ARTICLE Received 26 May 2015 | Accepted 24 Jun 2016 | Published 10 Aug 2016 DOI: 10.1038/ncomms12354 OPEN Ordered chromatin changes and human X chromosome reactivation by cell fusion-mediated pluripotent reprogramming Irene Cantone1, Hakan Bagci1, Dirk Dormann2, Gopuraja Dharmalingam3, Tatyana Nesterova4, Neil Brockdorff4, Claire Rougeulle5, Celine Vallot5, Edith Heard6, Ronan Chaligne6, Matthias Merkenschlager1 & Amanda G. Fisher1 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. 1 Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK. 2 Microscopy Laboratory, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK. 3 Bioinformatics and Computing facility, MRC Clinical Sciences Centre, Imperial College, London W12 0NN, UK. 4 Developmental Epigenetics Group, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. 5 Epigenetics and Cell Fate, CNRS UMR7216, Universite´ Paris Diderot, Sorbonne Paris Cite´, 75013 Paris, France. 6 Mammalian Developmental Epigenetics Group, Institut Curie, 26 rue d’Ulm, 75248 Paris, France. Correspondence and requests for materials should be addressed to A.G.F. (email: amanda.fi[email protected]). NATURE COMMUNICATIONS | 7:12354 | DOI: 10.1038/ncomms12354 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12354 chromosome inactivation (XCI) is an exemplar of also noted that in several human–rodent hybrid lines the epigenetically regulated silencing used by mammals distribution of XIST RNA was also perturbed3,34,35, raising Xto compensate for gene dosage between males (XY) and concerns that there might be an intrinsic incompatibility females (XX)1. XCI is a multi-factorial and multi-step process between human and mouse/hamster/rat cells. Here we used cell that is established as pluripotent cells of the embryo differentiate fusion to examine the earliest events in human XCR. Rather and can be reversed when somatic cells re-acquire pluripotency2. than examining conventional human–rodent hybrids that Non-coding Xist RNA initiates inactivation by coating contain a limited human chromosome contribution36,we the presumptive inactive X chromosome (Xi)3 and creating analysed XCI in newly formed heterokaryons before and just a nuclear domain from which RNA polymerase II and after the first mitosis. This system has the advantages of being activating chromatin marks are excluded4,5. Several repressive able to monitor the immediate events in reprogramming with histone and DNA modifications including histone H3 lysine higher efficiencies than the iPSC system and to track cells 27 tri-methylation (H3K27me3) and 5-methyl-cytosine are undergoing reprogramming more easily. incorporated within the chromatin of the Xi and result in To investigate the dynamics and extent of human XCR induced the stabilization of gene silencing6–8. Prior studies that by pluripotent reprogramming we fused human female fibroblasts have examined the contribution of different XCI factors to this (hF) with mouse embryonic stem cells (mESCs). Previous silencing have shown that removal of XIST/Xist9,10 or H3K27me3 studies have shown that following cell fusion an ensemble of (refs 11,12) in somatic cells is insufficient to reverse XCI but can mESC-factors is available to the human nucleus and that this lead to sporadic gene reactivation when coupled to experimental stimulates a rapid reactivation of the human pluripotency perturbation of histone acetylation or DNA methylation13. network, accompanied by global chromatin changes and the Collectively such experiments contribute to the classical view functional resetting of lineage potential37,38. Here we show that that components such as XIST/Xist and polycomb-mediated immediately after cell fusion, human and mouse genomes remain histone modifications are required for the initiation but not separate until the first mitosis when hybrid cells arise39, and that the maintenance of Xi silencing. Recently, however, it was human pluripotency genes are re-expressed ahead of cell division. shown that loss of Xist, when combined with multiple cell During this early period, we show that human nuclei undergo a divisions, could lead to the consistent reactivation of some progressive loss of H3K27me3 and XIST from the Xi and genes along the Xi chromosome14. This suggests that while selectively re-express certain human Xi genes. These data suggest control of Xi silencing is multi-layered and partially redundant, that XIST loss although necessary may be insufficient for Xi different domains on the Xi may vary in susceptibility to reactivation, and reveal that that reprogramming of human epigenetic reactivation. female somatic cells can induce the reactivation of specific Xi In somatic cells the induction of pluripotency can result in genes ahead of mitosis. reactivation of the silent Xi. A strong correlation between pluripotency and X chromosome reactivation (XCR) has been shown in mouse in experimental reprogramming studies, as Results well as in vivo during normal pre-implantation and primordial Pluripotent reprogramming of human female fibroblasts. germ cell development15. Model systems where XCI and its In order to investigate human XCR during pluripotent reversal can be induced have allowed the molecular interactions reprogramming we first examined the epigenetic signatures of the between the pluripotency network and XCI to be dissected. two X chromosomes in female diploid fibroblasts by fluorescence For example, several mouse pluripotency-associated transcription in situ hybridization (FISH), 4,6-diamidino-2-phenylindole factors including Oct4, Nanog and Rex1 have been shown to (DAPI) staining and the distribution of modified histones control XCI by regulating the transcription of Xist or its (Fig. 1a). In the nuclei of female hF, the Xi is condensed during antagonist Tsix2,16–18. In addition, a better understanding of the interphase and forms a heterochromatin compartment identified contribution of different factors in maintaining Xi gene silencing as the DAPI-dense Barr body. This compartment is coated by has been achieved by identifying the intermediate steps and XIST RNA and enriched in H3K27me3, as well as other histone the dynamics of XCR during somatic cell reprogramming19. modifications associated with silencing6. Before reprogramming, Comparable studies in human cells have been more problematic. karyotype analysis and DNA-FISH revealed that most fibroblasts This arises from the fact that human embryonic stem cells had two X chromosomes (490%) and labelling with (hESCs) and reprogrammed human induced pluripotent stem anti-H3K27me3 antibodies clearly identified a single Barr body cells (hiPSCs) with two active X chromosomes are notoriously in 86% of cells (Fig. 1a). Further confirmation was provided by unstable in culture20–24. Cultured human pluripotent cells are simultaneous RNA-FISH labelling with probes recognizing prone to undergo unscheduled re-initiation of XCI25,26, followed XIST and ATRX (an X-linked gene), where XIST RNA painted by epigenetic ‘erosion’ and inappropriate loss of silencing27. the Xi and nascent ATRX transcripts marked the location of Dynamic studies of XCR in human cells have, therefore, been the active X chromosome (Xa) (Fig. 1a, middle panels). hampered by epigenetic instability in culture and the consequent Fibroblasts were then immortalized by human TERT selective pressures that environmental conditions, such as transduction to alleviate senescence24 and the Xi status was oxygen tension, impose (reviewed in ref. 28). To circumvent revalidated before reprogramming. these limitations, we have utilized cell–cell fusions of somatic Female hF and male mESCs were fused in equal ratio and and ESCs to generate transient human–mouse heterokaryons in the kinetics of reprogramming and XCR was followed in which the earliest steps of pluripotency-induced reprogramming heterokaryon and hybrid cells generated over the next 6 days. could be followed. To identify and enrich for fused (hFxmESC) cells we used two Previously, studies of fusions between cells of different types different approaches. In the first approach, Hprt-null mESCs that had provided some of the first evidence that XCR was associated carry a puromycin resistance gene (PuroR) were fused with with pluripotency29. In early studies, in which human somatic primary or TERT-immortalized human fibroblasts and and rodent pluripotent and embryonic