Conserved cis-regulatory elements are critical for the circadian regulation of human ion channel gene promoters in the heart

Background: The circadian clock plays a fundamental role in regulating cardiovascular function. RNA sequencing of mouse ventricles identified ~13,000 transcripts, with a select few ion channel genes ( Kcnh2, Gja1 ) exhibiting 24-hour oscillations in expression. Hypothesis: Core circadian clock and associated proteins interact with conserved cis-regulatory elements in the human KCNH2 and GJA1 promoters to regulate their circadian and overall expression. Objective: To identify and characterize the cis-regulatory elements that regulate both circadian and overall promoter activity of the human KCNH2 and GJA1 promoters. Methods: Real-time bioluminescence (LumiCycle, Actimetrics) and Dual Glo luciferase assays were used to analyze cloned promoter-luciferase reporter constructs expressed in C2C12 myoblasts. Bioluminescence was recorded at 10-minute intervals for 7 to 10 days to analyze the period, phase, and amplitude of promoter activity. Promoter variants were generated via targeted deletions and mutagenesis to introduce naturally occurring single-nucleotide polymorphisms (SNPs). Results: The KCNH2 promoter exhibited robust 24-hour oscillations in C2C12 cells. Deletion and mutational analyses identified a highly conserved tandem E-box element within 1 kb of the exon 1 start site as critical for both circadian and overall KCNH2 promoter activity. SNPs within this element produced variant-specific effects on promoter activity. Deletion analysis of the GJA1 promoter revealed a region whose removal significantly enhanced both circadian and overall expression, indicating a suppressive function. Conclusions: Our data demonstrate that conserved cis-regulatory elements regulate the rhythmic promoter activity of KCNH2 and GJA1. We identified a critical tandem E-box element in KCNH2 , and a suppressor region in GJA1. SNPs in the KCNH2 E-box element differentially affect promoter activity, suggesting potential physiological consequences of genetic variation in this region. These findings advance our understanding of the molecular mechanisms linking circadian rhythms to cardiac electrophysiology. This project is funded by NIH R01HL153042 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.