De-differentiation by Adenovirus E1A Due to Inactivation of Hippo Pathway Effectors YAP and TAZ [ChIP-seq]. De-differentiation by Adenovirus E1A Due to Inactivation of Hippo Pathway Effectors YAP and TAZ [ChIP-seq]

Adenovirus-transformed cells have a de-differentiated phenotype. Eliminating E1A in transformed human embryonic kidney cells de-repressed ~2600 genes, generating a gene expression profile closely resembling mesenchymal stem cells (MSC). This was associated with a dramatic change in cell morphology from one with scant cytoplasm and a globular nucleus to one with increased cytoplasm, extensive actin stress fibers and actomyosin-dependent flattening against the substratum. E1A-induced histone hypoacetylation by p300/CBP at H3K27/18 was reversed. Most of the increase in H3K27/18ac was near TEAD transcription factors associated with their co-activators YAP and TAZ regulated by the Hippo pathway. E1A causes YAP/TAZ cytoplasmic sequestration. After eliminating E1A, YAP/TAZ were transported into nuclei where they associated with poised enhancers with DNA-bound TEAD4 and H3K4me1. This activation of YAP/TAZ required RHO-family GTPase signaling and caused histone acetylation by p300/CBP, chromatin remodeling, and cohesin loading to establish MSC-associated enhancers and then super-enhancers. Consistent results were also observed in rat embryo kidney cells, human fibroblasts and human respiratory tract epithelial cells. These results together with earlier studies suggest that YAP/TAZ function in a developmental check-point controlled by signaling from the actin cytoskeleton that prevents differentiation of a progenitor cell until it is in the correct cellular and tissue environment. Overall design: Examination of YAP, H3K18ac, H3K27ac, H3K4me1, TEAD1, TEAD4, RAD21, chromatin accessibility and mRNA by ChIP-seq, ATAC-seq, and mRNA-seq

腺病毒转化细胞呈现去分化表型。在转化的人类胚胎肾细胞中敲除E1A后，约2600个基因被去抑制，其基因表达谱与间充质干细胞 (mesenchymal stem cells, MSC) 高度相似。这一现象伴随细胞形态发生显著改变：从胞质稀少、细胞核呈球状的形态，转变为胞质增多、形成大量肌动蛋白应力纤维，并依赖肌动球蛋白贴附于基质表面的扁平形态。E1A介导的p300/CBP对H3K27/18位点的组蛋白低乙酰化被逆转。H3K27/18位点乙酰化水平的上调大多富集于结合了其共激活因子YAP与TAZ的TEAD转录因子附近，而YAP与TAZ受Hippo信号通路调控。E1A可导致YAP/TAZ被隔离于细胞质中。敲除E1A后，YAP/TAZ被转运至细胞核内，与结合了DNA的TEAD4以及带有H3K4me1标记的 poised增强子 (poised enhancers) 结合。这一YAP/TAZ的激活过程依赖RHO家族GTP酶信号通路，并通过p300/CBP介导组蛋白乙酰化、染色质重塑以及黏连蛋白 (cohesin) 加载，从而建立与间充质干细胞相关的增强子及超级增强子。在大鼠胚胎肾细胞、人类成纤维细胞以及人类呼吸道上皮细胞中也观察到了一致的结果。上述结果结合前期研究表明，YAP/TAZ在由肌动蛋白细胞骨架信号调控的发育检验点中发挥功能，该检验点可阻止祖细胞 (progenitor cell) 分化，直至其处于正确的细胞及组织微环境中。实验设计：通过染色质免疫共沉淀测序 (chromatin immunoprecipitation sequencing, ChIP-seq)、转座酶可及性测序 (assay for transposase-accessible chromatin using sequencing, ATAC-seq) 以及mRNA测序 (mRNA-seq)，检测YAP、H3K18ac、H3K27ac、H3K4me1、TEAD1、TEAD4、RAD21的结合情况、染色质可及性以及基因表达水平。