Dynamic reversal of random X-Chromosome inactivation during iPSC reprogramming

Abstract

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. Although XCI has long been studied, providing important insights into gene regulation, the dynamics and mechanisms underlying the reversal of stable chromatin silencing of X-linked genes are much less understood. Here, we use allele-specific transcriptomics to study XCR during mouse iPSC reprogramming in order to elucidate the timing and mechanisms of chromosome-wide reversal of gene silencing. We show that XCR is hierarchical, with subsets of genes reactivating early, late, and very late during reprogramming. Early genes are activated before the onset of late pluripotency genes activation. Early genes are located genomically closer to genes that escape XCI, unlike genes reactivating late. Early genes also show increased pluripotency transcription factor (TF) binding. We also reveal that histone deacetylases (HDACs) restrict XCR in reprogramming intermediates and that the severe hypoacetylation state of the inactive X Chromosome (Xi) persists until late reprogramming stages. Altogether, these results reveal the timing of transcriptional activation of monoallelically repressed genes during iPSC reprogramming, and suggest that allelic activation involves the combined action of chromatin topology, pluripotency TFs, and chromatin regulators. These findings are important for our understanding of gene silencing, maintenance of cell identity, reprogramming, and disease.

Chromosome Compartments on the Inactive X Guide TAD Formation Independently of Transcription during X-Reactivation

“…X-reactivation can also be studied in vitro: induced pluripotent stem cells (iPSCs) generated from somatic cells through reprogramming have two active X chromosomes (Janiszewski et al, 2019;Maherali et al, 2007a;Pasque et al, 2014;Payer et al, 2013;Stadhouders et al, 2018) , linking the X-reactivation process to de-differentiation into the naïve pluripotent stem cell state (Payer and Lee, 2014). However, the mechanisms that govern the transition have not yet been elucidated.…”

“…However, the mechanisms that govern the transition have not yet been elucidated. It is unclear how the interplay of sequence, 3D-structure, chromatin status, and transacting factors affects the reactivation of X-linked genes and why X-reactivation in vitro during iPSC reprogramming is a slow and gradual progress as recently proposed (Janiszewski et al, 2019). In particular, it is unknown, if the dramatic topological rearrangement of the X chromosome from an inactive state with two mega-domains into an autosome-like active state consisting of A/B-compartments and TADs (Deng et al, 2015;Giorgetti et al, 2016;Minajigi et al, 2015;Rao et al, 2014) occurs before X-linked genes are reactivated, or rather follows transcription as observed during XCI (Collombet et al, 2020).…”

Defining totipotency using criteria of increasing stringency

“…Processing raw read counts was performed as described in 66 . Briefly, the DESeq2 package and the associated protocol 67 was used.…”

X-Chromosome Inactivation during Preimplantation Development and in Pluripotent Stem Cells

“…In vitro reprogramming of somatic cells into iPSCs entails profound changes in genome organization, DNA methylation, histone acetylation and methylation, and gene expression [reviewed in Apostolou and Hochedlinger, 2013] and, among these, XCR is mandatory for the faithful reprogramming of the founder cells to pluripotency. In mouse cells, XCR is a progressive and slow event [Stadtfeld et al, 2008;Payer et al, 2013] that takes about 1 week to occur [Janiszewski et al, 2019], and it is strongly linked to the sequential hierarchical activation of pluripotency-associated genes (Esrrb, Sall4, and Lin28) [Buganim et al, 2012;Pasque et al, 2014]. The expression of the reprogramming factors Oct4, Sox2, Klf4, and cMyc (OSKM) in female fibroblasts is not sufficient for Xist repression, suggesting an active role of the other pluripotency-associated factors.…”

“…programming [Janiszewski et al, 2019]. Then, only after reactivation of Nanog, Xist starts to be repressed, Tsix is progressively activated and CpG islands demethylated on the Xi [Payer et al, 2013;Pasque et al, 2014].…”