Generation of induced pluripotent stem cells from specialized cell types provides an excellent model to study how cells maintain their stability, and how they can change identity, especially in the context of disease. Previous studies have shown that chromatin safeguards cell identity by acting as a barrier to reprogramming. We investigated mechanisms by which the histone macroH2A variants inhibit reprogramming and discovered that they work as gate keepers of the mesenchymal cell state by blocking epithelial transition, a step required for reprogramming of mouse fibroblasts. More specifically, we found that individual macroH2A variants regulate the expression of defined sets of genes, whose overall function is to stabilize the mesenchymal gene expression program, thus resisting reprogramming. We identified a novel gene network (MSCN, mesenchymal network) composed of 63 macroH2A-regulated genes related to extracellular matrix, cell membrane, signaling and the transcriptional regulators Id2 and Snai2, all of which function as guardians of the mesenchymal phenotype. ChIP-seq and KD experiments revealed a macroH2A variant-specific combinatorial targeting of the genes reconstructing the MSCN, thus generating robustness in gene expression programs to resist cellular reprogramming.

Cellular reprogramming of somatic cells towards induced pluripotency is a multistep stochastic process mediated by the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM), which orchestrate global epigenetic and transcriptional changes. We performed a large-scale analysis of integrated ChIP-seq, ATAC-seq and RNA-seq data and revealed the spatiotemporal highly dynamic pattern of OSKM DNA binding during reprogramming. We found that OSKM show distinct temporal patterns of binding to different classes of pluripotency-related enhancers. Genes involved in reprogramming are regulated by the coordinated activity of multiple enhancers, which are sequentially bound by OSKM for strict transcriptional control. Based on these findings, we developed an unbiased approach to identify Reprogramming-Inducible Enhancers (RIEs), constructed enhancer-traps and isolated cells undergoing reprogramming in real time. We used a representative RIE taken from the Upp1 gene fused to Gfp and isolated cells at different time-points during reprogramming and found that they have unique developmental capacities as they are reprogrammed with high efficiency due to their distinct molecular signatures. In conclusion, our experiments have led to the development of an unbiased method to identify and isolate reprogrammable cells in real time by exploiting the functional dynamics of OSKM, which can be used as efficient reprogramming biomarkers.

Cellular reprogramming converts differentiated cells into a pluripotent state through extensive chromatin remodeling. The histone variant macroH2A has classically viewed as an epigenetic barrier stabilizing somatic identity and restricting pluripotency gene activation. Here, we show that during reprogramming, macroH2A1 nucleosomes undergo functional repurposing. Early in the process, mH2A1 nucleosomes act as a barrier to cellular plasticity, but later facilitate the establishment and maintenance of pluripotency by reshaping the epigenetic landscape. High-resolution chromatin profiling reveals that mH2A1.2 nucleosomes undergo rapid, large-scale repositioning, dissociating from promoters and reassembling ~30 bp away, frequently near NRF-1 binding sites. This repositioning occludes E2F4 binding, relieving cell-cycle arrest thereby enabling reprogramming. Likewise, mH2A1.1 nucleosomes display extensive mobility that culminates in deposition at pluripotency genes in ESCs, where they sustain their expression by assembling promoter transcriptional hubs. In this later role, mH2A1.1 functions as a chromatin bookmark stabilizing the Nanog, Sox2, and Oct4 network. These findings suggest that mH2A1 nucleosomal mobility underlines its context-dependent functional repurposing from reprogramming inhibitor to facilitator, illustrating how chromatin components evolve dynamic roles to coordinate cell-state transitions.

Cellular reprogramming of somatic cells towards induced pluripotency is a multistep stochastic process mediated by the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM), which orchestrate global epigenetic and transcriptional changes. We performed a large-scale analysis of integrated ChIP-seq, ATAC-seq and RNA-seq data and revealed the spatiotemporal highly dynamic pattern of OSKM DNA binding during reprogramming. We found that OSKM show distinct temporal patterns of binding to different classes of pluripotency-related enhancers. Genes involved in reprogramming are regulated by the coordinated activity of multiple enhancers, which are sequentially bound by OSKM for strict transcriptional control. Based on these findings, we developed an unbiased approach to identify Reprogramming Inducible Enhancers (RIEs), constructed enhancer-traps and isolated cells undergoing reprogramming in real-time. We used a representative RIE taken from the Upp1 gene fused to GFP and isolated cells at different time points during reprogramming and found that they have unique developmental capacities as they are reprogrammed with high efficiency due to their distinct molecular signatures. In conclusion, our experiments have led to the development of an unbiased method to identify and isolate reprogrammable cells in real-time by exploiting the functional dynamics of OSKM, which can be used as efficient reprogramming biomarkers.