Mutual dependence of the MRTF-SRF and YAP-TEAD pathways in cancer-associated fibroblasts is indirect and mediated by cytoskeletal dynamics.

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 TGFß signaling. Thus, the MRF-SRF and YAP-TEAD pathways interact indirectly through their ability to control cytoskeletal dynamics.

Author Correction: Enhancer decommissioning by LSD1 during embryonic stem cell differentiation.

In this Letter, the western blot for LSD1 in the right panel of Fig. 2b ('TCP +') was inadvertently duplicated from the tubulin blot immediately below. The actual tubulin western blot shows the same result, with no significant change to the levels of tubulin (see Fig. 1 of this Amendment). In addition, the western blots for LSD1 and HDAC1 of Fig. 3b and c have been corrected to include vertical black lines to delineate the juxtaposition of lanes that were non-adjacent in the original blotting experiment (see Fig. 2 of this Amendment). Supplementary Figs. 4a, 6b and 9b have also been corrected to delineate non-adjacent lanes with vertical black lines (see Supplementary Information of this Amendment). The complete raw data images from these western blotting experiments can also be found in the Supplementary Information of this Amendment. The original Letter has not been corrected.

Enhancer decommissioning by LSD1 during embryonic stem cell differentiation.

Transcription factors and chromatin modifiers are important in the programming and reprogramming of cellular states during development. Transcription factors bind to enhancer elements and recruit coactivators and chromatin-modifying enzymes to facilitate transcription initiation. 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 deacetylase 1 and 2 are essential for normal T-cell development and genomic stability in mice.

Histone deacetylase 1 and 2 (HDAC1/2) regulate chromatin structure as the catalytic core of the Sin3A, NuRD and CoREST co-repressor complexes. To better understand the key pathways regulated by HDAC1/2 in the adaptive immune system and inform their exploitation as drug targets, we have generated mice with a T-cell specific deletion. Loss of either HDAC1 or HDAC2 alone has little effect, while dual inactivation results in a 5-fold reduction in thymocyte cellularity, accompanied by developmental arrest at the double-negative to double-positive transition. Transcriptome analysis revealed 892 misregulated genes in Hdac1/2 knock-out thymocytes, including down-regulation of LAT, Themis and Itk, key components of the T-cell receptor (TCR) signaling pathway. Down-regulation of these genes suggests a model in which HDAC1/2 deficiency results in defective propagation of TCR signaling, thus blocking development. Furthermore, mice with reduced HDAC1/2 activity (Hdac1 deleted and a single Hdac2 allele) develop a lethal pathology by 3-months of age, caused by neoplastic transformation of immature T cells in the thymus. Tumor cells become aneuploid, express increased levels of c-Myc and show elevated levels of the DNA damage marker, γH2AX. These data demonstrate a crucial role for HDAC1/2 in T-cell development and the maintenance of genomic stability.

Histone deacetylase 1 (HDAC1), but not HDAC2, controls embryonic stem cell differentiation.

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.

Lysine-specific demethylase 1 regulates the embryonic transcriptome and CoREST stability.

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 (ES) cells. Analysis of postimplantation gene trap embryos revealed that LSD1 expression, and therefore function, is restricted to the epiblast. Conditional deletion of LSD1 in mouse ES cells, the in vitro counterpart of the epiblast, revealed a reduction in CoREST protein and associated HDAC activity, resulting in a global increase in histone H3-Lys56 acetylation, but not H3-Lys4 methylation. Despite this biochemical perturbation, ES cells with LSD1 deleted proliferate normally and retain stem cell characteristics. Loss of LSD1 causes the aberrant expression of 588 genes, including those coding for transcription factors with roles in anterior/posterior patterning and limb development, such as brachyury, Hoxb7, Hoxd8, and retinoic acid receptor γ (RARγ). The gene coding for brachyury, a key regulator of mesodermal differentiation, is a direct target gene of LSD1 and is overexpressed in e6.5 Lsd1 gene trap embryos. Thus, LSD1 regulates the expression and appropriate timing of key developmental regulators, as part of the LSD1/CoREST/HDAC complex, during early embryonic development.