title: Target gene analysis by microarrays and chromatin immunoprecipitation identifies HEY proteins as highly redundant bHLH repressors.
abstract: HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.
authors.affiliation: Developmental Biochemistry, Theodor-Boveri-Institute, Biocenter, University of Wuerzburg, Wuerzburg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Heisig
authors.forename: David
authors.initials: D
authors.lastname: Weber
authors.forename: Eva
authors.initials: E
authors.lastname: Englberger
authors.forename: Anja
authors.initials: A
authors.lastname: Winkler
authors.forename: Susanne
authors.initials: S
authors.lastname: Kneitz
authors.forename: Wing-Kin
authors.initials: WK
authors.lastname: Sung
authors.forename: Elmar
authors.initials: E
authors.lastname: Wolf
authors.forename: Martin
authors.initials: M
authors.lastname: Eilers
authors.forename: Chia-Lin
authors.initials: CL
authors.lastname: Wei
authors.forename: Manfred
authors.initials: M
authors.lastname: Gessler
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024721:E-Box Elements / Q000235:genetics; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D032383:Myocytes, Cardiac / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D051880:Receptors, Notch; D012097:Repressor Proteins; C485631:HEY1 protein, human; C405693:HEY2 protein, human; C497364:HEYL protein, human