs SSEA1+/GFP+, ESCs SSEA1+/GFP+.