title: Mechanisms of epigenetic and cell-type specific regulation of Hey target genes in ES cells and cardiomyocytes.
abstract: Hey bHLH transcription factors are critical effectors of Notch signaling. During mammalian heart development they are expressed in atrial and ventricular cardiomyocytes and in the developing endocardium. Hey knockout mice suffer from lethal cardiac defects, such as ventricular septum defects, valve defects and cardiomyopathy. Despite this functional relevance, little is known about the regulation of downstream targets in relevant cell types. The objective of this study was to elucidate the regulatory mechanisms by which Hey proteins affect gene expression in a cell type specific manner. We used an in vitro cardiomyocyte differentiation system with inducible Hey1 or Hey2 expression to study target gene regulation in cardiomyocytes (CM) generated from murine embryonic stem cells (ESC). The effects of Hey1 and Hey2 are largely redundant, but cell type specific. The number of regulated genes is comparable between ESC and CM, but the total number of binding sites is much higher, especially in ESC, targeting mainly genes involved in transcriptional regulation and developmental processes. Repression by Hey proteins generally correlates with the extent of Hey-binding to target promoters, Hdac recruitment and lower histone acetylation. Functionally, treatment with the Hdac inhibitor TSA abolished Hey target gene regulation. However, in CM the repressive effect of Hey-binding is lost for a subset of genes. These also lack Hey-dependent histone deacetylation in CM and are enriched for binding sites of cardiac specific activators like Srf, Nkx2-5, and Gata4. Ectopic Nkx2-5 overexpression in ESC blocks Hey-mediated repression of these genes. Thus, Hey proteins mechanistically repress target genes via Hdac recruitment and histone deacetylation. In CM Hey-repression is counteracted by cardiac activators, which recruit histone acetylases and prevent Hey mediated deacetylation and subsequent repression for a subset of genes.
authors.affiliation: Developmental Biochemistry, Theodor Boveri Institute (Biocenter), University of Wuerzburg, Am Hubland, D-97074 Wuerzburg, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Weber
authors.affiliation: Developmental Biochemistry, Theodor Boveri Institute (Biocenter), University of Wuerzburg, Am Hubland, D-97074 Wuerzburg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Heisig
authors.affiliation: Physiological Chemistry, Theodor Boveri Institute (Biocenter), University of Wuerzburg, Am Hubland, D-97074 Wuerzburg, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Kneitz
authors.affiliation: Biochemistry and Molecular Biology, Theodor Boveri Institute (Biocenter), University of Wuerzburg, Am Hubland, D-97074 Wuerzburg, Germany.
authors.forename: Elmar
authors.initials: E
authors.lastname: Wolf
authors.affiliation: Biochemistry and Molecular Biology, Theodor Boveri Institute (Biocenter), University of Wuerzburg, Am Hubland, D-97074 Wuerzburg, Germany; Comprehensive Cancer Center Mainfranken, University of Wuerzburg, Am Hubland, D-97074 Wuerzburg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Eilers
authors.affiliation: Developmental Biochemistry, Theodor Boveri Institute (Biocenter), University of Wuerzburg, Am Hubland, D-97074 Wuerzburg, Germany; Comprehensive Cancer Center Mainfranken, University of Wuerzburg, Am Hubland, D-97074 Wuerzburg, Germany; Comprehensive Heart Failure Center, University of Wuerzburg, Am Hubland, D-97074 Wuerzburg, Germany. Electronic address: gessler@biozentrum.uni-wuerzburg.de.
authors.forename: Manfred
authors.initials: M
authors.lastname: Gessler
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D044127:Epigenesis, Genetic; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006655:Histone Deacetylases / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000378:metabolism; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012097:Repressor Proteins / Q000378:metabolism*; D034421:Sus scrofa; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D018797:Cell Cycle Proteins; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases; D006657:Histones; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C120420:Hey1 protein, mouse; C485778:Hey2 protein, mouse; C484469:Nkx2-5 protein, mouse
keywords: Cardiomyocyte differentiation; Epigenetic regulation; Notch signaling; Transcriptional repression