Sequence variants of the CRH 5'-flanking region: effects on DNA-protein interactions studied by EMSA in PC12 cells

Recently, studies in adult rheumatoid arthritis patients have shown an association with four single-nucleotide polymorphisms (SNPs) in the 3.7-kb regulatory region of human corticotropin-releasing hormone (hCRH) gene located at positions -3531, -3371, -2353, and -684 bp. Three of these novel polymorphisms are in absolute linkage disequilibrium, resulting in three combined alleles, named A1B1, A2B1, and A2B2. To study whether the described polymorphic nucleotide sequences in the 5' region of the hCRH gene interfere with binding of nuclear proteins, an electric mobility shift assay (EMSA) was performed. At position -2353 bp, a specific DNA protein complex was detected for the wild-type sequence only, possibly interfering with a binding site for the activating transcription factor 6 (ATF6). In contrast, no difference could be detected for the other SNPs. However, at position -684, a quantitative difference in protein binding due to cAMP incubation could be observed. To further investigate whether these SNPs in the CRH promoter are associated with an altered regulation of the CRH gene, we performed a luciferase reporter gene assay with transiently transfected rat pheochromocytoma cells PC12. Incubation with 8-Br-cAMP alone or in combination with cytokines enhanced significantly the promoter activity in PC12 cells. The promoter haplotypes studied exhibited a differential capacity to modulate CRH gene expression. In all our experiments, haplotype A1B1 showed the most pronounced influence on promoter activity. Taken together, our results demonstrate a differential binding capacity of nuclear proteins of the promoter polymorphisms resulting in a different gene regulation. Most probably the SNP at position -2,353 plays a major role in mediating these differences.