An ancestral variant of Secretogranin II confers regulation by PHOX2 transcription factors and association with hypertension

Abstract

Granins regulate secretory vesicle formation in neuroendocrine cells and granin-derived peptides are co-released with neurotransmitters as modulatory signals at sympathetic sites. We report evidence for association between a regulatory polymorphism in Secretogranin II (SCG2) and hypertension in African-American subjects. The minor allele is ancestral in the human lineage and is associated with disease risk in two case-control studies and with elevated blood pressure in a separate familial study. Mechanistically, the ancestral allele acts as a transcriptional enhancer in cells that express endogenous Scg2, whereas the derived allele does not. ARIX (PHOX2A) and PHOX2B are identified as potential transactivating factors by oligonucleotide affinity chromatography and mass spectrometry and confirmed by chromatin immunoprecipitation. Each of these transcription factors preferentially binds the risk allele, both in vitro and in vivo. Population genetic considerations suggest positive selection of the protective allele within the human lineage. These results identify a common regulatory variation in SCG2 and implicate granin gene expression in the control of human blood pressure and susceptibility to hypertension.

Figure 1. SCG2 sequence variation

Top, sequence conservation is plotted for 100 bp sliding windows, visualized by VISTA. Arrows indicates initial resequencing reads for variant detection in clinical samples. Boxes indicate exons, coding region in black. Identified sequence variations are indicated by vertical bars. Variant sites whose minor alleles are common (>0.05), rare (<0.05, but seen more than once) or singleton in our data are indicated by bars of different height. X indicates SNPs for which the minor allele appears to be ancestral by comparison with other primates. Computationally constructed haplotypes from the four SNPs with minor allele frequency ≥0.05 (positions 736, 1253, 4366 and 5024 relative to the reported cap site) are indicated along with their distribution frequencies in four populations.

Figure 2. SCG2 intron haplotype network and sequences around SCG2 variant sites. show conservation of the minor allele in non-human primates

(A) Each haplotype is represented by a circle whose area represents relative haplotype frequency. Branches in the minimum spanning tree represent one nucleotide substitution (except for the branch to the inferred last common ancestor with chimpanzees, LCA). Dashed line indicates alternative topologies of equal length. Each number represents a haplotype that is corresponding to haplotypes in Supplemental Table S5. (B, C) Alignments of orthologous sequences around SNP_736 (B) and SNP_964 (C). Asterisks mark the human polymorphism sites. Primate sequences were obtained by PCR-directed sequencing; rat and mouse sequences were obtained from UCSC public database. Sequences were aligned in ClustalW.

Figure 3. SCG2 SNP_736 affects enhancer activity and nuclear extract factor binding

(A) Luciferase reporter assays show allele-specific enhancer activity. PC12 cells were co-transfected with a reporter driven by basal SV40 promoter plus SCG2 intronic fragment from the indicated haplotype and a second reporter as a transfection control in three independent experiments. One-factor ANOVA with Bonferroni correction indicates significant differences for haplotype GC vs. AT (*, P= 0.038) and for haplotype GC vs. AC (**, P= 0.003). (B) EMSA shows allele-selective binding competition assays from PC12 cells. 5, 10, 20-fold concentrations of the indicated unlabeled competitor were present in the binding reaction. (C) RT-PCR detection of SCG2 (top panel) in human and rat cell lines. NXF1 (bottom) is shown as a positive control. (D) Electrophoretic mobility shift assay (EMSA) with a 19 bp probe centered on SNP_736 identifies a complex specific to SCG2-expressing cell types. M, size marker; N, no extract; L, EBV-transformed lymphoblasts; F, EBV-transformed fibroblasts; 293, Human embryonic kidney 293 cells; Ne, Human neuroblastoma cell line IMR-32; P, Rat pheochromacytoma cell line PC12.

Figure 4. Properties of allele-specific binding activity and identification of Phox2a and Phox2b

(A) Elution profile of nuclear extracts after heparin column chromatography. (B) Allele-selective SCG2 SNP_736 binding activity is enriched in 0.5M NaCl fraction. Equal protein (10μg) was loaded for each fraction. N, no extract; C1, Crude nuclear extract before dialysis; C2, Crude nuclear extract after dialysis; F, Flow-through fraction. (C) Allele-selective binding by PC12 nuclear extract is salt-sensitive. N, no extract; G, G allele probe; A, A allele probe; C, Control probe (TGTACCTAATCACATTTG). (D) EMSA activity is sensitive to factor-specific antibodies. Phox2a antibody produces a modest supershift fraction while pre-incubation with Phox2b antibody neutralizes allele-selective binding for SCG2 SNP_736. Each antibody result was replicated in at least two independent experiments. No, No antibody control; NA, Non-specific antibody (TFIIB).

Figure 5. Allele-selective Phox2 binding in cells

(A) Chromatin immunoprecipitation (ChIP) from IMR-32 human neuroblastoma cells. SCG2 intron site is selectively enriched by Phox2 antibodies compared with positive (DBH) and negative (GAPDH) controls. The general transcription factor TFIIB enriches each target site. (B) ChIP from PC12 cells transfected with human SCG2 reporter constructs carrying either SNP_736 allele (A or G). ChIP products were assayed by competitive PCR for transfected human SCG2 intron enhancer (top band) and endogenous rat c-fos promoter (bottom band) as an internal control. SCG2 was selectively enriched by both Phox2a and Phox2b antibodies and to a lesser extent by TFIIB. c-fos was consistently enriched only by TFIIB. (C) Pyrosequencing quantifies SNP_736 allele ratios. Peak height ratios on each pyrogram report G (left) and A (right) alleles in mixing experiments for the indicated proportions. (D) Representative pyrosequencing analysis of ChIP products and input chromatin from cells co-transfected with equal amounts of each SNP_736 allele. Both Phox2a and Phox2b antibodies selectively enrich the ancestral A allele. (E) Quantification of pyrosequencing peak height ratios of A/G in SCG2 intron SNP_736 enriched by ChIP with Phox2a or Phox2b antibodies, along with control antibody TFIIB after normalization to peak height ratio of A versus G in the ChIP input fraction, indicates allele-selective binding by endogenous Phox2 proteins in synthetically heterozygous cells.