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.
Highlights
NOTCH signaling is a key regulatory pathway for cardiovascular development and homeostasis [1]
The three HEY genes encode basic helixloop-helix transcription factors that are critical effectors to convey signaling by NOTCH receptors and similar signaling systems
By gene expression analysis and chromatin immunoprecipitation we have identified a large set of HEY target genes
Summary
NOTCH signaling is a key regulatory pathway for cardiovascular development and homeostasis [1]. Its receptors mainly act through transcriptional activation of target genes by a complex of the NOTCH intracellular domain, released by gamma-secretase, the transcription factor CBF1 (RBP-Jk) and the Mastermind coactivator proteins (Maml). Without NOTCH binding CBF1 has a repressive function and associates with additional corepressor proteins. Upon activation different and in part cell type specific target genes are induced, the most prominent ones encoding members of the HEY and HES family of bHLH repressor proteins. There are three HEY genes (HEY1, HEY2 and HEYL) and several HES genes, with HES1 being the closest relative. All are related to the Drosophila hairy and Enhancer-of-split genes, which are well known transcriptional repressor proteins
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have