Journal of Hypertension:
ORIGINAL PAPERS: Genetic aspects
Heredity and cardiometabolic risk: naturally occurring polymorphisms in the human neuropeptide Y2 receptor promoter disrupt multiple transcriptional response motifs
Wei, Zhiyuna,b,c,e; Zhang, Kuixinga,b,c; Wen, Gena,b,c; Balasubramanian, Karthikaa,b,c; Shih, Pei-an B.a,b,c; Rao, Fangwena,b,c; Friese, Ryan S.a,b,c; Miramontes-Gonzalez, Jose P.a,b,c; Schmid-Schoenbein, Geert W.a,b,c; Kim, Hyung-Sukd; Mahata, Sushil K.a,b,c; O’Connor, Daniel T.a,b,c
aDepartment of Medicine
bDepartment of Pharmacology
cDepartment of Bioengineering, the Institute for Genomic Medicine, University of California at San Diego, the VA San Diego Healthcare System, La Jolla, California
dDepartment of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, USA
eBio-X Institutes, Shanghai Jiao Tong University, China
Correspondence to Daniel T. O’Connor, MD, Department of Medicine (0838), UCSD School of Medicine and VASDHS, 9500 Gilman Drive, La Jolla, CA 92093-0838, USA. Tel: +1 858 5340661; fax: +1 858 5340626; e-mail: firstname.lastname@example.org
Abbreviations: NPY, neuropeptide Y; NPY2R, neuropeptide Y2 receptor; PYY, peptide YY; SNP, single nucleotide polymorphism
Received 7 June, 2012
Revised 28 August, 2012
Accepted 4 October, 2012
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (http://www.jhypertension.com).
Objectives: The neuropeptide Y2 G-protein-coupled receptor (NPY2R) relays signals from PYY or neuropeptide Y toward satiety and control of body mass. Targeted ablation of the NPY2R locus in mice yields obesity, and studies of NPY2R promoter genetic variation in more than 10 000 human participants indicate its involvement in control of obesity and BMI. Here we searched for genetic variation across the human NPY2R locus and probed its functional effects, especially in the proximal promoter.
Methods and results: Twin pair studies indicated substantial heritability for multiple cardiometabolic traits, including BMI, SBP, DBP, and PYY, an endogenous agonist at NPY2R. Systematic polymorphism discovery by resequencing across NPY2R uncovered 21 genetic variants, 10 of which were common [minor allele frequency (MAF) >5%], creating one to two linkage disequilibrium blocks in multiple biogeographic ancestries. In vivo, NPY2R haplotypes were associated with both BMI (P = 3.75E−04) and PYY (P = 4.01E−06). Computational approaches revealed that proximal promoter variants G-1606A, C-599T, and A-224G disrupt predicted IRF1 (A>G), FOXI1 (T>C), and SNAI1 (A>G) response elements. In neuroendocrine cells transfected with NPY2R promoter/luciferase reporter plasmids, all three variants and their resulting haplotypes influenced transcription (G-1606A, P < 2.97E−06; C-599T, P < 1.17E−06; A-224G, P < 2.04E−06), and transcription was differentially augmented or impaired by coexpression of either the cognate full-length transcription factors or their specific siRNAs at each site. Endogenous expression of transcripts for NPY2R, IRF1, and SNAI1 was documented in neuroendocrine cells, and the NPY2R mRNA was differentially expressed in two neuroendocrine tissues (adrenal gland, brainstem) of a rodent model of hypertension and the metabolic syndrome, the spontaneously hypertensive rat.
Conclusion: We conclude that common genetic variation in the proximal NPY2R promoter influences transcription factor binding so as to alter gene expression in neuroendocrine cells, and consequently cardiometabolic traits in humans. These results unveil a novel control point, whereby cis-acting genetic variation contributes to control of complex cardiometabolic traits, and point to new transcriptional strategies for intervention into neuropeptide actions and their cardiometabolic consequences.
The neuropeptide Y (NPY) receptor Y2 (NPY2R; OMIM 162642; IUPHAR Y2) is a G-protein-coupled receptor responding to hormones peptide YY (PYY)  and NPY to control appetite and cardiovascular homeostasis. There are five subtypes of NPY receptor identified in mammals, four of which are functional in humans. Subtypes Y1 and Y5 have known roles in the stimulation of feeding, whereas Y2 and Y4 seem to have roles in appetite inhibition. NPY2R is widely expressed in tissues pertinent to cardiometabolic control, including the arcuate nucleus, a major integrator of appetite control in the hypothalamus. In previous studies, NPY2R genetic variants were associated with obesity or BMI in several populations, including whites [2,3], Asians , and Africans . Indeed, studies of NPY2R promoter genetic variation in more than 10 000 individuals [2,3,6] indicate its involvement in control of obesity or BMI (on-line Table 1, http://links.lww.com/HJH/A209). NPY2R also cooperates with NPY in stress-induced obesity and the metabolic syndrome . NPY2R genetic variants associate with such human cardiometabolic traits as high-density lipoprotein cholesterol , SBP , type 2 diabetes in men , and left ventricular hypertrophy . Hypothalamus-targeted NPY2R-knockout mice showed a decrease in body weight despite an increase in food intake . In the rat (http://rgd.mcw.edu), the Npy2r genetic locus underlies the confidence interval of a quantitative trait locus (QTL) for blood pressure (BP): BP QTL-90 (Bp90) . Such diverse evidence indicates that NPY2R plays an indispensable role in the cardiometabolic syndrome.
In these studies, we first documented the role of heredity in cardiometabolic traits, using twin pair variance components, and then systematically searched for naturally occurring genetic variation across the human NPY2R locus. Because several of the discovered common variants occurred in a likely functional domain (the promoter), we probed their mechanistic consequences, beginning with bioinformatic motif analysis and proceeding to transfected promoter/luciferase reporter plasmids, site-directed mutagenesis, and characterization of trans-acting factors. We developed evidence that variation in the NPY2R promoter, especially at common variants G-1606A, C-599T, and A-224G, disrupt particular motifs (IRF1, FOXI1, and SNAI1 elements, respectively), creating differential cis-interactions and trans-interactions, to alter transcriptional activity and ultimately BP, body mass, and associated risk traits in the population.
PARTICIPANTS AND METHODS
Systematic polymorphism discovery at the NPY2R locus
We studied the NPY2R locus in n = 80 participants (2n = 160 chromosomes) as described below under ‘Human participants’. Genomic DNA was prepared from leukocytes as described previously . Public draft human genome sequences were obtained from the University of California, Santa Cruz Genome Bioinformatics website (http://genome.ucsc.edu) and used as a scaffold for primer design. The base position numbers were according to the National Center for Biotechnology Information (NCBI) NPY2R source clone, RefSeq gene/transcript NM_000910.2. Promoter positions were numbered upstream of (−) the NPY2R exon-1 start (cap) site. PCR primers were designed by primer-3  (http://frodo.wi.mit.edu/primer3/) to capture approximately 2000 bp of the proximal promoter, between approximately 500 bp to approximately2000 bp over each of the two exons (including 5′-UTR, 3′-UTR, and exon/intron borders), and regions highly conserved across species. Target regions were amplified and then dideoxy-sequenced using an ABI-3100 capillary sequencer (Applied Biosystems, Carlsbad, California, USA). Polymorphism (typically as heterozygosity) was visualized on the Applied Biosystems (ABI) tracings using Codon Code Aligner (http://www.codoncode.com/aligner).
Resequencing the NPY2R locus
Human studies were approved by the University of California, San Diego (UCSD) Human Research Protection Program. Experiments were conducted with the understanding and consent of each participant. We studied the NPY2R locus in n = 80 participants (2n = 160 chromosomes) from four diverse biogeographic ancestry groups systematically sequenced for polymorphism discovery across the NPY2R locus: white (European ancestry, 2n = 46 chromosomes), black (sub-Saharan African ancestry, 2n = 50 chromosomes), Hispanic (Mexican American, 2n = 32 chromosomes), and east Asian (2n = 32 chromosomes).
UCSD twin pairs
Twin recruitment included access to a population birth record-based twin registry , as well as by newspaper advertisement, as described . Description of the 362 participants in the twin heritability and allelic association studies has been published . For human allelic and haplotype association studies, this twin group was expanded to 693 participants of European ancestry, derived from additional siblings from twinships and sibships, as previously described .
Statistics and informatics
Linkage disequilibrium and haplotypes
In the resequenced participants, patterns of linkage disequilibrium as well as haplotype frequencies were analyzed and visualized by the software Haploview (Broad Institute, Massachusetts, USA) . Linkage disequilibrium blocks were derived by the confidence interval criterion and visualized by r2 plot in Haploview from unphased diploid genotypes of n = 80 resequenced participants (2n = 160 chromosomes) from four diverse biogeographic ancestry groups systematically sequenced across the NPY2R locus. Common variants (minor allele frequency >5%) were used to establish linkage disequilibrium.
In the twins and siblings, haplotype-on-trait analyses were conducted by regression in R (reporting effect size as β, or slope per allele, as well as its SEM) with Haplo.glm in Haplo.stats  (http://mayoresearch.mayo.edu/schaid_lab/software.cfm). Trait-associated haplotype GTT was present on 11.1 of chromosomes analyzed.
Bioinformatics: computational prediction of transcription factor-binding motifs overlying NPY2R promoter common variants
Multiple sequence alignments were performed by Clustal-W  (http://www.ebi.ac.uk/Tools/clustalw2/). Potential transcription factor binding motifs were predicted from the JASPAR  (http://jaspar.genereg.net/) and ConSite  (http://asp.ii.uib.no:8090/cgi-bin/CONSITE/consite/) databases.
Heritability and pleiotropy (shared genetic determination or genetic covariance, (ρG)
Estimates of heritability (h2) (h2 = VG/VP, wherein VG is additive genetic variance and VP is total phenotypic variance) were obtained using the twin-pair variance-component methodology implemented in the Sequential Oligogenic Linkage Analysis Routines (SOLAR) package  available at (http://txbiomed.org/departments/genetics/). This method maximizes the likelihood assuming a multivariate normal distribution of phenotypes in twin pairs (monozygotic versus dizygotic) with a mean dependent on a particular set of explanatory covariates. The null hypothesis (H0) of no heritability is tested by comparing the full model, which assumes genetic variation (VG), and a reduced model, which assumes no genetic variation, using a likelihood ratio test. Heritability estimates were adjusted for age and sex because of the effects of these covariates on several traits. Pleiotropy (genetic covariance for two correlated, heritable traits; i.e., the cross-product of trait heritabilities)  was estimated as the parameter ρG in SOLAR . SOLAR also estimated the environmental covariance, as parameter ρE.
Functional studies of NPY2R genetic variation
Human phenotyping: peptide YY
Human PYY (total) was measured using a Linco (Millipore, St. Charles, Missouri, USA) HRP-TMB ELISA kit (catalog # EZHPYYT66K). EDTA-anticoagulated plasma was frozen and stored at −70°C prior to assay; this ELISA measures the enzyme by absorbance at 450 nm. The assay sensitivity is 1.4 pg/ml plasma, with an intra-assay coefficient of variability (CV) of 0.9–5.8%, and interassay CV of 3.7–16.5%. The assay equivalently recognizes PYY1–36 and PYY3–36, but does not cross-react (at up to 50 nmol/l) with NPY, ghrelin, gastric inhibitory polypeptide, glucagon, glucagon-like peptide-1, leptin, insulin, C-peptide, amylin, or adiponectin. PYY distribution in human individuals was tested by the one-sample, two-tailed nonparametric Kolmogorov–Smirnov test in SPSS (IBM Corporation, New York, USA); untransformed PYY deviated from normality (P = 0.002), whereas log-transformed PYY did not display such deviation (P = 0.291). Estimates of heritability (by variance components in SOLAR; see above) did not differ when performed on untransformed versus log-transformed PYY data (see RESULTS).
Human single nucleotide polymorphism genotyping and marker-on-trait association
Single nucleotide polymorphism (SNP) genotypes at rs6851222 (Promoter G-1606A), rs6857715 (Promoter C-599T), and rs1047214 (Exon-2 T/C Ile195Ile) were chosen to span the NPY2R locus, and typed by the TaqMan method on an ABI-7900HT Fast Real-Time PCR System, with labeled probes synthesized at Applied Biosystems. Each SNP was in Hardy Weinberg equilibrium (all P > 0.05). Haplotypes were derived from diploid genotype data, and haplotype-on-trait analyses were conducted by regression , or by Generalized Estimating Equations, with analyses adjusted for age, sex, and biogeographic ancestry.
NPY2R promoter haplotype/luciferase reporter design and construction
Human NPY2R promoter fragments, corresponding to NPY2R-2323/+130 bp in NPY2R (NCBI NPY2R source clones: RefSeq gene/transcript NM_000910.2), were PCR-amplified from genomic DNA (after resequencing) and cloned into the polylinker (between KpnI and BglII sites) of the promoterless firefly luciferase reporter plasmid pGL3-Basic (Promega, Madison, Wisconsin, USA), as described . Site-directed mutagenesis (QuikChange; Stratagene, Santa Clara, California, USA) created the required variant at position −1606, −599, and −224 (Supplemental Digital Content Fig. S1, http://links.lww.com/HJH/A209). Supercoiled plasmids were purified using NucleoBond Xtra Maxi kits (740414.10; Machery-Nagel, Bethlehem, Pennsylvania, USA) prior to transfection, and verified by sequencing. Promoter positions are numbered upstream (−) of the transcriptional start (cap) site.
Luciferase reporter assays of NPY2R promoter variants
PC12 rat pheochromocytoma cells were transfected (at 60–80% confluence, 1 day after 1 : 4 splitting in 24-well plate) with 500 ng of supercoiled promoter/firefly luciferase reporter plasmid per well, by the liposome method (Transfectin; Bio-Rad, Hercules, California, USA). The firefly luciferase activity in cell lysates was measured 24 h after transfection, using the luciferase assay system (Promega), and the results were expressed as the ratio of firefly activity/total protein in the lysate, as described . Each experiment included at least three replicates. Results were expressed as mean ± SEM. Statistical significance was calculated using Student's t-test or ANOVA, and significance was established at the P value less than 0.05 level. Inspection of the NCBI Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) indicates that transcripts for NPY2R are abundantly expressed in the adrenal gland (GEO dataset GDS3556 and GDS2374) as well as PC12 chromaffin cells (GDS2555).
Exogenous/cotransfected transcription factors
Eukaryotic expression plasmids containing cDNAs encoding transcription factors IRF1 (rat; clone ID 7099391), FOXI1 (human; clone ID 5185923) and SNAI1 (human; clone ID 4537122) were from Open Biosystems (Huntsville, Alabama, USA). cDNAs were obtained in either pExpress-1 or cytomegalovirus promoter (pCMV)-SPORT6 plasmids, and subcloned if needed into a eukaryotic pCMV expression vector (pcDNA-3.1). One hundred nanograms of each transcription factor expression plasmid, or 100 ng pcDNA-3.1 empty vector (control), was cotransfected into PC12 cells, along with 500 ng of NPY2R promoter/luciferase reporter, wild-type versus variants. After 24 h, cells were lysed and luciferase activities were assayed as described above and normalized by total protein. Response of the NPY2R promoter to exogenous transcription factor was revealed by comparison of the normalized luciferase activity between the transcription factor-transfected group and the mock-transfected (empty vector, pcDNA 3.1) group.
Silencer select predesigned siRNAs targeting IRF1 (rat; siRNA ID s127967), FOXI1 (rat; siRNA ID s220491), or SNAI1 (rat; siRNA ID s137986) were from Ambion (Applied Biosystems). Silencer select negative control #1 siRNA (part number 4390843) was used as the negative control. Six nanomoles per litre final concentration of each transcription factor siRNA, or negative control siRNA, was cotransfected into PC12 cells, along with 500 ng of NPY2R promoter/luciferase reporter wild-type versus variant. After 24 h, cells were lysed and luciferase activities were assayed as described above and normalized by total protein. Response of the NPY2R promoter to exogenous siRNAs was revealed by comparison of the normalized luciferase activity between the transcription factor siRNA-transfected group and the mock-transfected (negative control siRNA) group.
Quantification of endogenous transcripts by real-time PCR: NPY2R itself and transcripts for factors whose binding motifs are disrupted by NPY2R promoter variants (IRF1, FOXI1, SNAI1)
Total RNA was extracted from cells (neuroendocrine PC12) or organs under each experimental state, using an ABI 6700 automated nucleic acid workstation, and quantitative real-time PCR (RT-PCR) was performed on mRNA→cDNA with the ABI-7700 TaqMan platform, using fluorescent reporter-tagged oligonucleotide primers, and normalization of data to β-actin expression in the same sample. Threshold cycle (Ct) is determined for both the specific target mRNA/cDNA as well as β-actin, and the difference in Ct (for target mRNA versus β-actin mRNA) is normalized to the average for that state (e.g., control versus experimental), by the ΔΔCt method .
Experimental animals: spontaneously hypertensive rat and Wistar–Kyoto rat
Animal studies were performed with age-matched, adult (12–17 weeks) male spontaneously hypertensive rat (SHR) and Wistar–Kyoto (WKY) rat strains from Charles River Laboratories (Wilmington, Massachusetts, USA). Features of the Charles River colonies, including BP monitoring, are given at (http://www.criver.com/EN-US/PRODSERV/BYTYPE/RESMODOVER/RESMOD/Pages/SHRRat.aspx). Isoflurane was used for terminal anesthesia of SHR and WKY rats. Adrenal glands and brainstem were isolated from each rat (n = 9 per group), immediately frozen in liquid nitrogen, and then stored at −80°C prior to RNA extraction and RT-PCR. Rats were studied according to a protocol approved by the Animal Subjects Committee of the University of California at San Diego, and research was conducted in accordance with institutional guidelines.
Heredity, pleiotropy, and cardiometabolic traits in humans
Twin pair variance component analyses indicate that multiple cardiometabolic traits display substantial and significant (P = 0.0001) heritability (h2) (Fig. 1a), including BMI (h2 = 86 ± 2%), SBP (h2 = 46 ± 6%), DBP (h2 = 52 ± 6%), and circulating PYY (h2 = 51 ± 6%), the principal endogenous ligand for the NPY2R. Heritability estimates for BMI, SBP, and DBP were consistent with previously reported values . Using the twin method, we also investigated genetic pleiotropy (shared genetic determination or genetic covariance) between BMI and other cardiometabolic traits (Fig. 1b). BMI displayed significant genetic covariance with SBP (P = 9.31E−05), DBP (P = 7.74E−04), and PYY (P = 3.0E−02); by contrast, environmental covariance (or shared environmental determination, ρE) was not significant for these same traits.
Polymorphism discovery across NPY2R
Located on chromosome 4q31, NPY2R spans two exons (one coding) with one intervening sequence (intron). We resequenced approximately 1800 bp of proximal promoter, each of exon-1 and exon-2 [down to the first polyadenylation site (bold): 5′-TACTAAATAAAACAAT-3′], and adjacent intron/exon borders (Fig. 2) in 2n = 160 chromosomes derived from four biogeographic ancestry groups (Table 1). We identified 21 variants (18 SNPs, 3Ins/Del) in these individuals. Of these variants, 10 are common [minor allele frequency (MAF) >5%], including two in the open reading frame within coding exon-2 (both synonymous), T + 5895C (Ile195Ile) and T + 6242C (Ile312Ile), whereas the rest are located in the proximal promoter.
Biogeographic ancestry and NPY2R linkage disequilibrium
NPY2R common allele frequencies did not differ across the four biogeographic ancestry groups (Table 1). To visualize patterns of marker-on-marker association, pair-wise linkage disequilibrium correlations among the eight common (MAF >5%) SNPs were quantified by the confidence interval method across the NPY2R locus. In each biogeographic ancestry group, two blocks of linkage disequilibrium were maintained, with one in the promoter region (Fig. 3a).
Neuropeptide Y2 receptor haplotype effects on traits
We ‘tagged’ the human NPY2R gene with three SNPs spanning the locus (Fig. 3b): haplotype GTT (found on 11.1% of chromosomes) was associated significantly with both BMI (P = 3.75E−04) and PYY secretion (P = 4.01E−06), and the principal effect accrued to GTT homozygotes (with two copies of that haplotype per diploid genome); the GTT effect size (or slope) was positive for BMI (1.93 ± 0.48 kg/m2 per copy), although negative for PYY (−26.3 ± 5.65 pg/ml per copy). Perhaps these pleiotropic effects of haplotype GTT involve increased response to PYY, with consequent fall in this anorexigenic hormone and ultimately an increase in BMI.
Endogenous NPY2R mRNA expression in a disease model in rodents: spontaneously hypertensive rat/Wistar–Kyoto rat
NPY2R mRNA expression was increased significantly in two key neuroendocrine tissues of the SHR (Fig. 4a): both the adrenal gland (by ∼2.6-fold, P = 0.002) and the brainstem (by ∼1.5-fold, P = 0.027).
Genetic variation in the proximal human NPY2R promoter: consensus motifs
Core promoter: nonpolymorphic motifs
Motifs identified did not include a consensus TATA box near the transcriptional start site; the closest partial TATA (i.e., T/A-rich) match on the (+) strand was 5′-(−113 bp)-AAAcTT-(−108 bp)-3′, whereas the nearest potential CAAT box was on the (−) strand at 5′ (−420 bp)-CCAAT(−424 bp)-3′. There was no proximal cAMP response element. The 13 G/C-rich (consecutive G/C ≥6 bp) regions were noted in the proximal promoter, as were 4 E-boxes (CANNTG). One of the G/C-rich domains constituted a consensus match for a B recognition element , on the very proximal (+) strand at 5′-(−49 bp)GGGCGCC(−43 bp)-3′. The closest potential initiator (Inr) elements  (consensus 5′-YYA+1NWYY-3′) were located at 5′-(−244 bp)CCAGTCC(−238 bp)-3′ (+ strand) and 5′-(+151 bp)TTACACT(+145 bp)-3′ (− strand). None of these core elements were polymorphic across 2n = 160 human chromosomes.
We identified 16 polymorphisms in the promoter (Table 1), eight of which were common (MAF >5%). Of note, the very proximal ‘core’ promoter (−186/+85 bp) was devoid of common variation. At promoter variants G-1606A, C-599T, and A-224G, we identified motifs likely to be disrupted by the sequence change (see below).
NPY2R promoter haplotypes affect gene expression
Constructed from three common SNPs (G-1606A, C-599T and A-224G) that were predicted to be functional (see below), eight haplotypes were created by site-directed mutagenesis from the most common promoter haplotype (alleles: G-1606, C-599 and A-224; 55.4% of chromosomes in our sample). NPY2R promoter/luciferase reporters with various haplotypes had significantly different expression activities (one-way ANOVA: P = 1.12E−23; Fig. 5a). We used two-way ANOVA to probe individual SNP effects on gene expression: each individual SNP, as well as their binary and ternary interactions, displayed significant influences on reporter expression (P = 5.00E−06; Fig. 5b).
Neuropeptide Y2 receptor G-1606A polymorphism: role of an IRF1 activator-binding site
G-1606A is located in a region highly conserved across sequenced primates (Fig. 4a), with the G allele ancestral in the human lineage, as judged by the chimp sequence (Fig. 6a). In this conserved local region, there is a partial consensus match for an IRF1 site (VAAARYGAAASY; −1606 in bold) with an improved match for the A allele (10/12 bp match) over the G allele (9/12 bp match) (Fig. 4a).
Exogenous IRF1 transcription factor: increased NPY2R promoter-driven reporter expression, A>G allele
During NPY2R promoter/luciferase reporter transfection/expression into chromaffin cells (cotransfection with empty vector pcDNA 3.1; Fig. 6b), the A allele displayed greater expression than the G allele (A>G). Cotransfection/expression of the IRF1 transcription factor increased reporter expression and amplified the difference in expression between the two alleles (Fig. 4b, P = 0.001).
Exogenous IRF1 siRNA: decreased NPY2R promoter-driven reporter expression, A>G allele
During NPY2R promoter/luciferase reporter cotransfection with negative control siRNA into chromaffin cells (Fig. 6c), the A allele once again displayed greater expression than the G allele (A>G). Cotransfection of IRF1 siRNA decreased reporter expression and attenuated the difference of expression between the two alleles (Fig. 6C, P = 8.72E−06).
Neuropeptide Y2 receptor C-599T polymorphism: role of an activator FOXI1 binding site
C-599T is located in a region highly conserved across sequenced primates (Fig. 7a), with the T allele ancestral in the human lineage, as judged by the chimp sequence (Fig. 7a). In this conserved local region, there is a total consensus match for a FOXI1 site (TRTTTRKWD; −599 in bold) with an improved match for the T allele (9/9 bp match) over the C allele (8/9 bp match) (Fig. 7a).
Exogenous FOXI1 transcription factor: increase in NPY2R promoter-driven reporter expression, T>C allele
During NPY2R promoter/luciferase reporter transfection into chromaffin cells (cotransfection with empty vector pcDNA 3.1; Fig. 7b), the T allele displayed greater expression than the C allele (T>C). Cotransfection/expression of FOXI1 transcription factor increased reporter expression and amplified the difference of expression between the two alleles (Fig. 7b, P = 5.57E−06).
Exogenous FOXI1 siRNA: decrease in NPY2R promoter-driven reporter expression, T>C allele
During NPY2R promoter/luciferase reporter, cotransfection with negative control siRNA into chromaffin cells (Fig. 7c), the T allele displayed greater expression than the C allele (T>C). Cotransfection of FOXI1 siRNA decreased reporter expression and attenuated the difference of expression between two alleles (Fig. 7c, P = 0.010).
Neuropeptide Y2 receptor A-224G polymorphism: role of a SNAI1 repressor binding site
A-224G is located in a region highly conserved across sequenced primates (Fig. 8a), with the A allele ancestral in the human lineage, as judged by the chimp sequence (Fig. 8a). In this conserved local region, there is a partial consensus match for an SNAI1 site (CAGGTG; −224 in bold) with an improved match for the A allele (5/6 bp match) over the G allele (4/6 bp match) (Fig. 8a).
Exogenous SNAI1 transcription factor: decrease in NPY2R promoter-driven reporter expression, A>G allele
During NPY2R promoter/luciferase reporter transfection into chromaffin cells (cotransfection with empty vector pcDNA 3.1; Fig. 8b), the G allele displayed greater expression than the A allele (G>A). Cotransfection of the SNAI1 transcription factor decreased reporter expression and amplified the difference of expression between the two alleles (Fig. 8b, P = 0.034).
Exogenous SNAI1 siRNA: increase in NPY2R promoter-driven reporter expression, A>G allele
During NPY2R promoter/luciferase reporter cotransfection with negative control siRNA into chromaffin cells (Fig. 8c), the G allele displayed greater expression than the A allele (G>A). Cotransfection of SNAI1 siRNA increased reporter expression and attenuated the difference of expression between two alleles (Fig. 8c, P = 0.019).
Endogenous mRNA expression in neuroendocrine cells: NPY2R and transcription factors whose binding is disrupted by NPY2R promoter common genetic variation (IRF1, FOXI1, SNAI1)
We used PC12 (rat pheochromocytoma) cells as an experimental system to test the effects of potentially allele-specific transcription factors, but are the receptor and these transcription factors endogenously expressed in this model system (Fig. 4b)? NPY2R itself, as well as the transcription factors IRF1 and SNAI1, displayed substantial expression in PC12 cells, whereas FOXI1 expression was undetectable.
NPY2R represents a central control point for the PYY/NPY regulatory pathway. In this study, we explored whether and how common genetic variations in the NPY2R promoter affect gene expression. We present evidence from several approaches (genomic, bioinformatic, transfection, trans-activation, and siRNA inhibition) in which we found that promoter variants G-1606A, C-599T, and A-224G conferred functional changes onto NPY2R expression, and that particular transcription factors were implicated. We, thus, present evidence of previously unexpected cis-variation in the regulation of NPY2R expression.
Cardiometabolic traits and NPY2R genetic variation
We found that multiple cardiometabolic traits are highly heritable, and also display shared genetic determination (Fig. 1). Associations between NPY2R SNPs and obesity are widely investigated in multiple populations, with substantial agreement that significant marker-on-trait effects occur . We too could replicate such effects, in that a haplotype across the NPY2R locus influenced both BMI and PYY (Fig. 3b). Thus, in this report we describe a potential genetic contributor to dysregulation of body mass: genetic variation at the NPY2R locus (Figs 2 and 3).
Neuropeptide Y2 receptor promoter variants G-1606A, C-599T, and A-224G
We focused on three promoter polymorphisms that are not only common (high MAF) but also predicted to influence transcription factor binding, by bioinformatic analyses. On the basis of this strategy, the G-1606A, C-599T, and A-224G were advanced to further investigation. Frequencies of their promoter haplotypes are shown in Fig. 5.
Of note for the physiological significance of these results, we detected abundant transcripts in neuroendocrine PC12 cells (Fig. 4b) for NPY2R itself, IRF1, and SNAI1. In addition, query of the NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo/) indicates that transcripts for NPY2R, IRF1 (binding G-1606A), FOXI1 (binding C-599T), and SNAI1 (binding A-224G) are expressed endogenously in PC12 chromaffin cells, by inspection of the following GEO transcript datasets: GDS3436 , GDS1038–1039 , and GDS2555 .
Results in context with the literature
Common genetic variation in the NPY2R promoter [2,3,6] has been associated with obesity or BMI traits in studies of over 10 000 individuals (on-line Table 1, http://links.lww.com/HJH/A209); in one case , the effect size (as Cramer's phi) suggested that NPY2R promoter genetic variation might account for up to approximately 9.3% of trait variance in the population. Among the three promoter variants evaluated in depth in our studies, C-599T (rs6857715) was implicated in one of these association studies: C-599T was associated with both adult and childhood obesity in a French sample , and this variant also had an effect on high-density lipoprotein cholesterol ; C-599T was a component of functional promoter haplotypes on gene expression (Figs 5 and 7) as well as the BMI/PYY-associated GTT haplotype in our population (Fig. 3b).
Furthermore, each of the three transcription factors (Figs 6–8) whose binding is altered by NPY2R promoter variants is already implicated in cardiometabolic function. A meta-analysis of genome-wide association studies revealed the influence of IRF1 on circulating C-reactive protein level, which is strongly associated with cardiovascular disease . IRF1 also plays a key role in development of insulitis and diabetes in a mouse model . FOXI1 may be necessary for expression of at least four subunits and proper assembly of the vacuolar H+-ATPase complex , whose activity has an impact on hypertension . SNAI1 transcriptionally controls cardiovascular progenitor cell formation through epicardial epithelial-mesenchymal transition , and such function is regulated by glucose metabolism .
Limitations of this study
A number of issues remain unexplored by our studies. For effects in very large sample sizes (>10 000 participants; on-line Table 1, http://links.lww.com/HJH/A209), we rely on the findings of other groups [2,3,6] that NPY2R promoter polymorphism influences obesity, especially for C-599T , although we did find evidence for such effects in our own population (Fig. 3b). Second, the cis-interactions/trans-interactions that we observed in transfected cells (Figs 6–8) are novel, and thus not yet established in vivo, although we did find evidence of differential expression of NPY2R in neuroendocrine tissues of the SHR (Fig. 4a), as well as endogenous expression of the pertinent transcripts in neuroendocrine cells (Fig. 4b).
Conclusions and perspectives
We conclude that cardiometabolic traits are highly heritable, that NPY genetic variation influences such traits (including BMI and PYY), and that within the NPY2R promoter, common polymorphisms are associated with alterations in transcriptional efficiency. The functional effects of polymorphism seem to arise from differential actions of specific transcription factors at the NPY2R promoter: IRF1 functioning as an activator disrupted by G-1606A bi-allelic variation, FOXI1 acting as an activator disrupted by C-599T, and SNAI1 acting as a repressor disrupted by A-224G. The results raise the potential for novel alterations in cis-interactions for control of PYY responses, thus, augmenting our understanding of molecular events underlying interindividual variation in energy balance, and the genetic predisposition toward obesity, a potent risk factor for cardiovascular disease.
Sources of funding are National Institutes of Health [HL58120; 1UL1RR031980 (UCSD Clinical and Translational Research Institute); MD000220 (UCSD Comprehensive Research Center in Health Disparities, CRCHD)], Department of Veterans Affairs.
Conflicts of interest
The authors have no conflicts of interest to declare.
Reviewers’ Summary Evaluations Reviewer 1
Neuropeptide Y receptors are activated by neuropeptide Y, peptide YY and pancreatic polypeptide. Subtypes Y1 and Y5 are involved in stimulation of feeding while Y2 and Y4 appear to be involved in satiety. By extension, there is interest in this pathway being involved in metabolic traits. Peptide YY is related to pancreatic peptide and is released postprandially primarily from the ileum and the colon and has a role in appetite suppression. This study shows that peptide YY levels have a high heritability of 51% and show that 3 promoter polymorphisms in the NPY2R influence transcriptional activity using luciferase reporter constructs with IRF1 and SNAI1 as putative transcription factors. A causal relation between these polymorpisms or peptide YY and cardiometabolic traits is not established and future studies should validate this finding and well powered association and functional studies.
Satiety and obesity are interdependently subject to gene x environment interactions. Significant genetic component, confirmed in twin's studies, actually illustrates a superior hereditary determination for obesity than for hypertension. While neuropeptide Y pathway has been associated in large studies with obesity, this paper provides novel evidence of functional relevance of polymorphisms within the promoter region of NPY2R in cis- as well as trans- modes. The fact that these genomic variances are present in a quarter of several populations is teaching us that their impact should be included in future preventive strategies of satiety, obesity and hypertension control.
1. Batterham RL, Cowley MA, Small CJ, Herzog H, Cohen MA, Dakin CL, et al. Gut hormone PYY(3-36) physiologically inhibits food intake. Nature 2002; 418:650–654.
2. Torekov SS, Larsen LH, Andersen G, Albrechtsen A, Glümer C, Borch-Johnsen K, et al. Variants in the 5′ region of the neuropeptide Y receptor Y2 gene (NPY2R) are associated with obesity in 5,971 white subjects. Diabetologia 2006; 49:2653–2658.
3. Lavebratt C, Alpman A, Persson B, Arner P, Hoffstedt J. Common neuropeptide Y2 receptor gene variant is protective against obesity among Swedish men. Int J Obes (Lond) 2006; 30:453–459.
4. Zhang J, Wang HJ, Ma J. Association between obesity and the polymorphism of neuropeptide Y2 receptor gene in children and adolescents. Zhonghua Liu Xing Bing Xue Za Zhi 2009; 30:695–698.
5. Friedlander Y, Li G, Fornage M, Williams OD, Lewis CE, Schreiner P, et al. Candidate molecular pathway genes related to appetite regulatory neural network, adipocyte homeostasis and obesity: results from the CARDIA Study. Ann Hum Genet 2010; 74:387–398.
6. Siddiq A, Gueorguiev M, Samson C, Hercberg S, Heude B, Levy-Marchal C, et al. Single nucleotide polymorphisms in the neuropeptide Y2 receptor (NPY2R) gene and association with severe obesity in French white subjects. Diabetologia 2007; 50:574–584.
7. Kuo LE, Kitlinska JB, Tilan JU, Li L, Baker SB, Johnson MD, et al. Neuropeptide Y acts directly in the periphery on fat tissue and mediates stress-induced obesity and metabolic syndrome. Nat Med 2007; 13:803–811.
8. Takiguchi E, Fukano C, Kimura Y, Tanaka M, Tanida K, Kaji H. Variation in the 5′-flanking region of the neuropeptide Y2 receptor gene and metabolic parameters. Metabolism 2010; 59:1591–1596.
9. Campbell CD, Lyon HN, Nemesh J, Drake JA, Tuomi T, Gaudet D, et al. Association studies of BMI and type 2 diabetes in the neuropeptide y pathway: a possible role for NPY2R as a candidate gene for type 2 diabetes in men. Diabetes 2007; 56:1460–1467.
10. Arnett DK, Devereux RB, Rao DC, Li N, Tang W, Kraemer R, et al. Novel genetic variants contributing to left ventricular hypertrophy: the HyperGEN study. J Hypertens 2009; 27:1585–1593.
11. Sainsbury A, Schwarzer C, Couzens M, Fetissov S, Furtinger S, Jenkins A, et al. Important role of hypothalamic Y2 receptors in body weight regulation revealed in conditional knockout mice. Proc Natl Acad Sci U S A 2002; 99:8938–8943.
12. Garrett MR, Rapp JP. Multiple blood pressure QTL on rat chromosome 2 defined by congenic Dahl rats. Mamm Genome 2002; 13:41–44.
13. Wen G, Mahata SK, Cadman P, Mahata M, Ghosh S, Mahapatra NR, et al. Both rare and common polymorphisms contribute functional variation at CHGA, a regulator of catecholamine physiology. Am J Hum Genet 2004; 74:197–207.
14. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Method Mol Biol 2000; 132:365–386.
15. Cockburn M, Hamilton A, Zadnick J, Cozen W, Mack TM. The occurrence of chronic disease and other conditions in a large population-based cohort of native Californian twins. Twin Res 2002; 5:460–467.
16. Zhang L, Rao F, Wessel J, Kennedy BP, Rana BK, Taupenot L, et al. Functional allelic heterogeneity and pleiotropy of a repeat polymorphism in tyrosine hydroxylase: prediction of catecholamines and response to stress in twins. Physiol Genomics 2004; 19:277–291.
17. Wessel J, Moratorio G, Rao F, Mahata M, Zhang L, Greene W, et al. C-reactive protein, an ‘intermediate phenotype’ for inflammation: human twin studies reveal heritability, association with blood pressure and the metabolic syndrome, and the influence of common polymorphism at catecholaminergic/beta-adrenergic pathway loci. J Hypertens 2007; 25:329–343.
18. Shih PA, Wang L, Chiron S, Wen G, Nievergelt C, Mahata M, et al. Peptide YY (PYY) gene polymorphisms in the 3’-untranslated and proximal promoter regions regulate cellular gene expression and PYY secretion and metabolic syndrome traits in vivo. J Clin Endocrinol Metab 2009; 94:4557–4566.
19. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005; 21:263–265.
20. Schaid DJ, Rowland CM, Tines DE, Jacobson RM, Poland GA. Score tests for association between traits and haplotypes when linkage phase is ambiguous. Am J Hum Genet 2002; 70:425–434.
21. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994; 22:4673–4680.
22. Wasserman WW, Sandelin A. Applied bioinformatics for the identification of regulatory elements. Nat Rev Genet 2004; 5:276–287.
23. Sandelin A, Wasserman WW, Lenhard B. ConSite: Web-based prediction of regulatory elements using cross-species comparison. Nucleic Acids Res 2004; 32(suppl 2):W249–W252.
24. Almasy L, Blangero J. Multipoint quantitative-trait linkage analysis in general pedigrees. Am J Hum Genet 1998; 62:1198–1211.
25. Falconer DS, Mackay TFC. Introduction to quantitative genetics. 4th ed.Harlow, Essex, UK:Longman; 1996.
26. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25:402–408.
27. Lagrange T, Kapanidis AN, Tang H, Reinberg D, Ebright RH. New core promoter element in RNA polymerase II-dependent transcription: sequence-specific DNA binding by transcription factor IIB. Genes Dev 1998; 12:34–44.
28. Javahery R, Khachi A, Lo K, Zenzie-Gregory B, Smale ST. DNA sequence requirements for transcriptional initiator activity in mammalian cells. Mol Cell Biol 1994; 14:116–127.
29. Naveilhan P, Hassani H, Canals JM, Ekstrand AJ, Larefalk A, Chhajlani V, et al. Normal feeding behavior, body weight and leptin response require the neuropeptide Y Y2 receptor. Nat Med 1999; 5:1188–1193.
30. Yamada M, Shida Y, Takahashi K, Tanioka T, Nakano Y, Tobe T. Prg1 is regulated by the basic helix-loop-helix transcription factor Math2. J Neurochem 2008; 106:2375–2384.
31. Impey S, McCorkle SR, Cha-Molstad H, Dwyer JM, Yochum GS, Boss JM, et al. Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions. Cell 2004; 119:1041–1054.
32. Lattanzi W, Bernardini C, Gangitano C, Michetti F. Hypoxia-like transcriptional activation in TMT-induced degeneration: microarray expression analysis on PC12 cells. J Neurochem 2007; 100:1688–1702.
33. Dehghan A, Dupuis J, Barbalic M, Bis JC, Eiriksdottir G, Lu C, et al. Meta-analysis of genome-wide association studies in >80 000 subjects identifies multiple loci for C-reactive protein levels. Circulation 2011; 123:731–738.
34. Nakazawa T, Satoh J, Takahashi K, Sakata Y, Ikehata F, Takizawa Y, et al. Complete suppression of insulitis and diabetes in NOD mice lacking interferon regulatory factor-1. J Autoimmun 2001; 17:119–125.
35. Vidarsson H, Westergren R, Heglind M, Blomqvist SR, Breton S, Enerback S. The forkhead transcription factor Foxi1 is a master regulator of vacuolar H-ATPase proton pump subunits in the inner ear, kidney and epididymis. PLoS ONE 2009; 4:e4471.
36. Wei Z, Biswas N, Wang L, Courel M, Zhang K, Soler-Jover A, et al. A Common Genetic Variant in the 3’-UTR of Vacuolar H+-ATPase ATP6V0A1 Creates a Micro-RNA Motif to Alter Chromogranin A (CHGA) Processing and Hypertension Risk. Circ Cardiovasc Genet 2011; 4:381–389.
37. Martínez-Estrada OM, Lettice LA, Essafi A, Guadix JA, Slight J, Velecela V, et al. Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin. Nat Genet 2010; 42:89–93.
38. Park SY, Kim HS, Kim NH, Ji S, Cha SY, Kang JG, et al. Snail1 is stabilized by O-GlcNAc modification in hyperglycaemic condition. EMBO J 2010; 29:3787–3796.
autonomic; genetics; hypertension; nervous system; obesity
Supplemental Digital Content
© 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins
Highlight selected keywords in the article text.