Transcription factors and chromatin modifiers are important in the programming and reprogramming of cellular states during development1,2. Transcription factors bind to enhancer elements and recruit coactivators and chromatin-modifying enzymes to facilitate transcription initiation3,4. During differentiation a subset of these enhancers must be silenced, but the mechanisms underlying enhancer silencing are poorly understood. Here we show that the histone demethylase lysine-specific demethylase 1 (LSD1; ref. 5), which demethylates histone H3 on Lys 4 or Lys 9 (H3K4/K9), is essential in decommissioning enhancers during the differentiation of mouse embryonic stem cells (ESCs). LSD1 occupies enhancers of active genes that are critical for control of the state of ESCs. However, LSD1 is not essential for the maintenance of ESC identity. Instead, ESCs lacking LSD1 activity fail to differentiate fully, and ESC-specific enhancers fail to undergo the histone demethylation events associated with differentiation. At active enhancers, LSD1 is a component of the NuRD (nucleosome remodelling and histone deacetylase) complex, which contains additional subunits that are necessary for ESC differentiation. We propose that the LSD1–NuRD complex decommissions enhancers of the pluripotency program during differentiation, which is essential for the complete shutdown of the ESC gene expression program and the transition to new cell states.

Histone deacetylases (HDAC) 1 and 2 are highly similar enzymes that help regulate chromatin structure as the core catalytic components of corepressor complexes. Although tissue-specific deletion of HDAC1 and HDAC2 has demonstrated functional redundancy, germ-line deletion of HDAC1 in the mouse causes early embryonic lethality, whereas HDAC2 does not. To address the unique requirement for HDAC1 in early embryogenesis we have generated conditional knockout embryonic stem (ES) cells in which HDAC1 or HDAC2 genes can be inactivated. Deletion of HDAC1, but not HDAC2, causes a significant reduction in the HDAC activity of Sin3A, NuRD, and CoREST corepressor complexes. This reduced corepressor activity results in a specific 1.6-fold increase in histone H3 K56 acetylation (H3K56Ac), thus providing genetic evidence that H3K56Ac is a substrate of HDAC1. In culture, ES cell proliferation was unaffected by loss of either HDAC1 or HDAC2. Rather, we find that loss of HDAC1 affects ES cell differentiation. ES cells lacking either HDAC1 or HDAC2 were capable of forming embryoid bodies (EBs), which stimulates differentiation into the three primary germ layers. However, HDAC1-deficient EBs were significantly smaller, showed spontaneous rhythmic contraction, and increased expression of both cardiomyocyte and neuronal markers. In summary, our genetic study of HDAC1 and HDAC2 in ES cells, which mimic the embryonic epiblast, has identified a unique requirement for HDAC1 in the optimal activity of HDAC1/2 corepressor complexes and cell fate determination during differentiation.corepressor | acetylation | deacetylation | chromatin C lass I histone deacetylases (HDAC1, -2, -3, and -8) are highly conserved enzymes present in the nucleus of all cells, where they help modulate levels of gene expression (1). The best characterized substrates of HDACs are the N-terminal tails of the core histones H2A, H2B, H3 and H4. Deacetylation of histone tails results in a "tightening" of chromatin, due to the electrostatic potential of unacetylated lysine residues to promote internucleosomal interactions (2, 3) and the loss of a binding site for components of the transcriptional machinery containing a bromodomain, e.g., TAFII1 (4).Among the class I HDACs, HDAC1 and HDAC2 are the most similar (83% amino acid identity), sharing an almost identical catalytic core domain and a conserved C-terminal tail (5). In mammalian cells HDAC1 and HDAC2 interact together (6) to form the catalytic core of a number of higher-order complexes including Sin3A, NuRD, CoREST, and NODE (reviewed in ref. 1). These complexes are then targeted to chromatin by sequencespecific (often cell-specific) transcription factors to repress transcription in cooperation with other chromatin modifiers, such as the lysine-specific demethylase, LSD1, in the CoREST complex (7-9). As part of these multiprotein complexes, the activity of HDAC1 and HDAC2 has been implicated in the regulation of cell cycle progression by tumor suppressors (10-12), differentiation (13, 14), cellular aging (15), and c...

Lysine-specific demethylase 1 (LSD1), which demethylates mono-and dimethylated histone H3-Lys4 as part of a complex including CoREST and histone deacetylases (HDACs), is essential for embryonic development in the mouse beyond embryonic day 6.5 (e6.5). To determine the role of LSD1 during this early period of embryogenesis, we have generated loss-of-function gene trap mice and conditional knockout embryonic stem ( The methylation of lysine residues within histones H3 and H4 helps to regulate the higher-order structure of chromatin in eukaryotic genomes. The consequences of lysine methylation on gene expression (unlike acetylation) can be either positive or negative, depending on the context of a particular lysine residue and the number of methyl moieties added (24, 27). Trimethylation of K9 on histone H3 (H3K9me3), for example, is generally associated with silenced genes and constitutive heterochromatin. In contrast, trimethylation of K4 on the same histone, H3 (H3K4me3), is associated with transcriptionally active regions. These methylated lysine residues provide the docking sites for the subsequent binding of chromatin-associated proteins with a cognate chromodomain, plant homeo domain (PHD) finger, or Tudor domain (42). Thus, the four methylated states of each specific lysine (unmodified or mono-, di-, or trimethylated) are interpreted by the association of other factors, such as the binding of HP1␣ to trimethyl H3 Lys9 (3, 29), which modify chromatin directly or indirectly. Lysine methylation in vivo is controlled by the opposing activities of lysine methyltransferases (KMTs) and lysine demethylases (KDMs). KDMs appear in two varieties: the amine oxidases, of which there are two (lysine-specific demethylase 1

Both the MRTF-SRF and the YAP-TEAD transcriptional regulatory networks respond to extracellular signals and mechanical stimuli. We show that the MRTF-SRF pathway is activated in cancer-associated fibroblasts (CAFs). The MRTFs are required in addition to the YAP pathway for CAF contractile and proinvasive properties. We compared MRTF-SRF and YAP-TEAD target gene sets and identified genes directly regulated by one pathway, the other, or both. Nevertheless, the two pathways exhibit mutual dependence. In CAFs, expression of direct MRTF-SRF genomic targets is also dependent on YAP-TEAD activity, and, conversely, YAP-TEAD target gene expression is also dependent on MRTF-SRF signaling. In normal fibroblasts, expression of activated MRTF derivatives activates YAP, while activated YAP derivatives activate MRTF. Cross-talk between the pathways requires recruitment of MRTF and YAP to DNA via their respective DNA-binding partners (SRF and TEAD) and is therefore indirect, arising as a consequence of activation of their target genes. In both CAFs and normal fibroblasts, we found that YAP-TEAD activity is sensitive to MRTF-SRF-induced contractility, while MRTF-SRF signaling responds to YAP-TEAD-dependent TGFβ signaling. Thus, the MRF-SRF and YAP-TEAD pathways interact indirectly through their ability to control cytoskeletal dynamics.