The cardiac pacemaker-specific channel Hcn4 is a direct transcriptional target of MEF2
The cardiac pacemaker-specific channel Hcn4 is a direct transcriptional target of MEF2
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DOI：
10.1093/cvr/cvp171
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发表时间：
2009-09-01
影响因子：
10.8
通讯作者：
Takano, Makoto
中科院分区：
文献类型：
--
作者：
Kuratomi, Shinobu;Ohmori, Yoko;Takano, Makoto
编码超极化激活的环核苷酸敏感通道（I-h）的Hcn 4是心脏窦房结的公认标志物。本研究的目的是在Hcn 4基因的基因组位点上鉴定调控Hcn 4转录的顺式元件，筛选出进化上保守的非编码序列（CNSs），这些序列通常参与基因表达的调控。VISTA Enhancer Browser在Hcn 4基因座内鉴定了16个区域，称为CNS 1-16。在原代新生大鼠心肌细胞中使用荧光素酶报告基因测定，我们发现CNS 13赋予Hcn 4启动子显著的增强子活性（超过30倍）。随后的突变分析显示，Hcn 4增强子功能依赖于位于CNS 13中的肌细胞增强因子-2（MEF 2）和激活蛋白-1（AP 1）结合序列。电泳迁移率变动分析和染色质免疫沉淀证实MEF 2和AP 1蛋白结合CNS 13。此外，MEF 2突变体的过表达抑制了CNS 13增强子的活性，降低了Hcn 4 mRNA的表达，并降低了心肌细胞I-h电流的幅度，这些结果表明，新的增强子CNS 13和MEF 2可能在Hcn 4的转录中起关键作用。
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.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.These results suggest that the novel enhancer CNS13 and MEF2 may play a critical role in the transcription of Hcn4 in the heart.