Abstract Body: Background: KCNH2 encodes a voltage-gated potassium channel (Kv11.1), which conducts
the rapid delayed rectifier potassium current (IKr) critical for ventricular repolarization in the heart.
Circadian clock factors regulate the expression of the KCNH2 mRNA transcript in the heart.
However, the molecular mechanisms for the circadian expression of KCNH2 transcripts have
yet to be defined.

Hypothesis: A conserved cis-regulatory element in the core KCNH2 promoter drives circadian
regulation of human KCNH2 (hKCNH2) promoter activity.

Objective: Identify the molecular determinants of the circadian regulation of hKCNH2 promoter
activity and determine the impact of single nucleotide polymorphisms (SNPs) in this region.

Methods: Real-time bioluminescence (LumiCycle, Actimetrics) and Dual Glo luciferase assays
were employed to identify critical cis elements that regulate the circadian expression of the
human KCNH2 promoter using promoter-luciferase reporter constructs. We studied several
deletion and mutant hKCNH2 promoter reporter vectors transiently transfected in a myogenic
cell line (C2C12 cells). Bioluminescence was measured at 10-minute intervals for 7-10 days.
Data were analyzed for circadian characteristics (period, phase and amplitude). Mutant
constructs included both engineered and constructs containing naturally occurring rare and
common SNPs.

Results: Lumicycle data demonstrated that the hKCNH2 promoter reporter showed robust
circadian and overall activity in C2C12 cells. Deletion and mutational analysis of the hKCNH2
promoter-reporter construct identified a highly conserved tandem E-box element within 1 Kb of
the exon 1 start site that was critical for circadian and overall regulation of hKCNH2 promoter
activity. Cells expressing hKCNH2 promoter-reporter constructs with different SNPs in this
tandem E-box element showed SNP-specific effects. Surprisingly, a relatively common SNP
decreased hKCNH2 circadian promoter activity by 66% (n = 6, p=0.0049).

Conclusion: We identified a conserved tandem E-box in the proximal promoter of hKCNH2
Exon1 necessary for circadian and overall hKCNH2 promoter activity. Several SNPs in this
region suggested a functional impact that might lead to decreased levels of IKr.