AIMS: Hcn4, which encodes the hyperpolarization-activated, cyclic nucleotide-sensitive channel (I(h)), is a well-established marker of the cardiac sino-atrial node. We aimed to identify cis-elements in the genomic locus of the Hcn4 gene that regulate the transcription of Hcn4. METHODS AND RESULTS: We screened evolutionarily conserved non-coding sequences (CNSs) that are often involved in the regulation of gene expression. The VISTA Enhancer Browser identified 16 regions, termed CNS 1-16, within the Hcn4 locus. Using the luciferase reporter assay in primary neonatal rat cardiomyocytes, we found that CNS13 conferred a prominent enhancer activity (more than 30-fold) on the Hcn4 promoter. Subsequent mutation analysis revealed that the Hcn4 enhancer function was dependent on myocyte enhancer factor-2 (MEF2) and activator protein-1 (AP1) binding sequences located in CNS13. Electrophoretic mobility shift assay and chromatin immunoprecipitation confirmed that MEF2 and AP1 proteins bound CNS13. Furthermore, overexpression of a dominant negative MEF2 mutant inhibited the enhancer activity of CNS13, decreased Hcn4 mRNA expression and also decreased the amplitude of I(h) current in myocytes isolated from the inflow tract of embryonic heart. CONCLUSION: These results suggest that the novel enhancer CNS13 and MEF2 may play a critical role in the transcription of Hcn4 in the heart.
AIMS: Hcn4, which encodes the hyperpolarization-activated, cyclic nucleotide-sensitive channel (I(h)), is a well-established marker of the cardiac sino-atrial node. We aimed to identify cis-elements in the genomic locus of the Hcn4 gene that regulate the transcription of Hcn4. METHODS AND RESULTS: We screened evolutionarily conserved non-coding sequences (CNSs) that are often involved in the regulation of gene expression. The VISTA Enhancer Browser identified 16 regions, termed CNS 1-16, within the Hcn4 locus. Using the luciferase reporter assay in primary neonatal rat cardiomyocytes, we found that CNS13 conferred a prominent enhancer activity (more than 30-fold) on the Hcn4 promoter. Subsequent mutation analysis revealed that the Hcn4 enhancer function was dependent on myocyte enhancer factor-2 (MEF2) and activator protein-1 (AP1) binding sequences located in CNS13. Electrophoretic mobility shift assay and chromatin immunoprecipitation confirmed that MEF2 and AP1 proteins bound CNS13. Furthermore, overexpression of a dominant negative MEF2 mutant inhibited the enhancer activity of CNS13, decreased Hcn4 mRNA expression and also decreased the amplitude of I(h) current in myocytes isolated from the inflow tract of embryonic heart. CONCLUSION: These results suggest that the novel enhancer CNS13 and MEF2 may play a critical role in the transcription of Hcn4 in the heart.
Authors: M Germana Sanna; Kevin P Vincent; Emanuela Repetto; Nhan Nguyen; Steven J Brown; Lusine Abgaryan; Sean W Riley; Nora B Leaf; Stuart M Cahalan; William B Kiosses; Yasushi Kohno; Joan Heller Brown; Andrew D McCulloch; Hugh Rosen; Pedro J Gonzalez-Cabrera Journal: Mol Pharmacol Date: 2015-10-22 Impact factor: 4.436
Authors: Rajan Sah; Pietro Mesirca; Marjolein Van den Boogert; Jonathan Rosen; John Mably; Matteo E Mangoni; David E Clapham Journal: Proc Natl Acad Sci U S A Date: 2013-07-22 Impact factor: 11.205
Authors: Xiaochun Long; Darla L Tharp; Mary A Georger; Orazio J Slivano; Monica Y Lee; Brian R Wamhoff; Douglas K Bowles; Joseph M Miano Journal: J Biol Chem Date: 2009-10-01 Impact factor: 5.157
Authors: Alicia D'Souza; Annalisa Bucchi; Anne Berit Johnsen; Sunil Jit R J Logantha; Oliver Monfredi; Joseph Yanni; Sukhpal Prehar; George Hart; Elizabeth Cartwright; Ulrik Wisloff; Halina Dobryznski; Dario DiFrancesco; Gwilym M Morris; Mark R Boyett Journal: Nat Commun Date: 2014-05-13 Impact factor: 14.919