Human NPY promoter variation rs16147:T>C as a moderator of prefrontal NPY gene expression and negative affect

Abstract

Studies in humans and animals suggest a role for NPY in the mediation of behavioral stress responses. Here, we examined whether the NPY promoter variant rs16147:T>C is functional for expression of NPY in a brain region relevant for behavioral control, anxiety and depression, the anterior cingulate cortex. In silico analysis of DNA structural profile changes produced by rs16147 variation suggests allelic differences in protein binding at the rs16147 site. This was confirmed by electrophoretic mobility shift assay, demonstrating that the rs16147 C-allele has strongly reduced affinity for a yet unknown factor compared to the T-allele. Analyzing 107 human post-mortem brain samples we show that allelic variation at rs16147 contributes to regulation of NPY mRNA and peptide levels in this region. Specifically, the C-allele leads to increased gene expression. In agreement with the molecular findings, rs16147:T>C is associated with anxiety and depressive symptoms in 314 young adults via a gene x environment interaction with early childhood adversity, replicating the recent finding of rs16147-C as a risk factor for stress related psychopathology. Our results show the importance of rs16147:T>C for regulation of NPY gene expression and brain function.

Figure 1

Altered DNA surface structure and DNA-protein interaction at rs16147. A) Allelic difference in DNA surface structure is shown at rs16147 by using predicted hydroxyl radical cleavage patterns (Greenbaum et al., 2007). Solid line = T-allele, dashed = C. B) Representative gel image of EMSA from ACC nuclear extracts show a single band (arrow) captured by T but not C allele-specific 33P-labeled oligonucleotide probes (probe specificities shown above lanes). Specificity of this interaction is demonstrated by adding unlabeled T- or C-allele and AP1 oligonucleotides as competitors (10 or 50 pmole, shown below the lanes). No brain extract control is shown on the left lane. C) Bar graphs showing quantitative analysis of the EMSAs. Data represent density values relative to the band generated by the T-allele oligonucleotide (lane 2). 1-way ANOVA: * p < 0.05, *** p < 0.001 vs. the T-allele band without competitor. D) No transcription factor binding sites are predicted within 20 bp up- and downstream of rs16147 (arrow) by evolutionary sequence constraint analysis (www.genomatix.de). Binding motifs for mammalian transcription factors are shown up to −500 bp upstream of the NPY transcriptional start site (red arrow).

Figure 2

Generalized regression model for NPY mRNA and peptide expression in ACC. Left: Regression estimates and their standard errors are shown for the predictors used in the model. Genotype for rs16147 was coded as TT=0, TC=1, CC=2. C.o.D. = cause of death, PMI = post mortem interval, RIN = RNA integrity number. NA = not applicable. CB mRNA = mRNA from cerebellum.. ⁺p < .10, *p < .05, **p<.01. Right: Individual NPY mRNA expression values in ACC are shown, number of subjects is given in parenthesis. Lines mark the mean ± upper and lower 95% confidence interval. NPY mRNA levels are expressed as ΔC_t(NPY-AluSx) representing the difference to the endogenous AluSx control on a log₂ scale. Higher values represent lower expression level.

Figure 3

Gene x environment interaction at NPY rs16147. A) Multiple (linear and logistic) regression models testing the effects of NPY rs16147 genotype, psychosocial adversity and their interaction on measures of depression and anxiety in young adults. Non-standardized regression coefficients b (SE) are shown for the main effects (second step), and for the interaction effects (third step) adjusted for sex; 1 coefficients from logistic regression; 2 coefficients from linear regression; +p < .10, *p < .05, **p<.01, ***p<.001. B) Mean BDI scores (SE), adjusted for sex, in young adults grouped by NPY rs16147 genotype and exposure to psychosocial adversity (divided by median split). C) Short Definition of the psychosocial adversity items (for detailed definition see Supp. Table S1). Numbers represent percentages of individuals exposed to the respective adversity condition. BDI = Beck Depression Inventory, STAIT = State-Trait Anxiety Inventory, T subscale.