title: 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.
authors.affiliation: Department of Medicine, Institute for Genomic Medicine, University of California at San Diego School of Medicine, La Jolla, CA, USA. juan@ucsd.edu
authors.forename: Juan L
authors.initials: JL
authors.lastname: Rodríguez-Flores
authors.forename: Kuixing
authors.initials: K
authors.lastname: Zhang
authors.forename: Sun Woo
authors.initials: SW
authors.lastname: Kang
authors.forename: Gen
authors.initials: G
authors.lastname: Wen
authors.forename: Sajalendu
authors.initials: S
authors.lastname: Ghosh
authors.forename: Ryan S
authors.initials: RS
authors.lastname: Friese
authors.forename: Sushil K
authors.initials: SK
authors.lastname: Mahata
authors.forename: Shankar
authors.initials: S
authors.lastname: Subramaniam
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Hamilton
authors.forename: Daniel T
authors.initials: DT
authors.lastname: O'Connor
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D002843:Chromatin / Q000378:metabolism; D019295:Computational Biology / Q000379:methods*; D017124:Conserved Sequence; D051766:Early Growth Response Protein 1 / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D014644:Genetic Variation; D006239:Haplotypes / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010625:Phenylethanolamine N-Methyltransferase / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D051381:Rats; D055760:SOXF Transcription Factors / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D002843:Chromatin; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C109188:SOX17 protein, human; D055760:SOXF Transcription Factors; D016329:Sp1 Transcription Factor; D010625:Phenylethanolamine N-Methyltransferase