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

Results

Pluripotent reprogramming of human female fibroblasts

In order to investigate human XCR during pluripotent reprogramming we first examined the epigenetic signatures of the two X chromosomes in female diploid fibroblasts by fluorescence in situ hybridization (FISH), 4,6-diamidino-2-phenylindole (DAPI) staining and the distribution of modified histones (Fig. 1a). In the nuclei of female hF, the Xi is condensed during interphase and forms a heterochromatin compartment identified as the DAPI-dense Barr body. This compartment is coated by XIST RNA and enriched in H3K27me3, as well as other histone modifications associated with silencing6. Before reprogramming, karyotype analysis and DNA-FISH revealed that most fibroblasts had two X chromosomes (>90%) and labelling with anti-H3K27me3 antibodies clearly identified a single Barr body in 86% of cells (Fig. 1a). Further confirmation was provided by simultaneous RNA-FISH labelling with probes recognizing XIST and ATRX (an X-linked gene), where XIST RNA painted the Xi and nascent ATRX transcripts marked the location of the active X chromosome (Xa) (Fig. 1a, middle panels). Fibroblasts were then immortalized by human TERTtransduction to alleviate senescence24 and the Xi status was revalidated before reprogramming.

Figure 1: Human female XaXi fibroblasts are reprogrammed via cell-fusion with mouse embryonic stem cells.

(a) Confocal images of normal XaXi female hF (IMR90) where the Xi is visible as a DAPI-dense region, enriched for H3K27me3 histones (arrowhead, top panels) and coated by XIST RNA (arrowhead, middle panels). The Xa is instead revealed by ATRX nascent transcript detection (open arrow, middle panels). Two X chromosomes were confirmed by labelling the ATRXlocus and the whole X chromosome using DNA-FISH (bottom panels; ≥95% in primary orTERT-overexpressing cells). Total number of cells (n) was >200 per experiment in at least two independent cultures. Scale bar, 5 μm. (b) Scheme representing the strategy for reprogramming human fibroblasts towards pluripotency. hF are fused with a male mESC line (E14Tg2a) that is sensitive to HAT (Hprt−/y) and resistant to puromycin (PuroR). Heterokaryons containing discrete nuclei from both partners were selected by HAT/puromycin and generate drug-resistant hybrids after the first cell division. (c) Histogram plots showing the expression of human pluripotency genes (OCT4, NANOG,CRIPTO and REX1) and fibroblast-specific genes (DCN, COL6A3) in the HAT/puromycin resistant cultures where unfused hF, mESCs, H1 hESCs and primary mouse embryonic fibroblasts (MEF) provide controls. Gene expression is reported as 2−ΔCt relative to GAPDHand represents the average of 3–10 independent experiments. Error bars indicate s.e.m. and (*) mark values that significantly differ from hF/day 0 (P≤0.05, two-sided t-test). See alsoSupplementary Fig. 1.