Characterization of the cardiac KCNE1 gene promoter.
BACKGROUND
KCNE1 encodes an essential cardiac slow delayed-rectifier potassium current (I(Ks)) beta-subunit (minK). Varying minK expression is important in disease-related remodeling and species-dependent expression. This study addressed 5'-regulatory elements that potentially control KCNE1 transcription.


RESULTS
The transcriptional start site of human KCNE1 (HKCNE1) was determined with 5'RACE. Of four isoforms, the putative promoter driving the isoforms constituting >80% expression in human hearts was further analyzed. A 1625-bp region 5' to the transcriptional start site was subcloned into luciferase-reporter plasmid (PGL3-Basic). The full promoter sequence increased luciferase expression 31-fold in neonatal rat cardiomyocytes (NRMs). A much smaller 327-bp core promoter maintained activity 21-29 fold. The core promoter conferred cardiomyocyte-preferential expression, with an activity in NRMs 4.9-fold greater than in Chinese Hamster Ovary cells (CHOs), compared to approximately 2.0 for the full-length promoter. Site-directed mutagenesis of all three GATA elements in the core promoter reduced its activity by >50% and attenuated cardiomyocyte-preferential expression. Mutagenesis of the second GATA element alone decreased promoter activity by approximately 50%. GATA4 knockdown with siRNA inhibited approximately 40% of core promoter activity in NRMs. Angiotensin-II increased HKCNE1 promoter activity, but only in the presence of intact GATA elements. The typically low-level I(Ks) expression in mouse and rabbit is related to low minK expression. Cloning of the mouse KCNE1 (MKCNE1) 5'-regulatory region showed approximately 50% sequence identity to human. MKCNE1 had only 1 GATA element in the region corresponding to the human core promoter and had less promoter activity (11.7 vs 29.0-fold PGL3-Basic for human).


CONCLUSIONS
Promoter elements in the HKCNE1 5'-end, particularly GATA binding sites, may be important in tissue, disease and species-related transcriptional regulation of I(Ks).
Mustapha
Zenab
Z
Research Center and Department of Medicine, Montreal Heart Institute and Université de Montréal, Montreal, Quebec, Canada.
Pang
Li
L
Nattel
Stanley
S
D020121:5' Untranslated Regions; D000818:Animals; D001145:Arrhythmias, Cardiac / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000235:genetics; D050980:GATA Transcription Factors / Q000235:genetics; D020868:Gene Silencing; D006801:Humans; D051379:Mice; D032383:Myocytes, Cardiac / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated / Q000032:analysis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics*; D012313:RNA / Q000032:analysis; D034622:RNA Interference; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D017931:DNA Primers; D050980:GATA Transcription Factors; C489622:KCNE1 protein, human; C092068:Kcne1 protein, mouse; D024642:Potassium Channels, Voltage-Gated; D020033:Protein Isoforms; D012313:RNA; D004247:DNA