Identification of a chronic obstructive pulmonary disease genetic determinant that regulates HHIP

Abstract

Multiple intergenic single-nucleotide polymorphisms (SNPs) near hedgehog interacting protein (HHIP) on chromosome 4q31 have been strongly associated with pulmonary function levels and moderate-to-severe chronic obstructive pulmonary disease (COPD). However, whether the effects of variants in this region are related to HHIP or another gene has not been proven. We confirmed genetic association of SNPs in the 4q31 COPD genome-wide association study (GWAS) region in a Polish cohort containing severe COPD cases and healthy smoking controls (P = 0.001 to 0.002). We found that HHIP expression at both mRNA and protein levels is reduced in COPD lung tissues. We identified a genomic region located ∼85 kb upstream of HHIP which contains a subset of associated SNPs, interacts with the HHIP promoter through a chromatin loop and functions as an HHIP enhancer. The COPD risk haplotype of two SNPs within this enhancer region (rs6537296A and rs1542725C) was associated with statistically significant reductions in HHIP promoter activity. Moreover, rs1542725 demonstrates differential binding to the transcription factor Sp3; the COPD-associated allele exhibits increased Sp3 binding, which is consistent with Sp3's usual function as a transcriptional repressor. Thus, increased Sp3 binding at a functional SNP within the chromosome 4q31 COPD GWAS locus leads to reduced HHIP expression and increased susceptibility to COPD through distal transcriptional regulation. Together, our findings reveal one mechanism through which SNPs upstream of the HHIP gene modulate the expression of HHIP and functionally implicate reduced HHIP gene expression in the pathogenesis of COPD.

Figure 1.

COPD GWAS locus on chromosome 4q31 and HHIP expression levels in lung tissues. (A) Regional diagram for the COPD association signal on chromosome 4q31 around the HHIP gene based on genotypes from HapMap. Linkage disequilibrium (r²) is represented by gray scale and calculated using the CEU reference panel in Haploview. Black bar indicates the LD block containing the COPD-susceptibility locus; grey bar indicates the HHIP gene and grey arrow indicates the transcription direction of the HHIP gene. Arrowheads mark the locations of published COPD GWAS associations: rs12504628, rs13147758, rs1828591, rs1980057 and rs13118928. (B and C) Expression of HHIP is decreased in COPD compared with control lung tissues as determined by real-time PCR TaqMan assay for mRNA levels (B) and by western blot for protein levels (C). Two probes used for the HHIP gene in the gene expression assay showed similar trends and results from one representative probe are shown in the left panel. Means ± standard errors are shown.

Figure 2.

Identification of a long-range enhancer for HHIP in the COPD GWAS locus on chromosome 4q31. (A). Long-range interaction between the COPD-susceptibility locus and the HHIP promoter in Beas-2B (bronchial epithelial) and MRC5 (lung fibroblast) cells detected by 3C-PCR. The graph demonstrates 3C interaction frequency of the constant fragment containing the HHIP promoter (orange bar) with other target fragments (black bars). The y-axis refers to 3C-PCR products normalized to the interaction frequency of fragments from the BAC clone. Geometric means and standard errors were from duplicate PCR reactions. Middle section: snapshot of LD block from UCSC genome browser with COPD GWAS variants. Lower section: 3C fragments at various lengths after BglII digestion. (B) ChIP-qPCR revealed relative enrichment of H3K4Me1 in the 3C interaction fragment (light blue column) compared with the control region (dark blue column). Geometric means and standard errors are shown from three independent repeats. Control region: 145668717–145668900; COPD GWAS SNP region: 145705250–145705431; (C) reporter assays revealed enhancer activity of COPD GWAS SNP region. Left panel: three pieces of the HHIP COPD GWAS SNP region were cloned to the 5′-end of the native HHIP promoter (control, orange bars) in the pGL3 basic reporter vector. Right panel: relative promoter activity in Beas-2B cells transfected with indicated constructs. Geometric means and standard errors were calculated from three independent repeats done in triplicate wells. GWAS regions—4K: 145701K–145705K; 3K: 145705K–145708K; 2.4K: 145705843–145708234; HHIP promoter: 145,786,102–145,787,277 based on hg18.

Figure 3.

COPD risk allele is associated with lower HHIP promoter activity. (A) The minimal COPD GWAS SNP region (∼500 bp around two key SNPs, light purple column) cloned at forward orientation (>>>) and reverse orientation (<<<) showed enhancer activity for the HHIP promoter (‘control’, orange) measured by dual-luciferase in Beas-2B cells. Shown are ratios of firefly luciferase expression to Renilla luciferase expression normalized to the mean ratio from the control construct. Geometric means and standard errors are shown for five independent repeats done in triplicate or quadruplicate. (B) The effects of rs1542725 and rs6537296 were evaluated in the reporter assays. Single-nucleotide alterations were introduced individually or combined into minimal enhancer constructs at forward (upper panel) and reverse (lower panel) orientations. Shown are ratios of firefly luciferase expression to Renilla luciferase expression normalized to the mean ratio from the risk haplotype (rs6537296A and rs1542725C). Geometric means and standard errors were calculated from eight (forward) or three (reverse) independent repeats.

Figure 4.

rs1542725 alters a Sp3 binding site. (A) Three probes used in EMSA are shown with nucleotides with underlines for rs1542725 and in italic for 6 bp central mutations spanning rs1542725. (B) P³² labeled oligonucleotide containing the sequence spanning rs1542725 with either C allele (rs1542725C, lanes 2–8) or T allele (rs1542725T, lane 1) was incubated with nuclear extract from Beas-2B cells. rs1542725C probe was incubated with identical competitor probe (WT) in lane 3, the NIC probe (NC) in lane 4 and the 6 bp mutant probe (MT) in lane 5. Sp1, Sp3 and unrelated Oct-1 control (Ctrl) antibodies were added in lanes 6, 7 and 8, respectively. Star (*) indicates the supershifted Sp3 band in lane 7. Arrows indicate specific nuclear proteins bound by probe.