Abstract
The hcn4 gene encodes the hyperpolarization activated nucleotide-gated cation channel, which is well known as cardiac pacemaker channel and specifically expressed in the sinoatrial node. However, its gene regulatory mechanism is still unclear. To examine the cis-regulatory sequences responsible for spatiotemporal hcn4 expression, we focused the evolutionally conserved non-coding sequences (CNS), which are often involved in the regulation of gene expression. The VISTA Enhancer Browser identified 16 conserved regions (CNS1-16) within the hcn4 gene locus, their enhancer effect on the activity of hcn4 minimal promoter were investigated using neonatal rat cardiac myocytes. The 160 bp fragment termed CNS13 mediated a prominent enhancer activity, more than 30-fold increase compared to the hcn4 promoter activity. We screened transcription factor binding motifs in the sequence of CNS13 by using the TRANSFAC database, and found putative AP1- and MEF2-biding sequences. A mutation in either the AP1- or MEF2-binding sequences significantly reduced the transcriptional activity of CNS13 reporter construct, and a double mutation completely abolished the enhancer activity in cardiac myocytes. We also confirmed the protein bindings to AP1- and MEF2-binding sequences within the CNS13 by EMSA. Our results suggest that a novel enhancer containing AP1- and MEF2-site may play a pivotal role for the hcn4 expression, and present a perspective for the mechanisms of cardiac electrophysiological differentiation. [J Physiol Sci. 2008;58 Suppl:S185]
