Cardiovascular Basic Helix Loop Helix Factor 1, a Novel Transcriptional Repressor Expressed Preferentially in the Developing and Adult Cardiovascular System

Michael T Chin,Koji Maemura,Shinya Fukumoto,Mukesh K Jain,Matthew D Layne,Masafumi Watanabe,Chung-Ming Hsieh,Mu-En Lee

doi:10.1074/jbc.275.9.6381

Abstract

We have cloned a cardiovascular-restricted basic helix-loop-helix factor that interacts with arylhydrocarbon receptor nuclear translocator (ARNT) in a yeast two-hybrid screen. Cardiovascular helix-loop-helix factor 1 (CHF1) is distantly related to the hairy family of transcriptional repressors. We analyzed its expression pattern during mouse embryo development. At day 8.5, the expression of CHF1 is first detected in the primitive ventricle of the primordial heart tube and persists throughout gestation. In rat hearts, this expression is down-regulated after birth, concurrent with terminal differentiation of cardiomyocytes. In the developing vasculature, CHF1 first appears in the dorsal aorta at day 9.0, which precedes the reported expression of smooth muscle cell markers, and persists into adulthood. In an in vitro system of smooth muscle cell differentiation, CHF1 mRNA was barely detectable in undifferentiated cells but was induced highly in differentiated smooth muscle cells. To determine whether CHF1 might affect the function of ARNT, we performed transfection studies. Co-transfection of CHF1 inhibited ARNT/EPAS1-dependent transcription by 85%, and this inhibition is dose-dependent. In electrophoretic mobility studies, CHF1 inhibited the binding of the ARNT/EPAS1 heterodimer to its target site. Our data suggest that CHF1 functions as a transcriptional repressor and may play an important role in cardiovascular development.

Highlights

Transcriptional control of cardiovascular development in vertebrates is a complex process requiring the coordinated expression of a variety of factors in a temporally and spatially defined manner)
In order to determine the mechanism by which Cardiovascular helix-loop-helix factor 1 (CHF1) inhibits vascular endothelial cell growth factor (VEGF) promoter activation, we performed EMSAs to determine the effect of CHF1 on the binding of the arylhydrocarbon receptor nuclear translocator (ARNT)/endothelial PAS domain protein 1 (EPAS1) heterodimer to the HIF1binding site
Deletion of amino acids 1–37 and 147–337, leaving primarily the Basic helix-loop-helix (bHLH) domain, abolished repression. These results suggest that the bHLH domain is necessary but not sufficient for repression and an intact Orange domain or other domains in the COOH terminus are required for repression of ARNT-dependent transcription

Summary

Introduction

Transcriptional control of cardiovascular development in vertebrates is a complex process requiring the coordinated expression of a variety of factors in a temporally and spatially defined manner (for review, see Refs. 1 and 2)). CHF1, A Novel bHLH Transcriptional Repressor using the bHLH/PAS domain of ARNT as a bait to probe a human heart cDNA library. We found that CHF1 was highly expressed in developing cardiomyocytes and vascular smooth muscle cells but was down-regulated in adult heart when cardiac myocytes became terminally differentiated.

Results

Conclusion