No abstract

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...

The transcriptional activator MyoD serves as a master controller of myogenesis. Often in partnership with Mef2 (myocyte enhancer factor 2), MyoD binds to the promoters of hundreds of muscle genes in proliferating myoblasts yet activates these targets only upon receiving cues that launch differentiation. What regulates this off/on switch of MyoD function has been incompletely understood, although it is known to reflect the action of chromatin modifiers. Here, we identify KAP1 (KRAB [Kr€ uppel-like associated box]-associated protein 1)/TRIM28 (tripartite motif protein 28) as a key regulator of MyoD function. In myoblasts, KAP1 is present with MyoD and Mef2 at many muscle genes, where it acts as a scaffold to recruit not only coactivators such as p300 and LSD1 but also corepressors such as G9a and HDAC1 (histone deacetylase 1), with promoter silencing as the net outcome. Upon differentiation, MSK1-mediated phosphorylation of KAP1 releases the corepressors from the scaffold, unleashing transcriptional activation by MyoD/Mef2 and their positive cofactors. Thus, our results reveal KAP1 as a previously unappreciated interpreter of cell signaling, which modulates the ability of MyoD to drive myogenesis.

Myocyte enhancer factor 2 (MEF2) is a family of transcription factors that regulates many processes, including muscle differentiation. Due to its many target genes, MEF2D requires tight regulation of transcription activity over time and by location. Epigenetic modifiers have been suggested to regulate MEF2-dependent transcription via modifications to histones and MEF2. However, the modulation of MEF2 activity by lysine methylation, an important posttranslational modification that alters the activities of transcription factors, has not been studied. We report the reversible lysine methylation of MEF2D by G9a and LSD1 as a regulatory mechanism of MEF2D activity and skeletal muscle differentiation. G9a methylates lysine-267 of MEF2D and represses its transcriptional activity, but LSD1 counteracts it. This residue is highly conserved between MEF2 members in mammals. During myogenic differentiation of C2C12 mouse skeletal muscle cells, the methylation of MEF2D by G9a decreased, on which MEF2D-dependent myogenic genes were upregulated. We have also identified lysine-267 as a methylation/demethylation site and demonstrate that the lysine methylation state of MEF2D regulates its transcriptional activity and skeletal muscle cell differentiation.

Pluripotent stem cells (PSCs) have distinct metabolic properties that support their metabolic and energetic needs and affect their stemness. In particular, high glycolysis is critical for the generation and maintenance of PSCs. However, it is unknown how PSCs maintain and acquire this metabolic signature. In this study, we found that core pluripotency factors regulate glycolysis directly by controlling the expression of glycolytic enzymes. Specifically, Oct4 directly governs Hk2 and Pkm2, which are important glycolytic enzymes that determine the rate of glycolytic flux. The overexpression of Hk2 and Pkm2 sustains high levels of glycolysis during embryonic stem cell (ESC) differentiation. Moreover, the maintenance of high glycolysis levels by Hk2 and Pkm2 overexpression hampers differentiation and preserves the pluripotency of ESCs in the absence of leukemia inhibitory factor. Overall, our study identifies a direct molecular connection between core pluripotency factors and ESC metabolic signatures and demonstrates the significance of metabolism in cell fate determination. STEM CELLS 2015;33:2699-2711 SIGNIFICANCE STATEMENTAlthough distinct metabolic properties are critical for the generation and maintenance of Pluripotent stem cells (PSCs), it is unknown how PSCs maintain and acquire this metabolic signature. In this study, we found that core pluripotency factors regulated glycolysis directly by targeting key glycolytic enzymes in embryonic stem cells (ESCs). Furthermore, we found that sustainment of high glycolytic flux delays ESC differentiation and enables certain populations of ESCs to retain the capacity for self-renewal and differentiation potential in the absence of LIF, demonstrating the significance of metabolism in stemness regulation and cell fate determination.