The cardiac pacemaker-specific channel Hcn4 is a direct transcriptional target of MEF2.
OBJECTIVE
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.


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.


CONCLUSIONS
These results suggest that the novel enhancer CNS13 and MEF2 may play a critical role in the transcription of Hcn4 in the heart.
Kuratomi
Shinobu
S
Department of Physiology, School of Medicine, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan.
Ohmori
Yoko
Y
Ito
Masayuki
M
Shimazaki
Kuniko
K
Muramatsu
Shin-Ichi
S
Mizukami
Hiroaki
H
Uosaki
Hideki
H
Yamashita
Jun K
JK
Arai
Yuji
Y
Kuwahara
Koichiro
K
Takano
Makoto
M
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004742:Enhancer Elements, Genetic; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D064326:MEF2 Transcription Factors; D009154:Mutation; D032383:Myocytes, Cardiac / Q000378:metabolism; D018006:Myogenic Regulatory Factors / Q000235:genetics / Q000378:metabolism*; D015221:Potassium Channels / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012849:Sinoatrial Node / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; C513836:HCN4 protein, rat; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D064326:MEF2 Transcription Factors; D018006:Myogenic Regulatory Factors; D015221:Potassium Channels; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D004247:DNA