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

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

Juan L Rodríguez-Flores et al. Mamm Genome. 2010 Apr.

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.

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Figures

Fig. 1

Common genetic variation in the proximal human PNMT promoter. Common core promoter motifs (e.g., TATA box, G/C-rich domains) are illustrated. The two common proximal variants (G-367A and G-161A) are depicted

Fig. 2

Allele- and factor-specific transcription enhancement and inhibition in the human PNMT promoter. ClustalW alignment of orthologous mammalian PNMT promoter motifs, including two human alleles, three primates, and six other mammals, with (a) -367 and (b) -161 positions in lowercase. Rat-human alignments shown with greater than 50% conserved motif in uppercase for (c) -367 and (d) -161. JASPAR position frequency matrix scoring of human sequence (boxes) using (e) SP1 on -367 and (f) SOX17 on -161. Polymorphic position in boldface type. Using the SOX17 JASPAR motif (YWYTGKB), there are an additional 22 nonpolymorphic SOX17 sequence matches at 7 of 8 or better stringency in the first 500 bp of proximal promoter. Using a conventional G/C-rich motif (Sn) consensus, there are an additional five nonpolymorphic matches spanning 11–18 bp each (see Fig. 1)

Fig. 3

SNP-interrupted conserved regulatory motifs in the human PNMT promoter: functional consequences. a Cartoon of PNMT promoter/reporter construct, one of four possible promoters based on variation at positions G-367A/G-161A (G.A, A.G, A.A, G.G), which were ligated 5′ of firefly luciferase in pGL3-Basic plasmid and transfected into PC12 cells. b Basal (unstimulated) promoter activity for transfected PNMT promoter haplotypes (G-367A/G-161A; G.A, A.G, A.A, G.G), measured in cell lysates after 48 h of transient transfection, mean of four wells ± 1 standard error. Statistics: Haplotype effect (1-way ANOVA, overall p = 0.009) is shown first. Two-way ANOVA is then shown, factoring by each SNP. Horizontal bars indicate post-hoc t tests between haplotypes. X axis: Promoter haplotype; Y axis: Promoter activity in RLU/protein. c Change in promoter activity for PNMT promoter haplotypes, measured in cell lysates after 48 h of transient transfection; mean of four wells ± 1 standard error. Y axis shows fold change of activity versus pGL3-Basic promotorless empty vector, with mock pcDNA3.1 (pCMV) cotransfection activity subtracted, and normalization by total protein (quantified by the Bradford assay). Along the X axis are grouped the transfected promoter haplotypes (G-367A.G-161A) and the pCMV-driven transcription factors cotransfected (SP1, EGR1, SOX17, and SP1 + SOX17). Horizontal bars indicate post-hoc t tests for haplotype comparisons within a cotransfection state. d ANOVA p values for overall effect, individual SNP effects, and SNP × SNP (G-367A × G-161A) interactions, during transcriptional activation by cotransfection

Fig. 4

Allele-specific electrophoretic mobility shifts. Electrophoresis at 90 V of double-stranded biotinylated oligonucleotides incubating in the presence/absence of PC12 nuclear DNA-binding protein extracts (water-soluble) and 1000 × molar excess unlabeled oligonucleotide. Visualization of bands by trans-blot to nylon membrane, streptavidin-horseradish-peroxidase conjugation, and incubation for 5 min with luminol solution, followed by exposure on X-ray film. Horizontal black bar separates G- (left) from A- (right) allele oligonucleotide experiments (label on top of image). Oligonucleotide sequences designed to match 29-bp motifs at a -367 and b -161, with polymorphic position in brackets (bottom). Center bar illustrates migration from top to bottom and highlights G-allele-specific shift for -367 oligonucleotide and A-allele-specific shift for -161 oligonucletotide

Fig. 5

Electrophoretic mobility shifts: Perturbation by transcription factor-specific antibodies. Electrophoresis at 90 V of double-stranded biotinylated oligonucleotides incubate in the presence/absence of PC12 nuclear DNA-binding protein extracts (water-soluble) and antibodies to a SP1 and EGR1 versus -367 oligonucleotides or b anti-SOX17 and anti-SP1 versus -161 oligonucleotides. No-antibody shift same as Fig. 4 used as a positive control. Nonspecific (preimmune) IgG (“Mock”) was a negative control. Same protocol as Fig. 4

Fig. 6

Haplotype-specific endogenous transcription factor binding in the human PNMT promoter. Chromatin ImmunoPrecipitation (ChIP) assay to determine binding of particular transcription factors to the motifs in vivo. a Cartoon of primer design and expected PCR amplification fragment size. Primers specific for amplification of b -367 were used to identify DNA-protein complexes precipitated with anti-EGR1, anti-SOX17, or nonspecific IgG (“Mock,” preimmune serum) antibodies, or raw sonicated DNA (“Input”) as a positive control. c Identical experiment testing anti-SOX17 with -161 primers

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