Allele-specific analysis of cell fusion-mediated pluripotent reprograming reveals distinct and predictive susceptibilities of human X-linked genes to reactivation

Abstract

BackgroundInactivation of one X chromosome is established early in female mammalian development and can be reversed in vivo and in vitro when pluripotency factors are re-expressed. The extent of reactivation along the inactive X chromosome (Xi) and the determinants of locus susceptibility are, however, poorly understood. Here we use cell fusion-mediated pluripotent reprograming to study human Xi reactivation and allele-specific single nucleotide polymorphisms (SNPs) to identify reactivated loci.ResultsWe show that a subset of human Xi genes is rapidly reactivated upon re-expression of the pluripotency network. These genes lie within the most evolutionary recent segments of the human X chromosome that are depleted of LINE1 and enriched for SINE elements, predicted to impair XIST spreading. Interestingly, this cadre of genes displays stochastic Xi expression in human fibroblasts ahead of reprograming. This stochastic variability is evident between clones, by RNA-sequencing, and at the single-cell level, by RNA-FISH, and is not attributable to differences in repressive histone H3K9me3 or H3K27me3 levels. Treatment with the DNA demethylating agent 5-deoxy-azacytidine does not increase Xi expression ahead of reprograming, but instead reveals a second cadre of genes that only become susceptible to reactivation upon induction of pluripotency.ConclusionsCollectively, these data not only underscore the multiple pathways that contribute to maintaining silencing along the human Xi chromosome but also suggest that transcriptional stochasticity among human cells could be useful for predicting and engineering epigenetic strategies to achieve locus-specific or domain-specific human Xi gene reactivation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1136-4) contains supplementary material, which is available to authorized users.

Reversal of X chromosome inactivation: lessons from pluripotent reprogramming of mouse and human somatic cells

“…In addition, a minority of heterokaryon and hybrid nuclei (7% at day 3 vs. 1% at day 0; unpublished data) have two diffused XIST clouds suggesting that cell fusionmediated reprogramming might be able to recapitulate some features of the in vivo epiblast cells. Consistently with this hypothesis, genome-wide expression analysis upon cell fusion-mediated reprogramming showed the reactivation of pluripotency genes associated with both primed and naïve human pluripotent cells [129] . In the future, single cell studies might indeed be used to segregate different human pluripotent states and help us refine culture conditions for naïve pluripotency.…”

“…To investigate the extent of Xi gene reactivation along the entire X chromosome, we derived human fibroblast clones with reciprocal Xi chromosomes (e.g. Xa 1 Xi 2 and Xi 1 Xa 2 ) and performed allele-specific RNA-seq analysis of these clones before and after reprogramming [129] . This analysis showed that cell fusion-mediated reprogramming induces partial human Xi reactivation with 10% of the sampled genes being consistently reactivated at different times after fusion and in different clones.…”

“…XCI state, however, cannot be fully recapitulated in these cultures [62,103] thus highlighting the need for further improvements. We propose that cell fusion-mediated reprogramming might be used to segregate different pluripotent states with distinct X chromosome epigenetic features in single cells, as it can induce the expression of genes associated with both the primed and naïve human pluripotent states [129] . This information might be useful for identifying pathways that stabilize naïve pluripotency and for refining culture conditions.…”

“…The advent of genome-wide techniques that can be used to assess allele-specific expression at the single cell level will be fundamental in defining different XCI states and investigating the susceptibility of distinct Xi loci to reactivation. In addition, it will be important to reach an agreement about whether Xi expression should be defined as percentage of expression relative to the Xa or based on more sophisticated statistical methods [129,[132][133][134] . Notably, the combination of molecular and cell biology techniques applied to human pluripotent cells before and after differentiation is required to distinguish naïve from primed and eroded pluripotency.…”

Human X chromosome inactivation and reactivation: implications for cell reprogramming and disease

“…Screening for chromatin modifiers that promote human Xi reactivation. The extent of expression of Xi genes in a representative clone before (hF) and after cell fusion-mediated reprogramming (hF Â mESC) reveals that some genes (highlighted in green) are susceptible to reactivation following reprogramming (for details see [71]). As pre-treatment of cells with drugs that reduced DNA methylation (þ5-aza-deoxyC) revealed a selective reactivation of additional 'Xi' genes (red dots), we propose to screen for candidate modifiers (þ agent Z) that restrict the expression of specific human X-linked genes.…”