Abstract 1920: Nkx2–5-dependent Transcriptional Regulation of ANF and BNP Through a Common Distal Enhancer
Background: Mutations in homeoprotein NKX2–5 are linked to human congenital heart disease most likely due to dysregulated expression of its downstream targets, yet its transcriptional regulation in vivo is largely unexplored. Nkx2–5 has been shown to regulate transcription of the cardiac fetal genes atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP), which are tandemly located on mouse chromosome 4 and separated by approximately 14 kb. Because the two genes are positively regulated by Nkx2–5, we hypothesized the presence of a common enhancer element regulating expression of the two genes.
Methods and Results: In tamoxifen-inducible Nkx2–5 knockout mice, expression of ANF and BNP was markedly downregulated shortly after deletion of the Nkx2–5. Using isolated neonatal cardiomyocytes from mice either expressing or lacking Nkx2–5, we performed chromatin immunoprecipitation assays followed by real-time PCR to identify Nkx2–5 specific binding elements. We examined the genomic region between −44 kb and +3 kb relative to the ANF transcription start site, and identified Nkx2–5 binding sites in the proximal promoters of ANF and BNP as well as in three additional 5′ distal regions, at −34, −31, −21 kb. Using the chromosome conformation capture methods, we determined that the two promoters are in relative close proximity with the element at −31 kb only in cardiomyocytes expressing Nkx2–5. These results suggest that Nkx2–5 proteins mediate the proximity between the −31kb element and the two proximal promoters. Luciferase reporter assays in the mouse cardiomyocytes demonstrated that the ANF proximal promoter contains Nkx2–5 responsive elements, and addition of the −31 kb element further increased the level of transcription in an Nkx2–5-dependent manner. While transcription from the BNP proximal promoter is not Nkx2–5-dependent, an addition of the −31 kb element renders transcription Nkx2–5-dependent.
Conclusion: Nkx2–5-dependent transcription of the ANF and BNP genes in mouse neonatal cardiomyocytes is regulated by proximal promoter elements and a common distal −31 kb element. Nkx2–5 mediates the formation of a chromatin loop that brings together the promoter elements and the distal enhancer element possibly forming an active chromatin hub.
This research has received full or partial funding support from the American Heart Association, National Center.