Editors' ChoiceDevelopment

Notch signals to Yap too

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Sci. Signal.  08 Sep 2015:
Vol. 8, Issue 393, pp. ec256
DOI: 10.1126/scisignal.aad3662

Phosphorylation of the transcriptional coactivator Yap, notably by the Hippo signaling pathway, causes its cytoplasmic retention and degradation. Yap activity is also regulated by Hippo-independent mechanisms, and it interacts with many DNA-binding proteins. Manderfield et al. found in developing vascular smooth muscle Yap interacted with the Notch intracellular domain (NICD). In mouse embryos lacking both Yap and the related protein Taz specifically in neural crest cells, the subset of these cells that gives rise to vascular smooth muscle migrated to their proper position but failed to differentiate. Compared with wild-type mouse embryonic fibroblasts (MEFs), MEFs lacking both Yap and Taz showed reduced expression of Jag1, which encodes a Notch ligand that is necessary for differentiation of neural crest into smooth muscle. Yap and NICD cooperatively stimulated the expression of luciferase reporters for the Notch targets Jag1 and Hes1 in HEK293T cells. Neither Yap nor NICD have intrinsic DNA-binding activity; they must partner with DNA-binding proteins, such as the Yap partner Tead or the NICD partner Rbp-J, to stimulate the expression of target genes. Coexpression of Tead1 did not further stimulate Yap- and NICD-dependent expression of the Jag1 reporter in cells also expressing plasmids encoding Yap and NICD, but mutating the Rbp-J binding site in the reporter prevented Yap- and NICD-dependent expression, as did the coexpression of Yap with mutations in one of two WW domains and NICD. Chromatin immunoprecipitation experiments demonstrated that overexpressed Yap and NICD both bound to the Jag1 reporter in HEK293T cells, but not when the Rbp-J binding site in the enhancer region of the Jag1 reporter was mutated. Endogenous Yap was present at both the Jag1 and Hes1 promoters in cultured breast cancer cells and in a cultured mouse aortic smooth muscle cell line. Overexpression of Hippo cascade kinases that promote Yap phosphorylation prevented Yap-mediated activation of the Jag1 reporter in HEK293T cells and inhibited expression of endogenous Jag1, Hes1, and smooth muscle markers in the smooth muscle cell line. Yap and NICD coimmunoprecipitated from extracts of both HEK293T cells and the cultured smooth muscle cells. These findings indicate that Yap forms a complex with NICD and that this complex is recruited to target gene promoters by Rbp-J.

L. J. Manderfield, H. Aghajanian, K. A. Engleka, L. Y. Lim, F. Liu, R. Jain, L. Li, E. N. Olson, J. A. Epstein, Hippo signaling is required for Notch-dependent smooth muscle differentiation of neural crest. Development 142, 2962–2971 (2015). [PubMed]