Hey Basic Helix-Loop-Helix Transcription Factors Are Repressors of GATA4 and GATA6 and Restrict Expression of the GATA Target Gene ANF in Fetal Hearts

Abstract

The Hey basic helix-loop-helix transcription factors are downstream effectors of Notch signaling in the cardiovascular system. Mice lacking Hey2 develop cardiac hypertrophy, often associated with congenital heart defects, whereas combined Hey1/Hey2 deficiency leads to severe vascular defects and embryonic lethality around embryonic day E9.5. The molecular basis of these disorders is poorly understood, however, since target genes of Hey transcription factors in the affected tissues remain elusive. To identify genes regulated by Hey factors we have generated a conditional Hey1 knockout mouse. This strain was used to generate paired Hey2-and Hey1/2-deficient embryonic stem cell lines. Comparison of these cell lines by microarray analysis identified GATA4 and GATA6 as differentially expressed genes. Loss of Hey1/2 leads to elevated GATA4/6 and ANF mRNA levels in embryoid bodies, while forced expression of Hey factors strongly represses expression of the GATA4 and GATA6 promoter in various cell lines. In addition, the promoter activity of the GATA4/6 target gene ANF was inhibited by Hey1, Hey2, and HeyL. Protein interaction and mutation analyses suggest that repression is due to direct binding of Hey proteins to GATA4 and GATA6, blocking their transcriptional activity. In Hey2-deficient fetal hearts we observed elevated mRNA levels of ANF and CARP. Expression of ANF and Hey2 is normally restricted to the trabecular and compact myocardial layer, respectively. Intriguingly, loss of Hey2 leads to ectopic ANF expression in the compact layer, suggesting a direct role for Hey2 in limiting ANF expression in this cardiac compartment.

Vertebrate heart growth is regulated by functional antagonism between Gridlock and Gata5

“…This cross-transcriptional interaction prevents excessive heart growth and thus ensures an optimal mass for the embryonic heart. The ability of transcriptional repression and physical association of murine Hey2/Hrt2 with Gata4 has been shown to lie in the same basic domain (25,26). In our study, we have found that the Grl carboxyl region containing the Orange domain is required for physical association with Gata5, whereas the bHLH domain is critical for inhibiting the Gata5 transcriptional activity.…”

“…Surprisingly, deletion of the carboxyl region, the putative Gata5 binding region, did not result in a complete loss of repressive activity. Because Hey2/Hrt2 protein does not affect repression through direct DNA binding (25)(26)(27), this result may be explained by the possibility that the bHLH domain might recruit other DNA-binding factors or Gata5-interacted proteins in HeLa cells that restore Grl repressive ability, but this may not occur in vivo and in cultured cardiomyocytes (Fig. 3M and SI Appendix, section VII).…”

Brugada syndrome risk loci seem protective against atrial fibrillation

“…Furthermore, HEY2-deficient embryos displayed a change in the expression patterns of GJA5, NPPA, and TBX5. 43,45,46 These genes have all previously been associated with AF. [47][48][49][50] Thus, several studies support our finding that BrS-associated genetic variants protect against AF.…”

Novel and functional DNA sequence variants within the GATA5 gene promoter in ventricular septal defects

“…Heterozygous loss of GATA6 gene result in reduced expression of NKX2-5 gene [10]. In addition, Hey basic helix-loop-helix transcription factor 2 (Hey2), a downstream effector of Notch signaling, binds to GATA6 and directly represses ANF gene expression [52]. Mice lacking Hey2 develop VSD and cardiomyopathy [53].…”