H3K9 methylation is a barrier during somatic cell reprogramming into iPSCs

Abstract

The induction of pluripotent stem cells (iPSCs) by defined factors is poorly understood stepwise. Here, we show that histone H3 lysine 9 (H3K9) methylation is the primary epigenetic determinant for the intermediate pre-iPSC state, and its removal leads to fully reprogrammed iPSCs. We generated a panel of stable pre-iPSCs that exhibit pluripotent properties but do not activate the core pluripotency network, although they remain sensitive to vitamin C for conversion into iPSCs. Bone morphogenetic proteins (BMPs) were subsequently identified in serum as critical signaling molecules in arresting reprogramming at the pre-iPSC state. Mechanistically, we identified H3K9 methyltransferases as downstream targets of BMPs and showed that they function with their corresponding demethylases as the on/off switch for the pre-iPSC fate by regulating H3K9 methylation status at the core pluripotency loci. Our results not only establish pre-iPSCs as an epigenetically stable signpost along the reprogramming road map, but they also provide mechanistic insights into the epigenetic reprogramming of cell fate.

The p53-induced lincRNA-p21 derails somatic cell reprogramming by sustaining H3K9me3 and CpG methylation at pluripotency gene promoters

“…Activation of the Oct4-GFP reporter was employed as readout for detecting the full acquisition of pluripotency. After infection with the shRNAs, the preiPSCs were passaged onto feeders and treated with vitamin C (Vc) for 8 days before GFP+ colony counting [10]. Notably, knockdown of 3 lincRNAs (-1463, -1526, and -p21) with the 2 independent shRNAs showed enhanced GFP+ colony formation compared to the control, while lincRNA-1307 knockdown had the opposite effects ( Figure 1D, lower panel and 1E).…”

“…Importantly, preiPSCs are stable and can be readily converted to fully reprogrammed iPSCs through a variety of approaches [10,21]. Because reprogramming cells consist of heterogeneous populations progressing at different speeds [3], preiPSCs provide a relatively well-defined model for dissecting the chain of events eventually leading to the full acquisition of pluripotency [10,11]. Using preiPSCs, we thus sought to identify lncRNAs that control the late stage of mouse somatic cell reprogramming.…”

“…We then selected 15 lincRNAs from classes I-VI for knockdown and functional analysis in a validated preiPSC to iPSC conversion model [10]. We generated 2 individual short hairpin RNA (shRNA) viral vectors for each of the 15 lincRNAs, and identified at least 1 effective shRNA (> 50% depletion) for 11 out of the 15 lincRNAs ( Figure 1D, upper panel).…”

“…LincRNA-p21 was predominantly nuclear ( Figure 3A), consistent with a potential role in epigenetic regulation. Then, we employed RNA immunoprecipitation (RIP) with antibodies against a panel of chromatin modifiers detrimental for reprogramming [9][10][11][12]23] using lysates from MEFs reprogrammed with OSKM on day 12. Notably, there was a substantial enrichment for lincRNA-p21 in DNMT1 RIP and a less substantial enrichment in SETDB1 RIP, but no enrichment in DN-MT3A/B and SUV39H1 RIPs, relative to IgG control ( Figure 3B).…”

“…Two fundamental reprogramming roadblocks are the activation of a p53-mediated response that promotes apoptosis and cell senescence, and the inefficient removal of preexisting somatic-like epigenetic marks. Accordingly, depletion of p53 [8] or the H3K9 methyltransferases Setdb1, Suv39h1 and Suv39h2 [9][10][11], and inhibition of the maintenance DNA methyltransferase DNMT1 [12] improve reprogramming efficiency.…”