Ordered chromatin changes and human X chromosome reactivation by cell fusion-mediated pluripotent reprogramming.

Irene Cantone,Claire Rougeulle,Matthias Merkenschlager,Amanda G Fisher,Gopuraja Dharmalingam,Tatyana Nesterova,Neil Brockdorff,Celine Vallot,Dirk Dormann,Ronan Chaligne,Edith Heard,Hakan Bagci

doi:10.1038/ncomms12354

Abstract

Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse, but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we use cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show that a rapid and widespread loss of Xi-associated H3K27me3 and XIST occurs in fused cells and precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30–50%). After cell division, RNA-FISH and RNA-seq analyses confirm that Xi reactivation remains partial and that induction of human pluripotency-specific XACT transcripts is rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation and reveal a complex hierarchy of epigenetic changes that are required to reactivate the genes on the human Xi chromosome.

Highlights

Erasure of epigenetic memory is required to convert somatic cells towards pluripotency
These data suggest that XIST loss necessary may be insufficient for Xi reactivation, and reveal that that reprogramming of human female somatic cells can induce the reactivation of specific Xi genes ahead of mitosis
Reprogramming induced the loss of Barr bodyassociated histone H3K27me[3] and delocalization of XIST from the human Xi, despite detection of XIST transcripts. These changes in pre-mitotic human chromatin appeared to be required for the subsequent re-expression of ATRX and HUWE1, but were insufficient to ensure the reactivation of many other X-linked genes including PHDA1

Summary

Introduction

Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Prior studies that have examined the contribution of different XCI factors to this silencing have shown that removal of XIST/Xist[9,10] or H3K27me[3] (refs 11,12) in somatic cells is insufficient to reverse XCI but can lead to sporadic gene reactivation when coupled to experimental perturbation of histone acetylation or DNA methylation[13]. Such experiments contribute to the classical view that components such as XIST/Xist and polycomb-mediated histone modifications are required for the initiation but not the maintenance of Xi silencing. These data suggest that XIST loss necessary may be insufficient for Xi reactivation, and reveal that that reprogramming of human female somatic cells can induce the reactivation of specific Xi genes ahead of mitosis

Methods

Results

Conclusion