Conserved regulatory motifs at phenylethanolamine N-methyltransferase (PNMT) are disrupted by common functional genetic variation: an integrated computational/experimental approach

Abstract

The adrenomedullary hormone epinephrine transduces environmental stressors into cardiovascular events (tachycardia and hypertension). Although the epinephrine biosynthetic enzyme PNMT genetic locus displays both linkage and association to such traits, genetic variation underlying these quantitative phenotypes is not established. Using an integrated suite of computational and experimental approaches, we elucidate a functional mechanism for common (minor allele frequencies > 30%) genetic variants at PNMT. Transcription factor binding motif prediction on mammalian PNMT promoter alignments identified two variant regulatory motifs, SP1 and EGR1, disrupted by G-367A (rs3764351), and SOX17 motif created by G-161A (rs876493). Electrophoretic mobility shifts of approximately 30-bp oligonucleotides containing ancestral versus variant alleles validated the computational hypothesis. Queried against chromaffin cell nuclear protein extracts, only the G-367 and -161A alleles shifted. Specific antibodies applied in electrophoretic gel shift experiments confirmed binding of SP1 and EGR1 to G-367 and SOX17 to -161A. The in vitro allele-specific binding was verified in cella through promoter reporter assays: lower activity for -367A haplotypes cotransfected by SP1 (p = 0.002) and EGR1 (p = 0.034); and enhanced inhibition of -161A haplotypes (p = 0.0003) cotransfected with SP1 + SOX17. Finally, we probed cis/trans regulation with endogenous factors by chromatin immunoprecipitation using SP1/EGR1/SOX17 antibodies. We describe the systematic application of complementary computational and experimental techniques to detect and document functional genetic variation in a trait-associated regulatory region. The results provide insight into cis and trans transcriptional mechanisms whereby common variation at PNMT can give rise to quantitative changes in human physiological and disease traits. Thus, PNMT variants in cis may interact with nuclear factors in trans to govern adrenergic activity.Electronic supplementary materialThe online version of this article (doi: 10.1007/s00335-010-9253-y) contains supplementary material which is available to authorized users.

Phenylethanolamine N-Methyltransferase Gene Promoter Haplotypes and Risk of Essential Hypertension

“…The in vitro allele-specific binding was verified in cell through promoter reporter assays: lower activity for −367A haplotypes co-transfected by SP1 (P = 0.002) and EGR1 (P = 0.034); and enhanced inhibition of −161A haplotypes (P = 0.0003) co-transfected with SP1 + SOX17. 20 These findings suggest that PNMT promoter haplotype AA for rs3764351 (G-367A) and rs876493 (G-161A) may inhibit the promoter activity and PNMT transcription, which seems to fit our finding that the AA haplotype is associated with reduced risk of essential hypertension. Since no significant difference in allele and genotype frequencies between hypertensive cases and normotensive controls was found for the component SNPs, it is likely an interaction between the two component SNPs leading to a synthetic association at the haplotype level.…”

Three-Dimensional Proteome-Wide Scale Screening for the 5-Alpha Reductase Inhibitor Finasteride: Identification of a Novel Off-Target

“…For instance, recent observations identified PNMT genetic variants and polymorphisms in human subjects. Interestingly, some haplotypes resulted in decreased activity or accelerated degradation and different abilities to produce epinephrine in the basal condition or during exercise. ,,, Probably, carriers of PNMT variants might react differently to FIN administration, inducing different responses. Finally, considering that FIN may cross the blood–brain barrier, it will be crucial to explore the possible influence of this drug on brain PNMT − and the subsequent pathology.…”

Cardiac phenylethanolamine N-methyltransferase: localization and regulation of gene expression in the spontaneously hypertensive rat

“…Genetic mapping has linked the PNMT gene to hypertension, with no sequence polymorphisms identified, suggesting that increases in PNMT may be due to altered transcriptional regulation of the gene (Koike et al, 1995). Furthermore, disruption of conserved regulatory motifs in the PNMT promoter by common functional genetic variation may lead to changes in physiology (Rodríguez-Flores et al 2010). Previously, we demonstrated altered transcriptional regulation of adrenal PNMT in the spontaneously hypertensive rat (SHR), likely contributing to the hypertension observed in this rodent model (Nguyen et al 2009).…”