Three-dimensional organization of the genome has emerged as an important player in transcriptional regulation [1][2][3][4][5][6][7] . In mammals, CTCF and the cohesin complex create sub-megabase structures with elevated internal chromatin contact frequencies, called topologically associating domains (TADs) [8][9][10][11] . Although TADs are generally considered important for transcriptional regulation, ablation of TAD organization by disrupting CTCF or the cohesin complex only caused modest gene expression changes [12][13][14][15] . In contrast, CTCF is required for cell cycle regulation 16 , early embryo development and for the formation of various adult cell types 17 . To uncouple the role of CTCF in cell state transitions and cell proliferation we studied the effect of CTCF be efficiently converted by exogenous CEBPA expression into functional induced macrophages (iMacs) with only one cell division on average (Fig. 1a; Supplementary Note 1) 26 . Using this system, we analyzed a time-series of transdifferentiating cells for genome-wide changes in 3D genome organization (in-situ Hi-C), enhancer activity (ChIP-seq of histone modifications), chromatin accessibility (ATAC-seq) and gene expression (RNA-seq).We first determined genome segmentation into A and B compartments on the basis of the first eigenvector values of a principal component analysis (PCA) on the Hi-C correlation matrix ('PC1 values'). Overall, although most of the genome remained stable, around 14% of A or B compartment regions were dynamic during transdifferentiation, showing transcriptional changed correlating with the altered compartmentalization state (Fig. 1b-f, Extended Data Fig. 1a-d; Supplementary Note 2). Next, we used chromosome-wide insulation potential 27 to identify between 3,100-3,300 TAD borders per time point (Fig. 1g). Boundaries were highly reproducible between biological replicates (Jaccard index>0.99) and enriched in binding sites for CTCF (Extended Data Fig. 1e). Genome-wide insulation scores analysed by PCA over time revealed progressive changes, reflecting a transdifferentiation trajectory (Extended Data Fig. 1f). While 70% of TAD borders were stable across all stages, 18% were lost or gained and 12% were transiently altered (Fig. 1g). CTCF binding was significantly more enriched at stable than at dynamic boundaries (Fig. 1h), as observed earlier 28 . Furthermore, while lost borders showed some CTCF occupancy in B cells that decreased in iMacs, gained borders were depleted for CTCF in both cell states (Fig. 1h), indicating CTCF-independent mechanisms driving local insulation. The dynamic rearrangement of TAD borders during transdifferentiation is illustrated by the DDX54 locus (Fig. 1i), in which a new boundary appears in iMacs without apparent changes in CTCF binding. Furthermore, border gain or loss did not correlate with changes in local gene expression (Extended Data Fig. 1g), indicating that transcription is not a driver of the observed changes. However, whereas motif analysis at ATAC-seq peaks within stable borders indee...
Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) requires activation of the pluripotency network and resetting of the epigenome by erasing the epigenetic memory of the somatic state. In female mouse cells, a critical epigenetic reprogramming step is the reactivation of the inactive X chromosome. Despite its importance, a systematic understanding of the regulatory networks linking pluripotency and X-reactivation is missing. Here we reveal the pathways important for iPSC reprogramming and X-reactivation using a genome-wide CRISPR screen. In particular, we discover that activation of the interferon γ (IFNγ) pathway early during reprogramming accelerates pluripotency acquisition and X-reactivation. IFNγ stimulates STAT3 signaling and the pluripotency network and leads to enhanced TET-mediated DNA demethylation, which consequently boosts X-reactivation. We therefore gain a mechanistic understanding of the role of IFNγ in reprogramming and X-reactivation and provide a comprehensive resource of the molecular networks involved in these processes.
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