The rs713586 risk variant dysregulates ADCY3 rather than DNAJC27, leading to obesity through ZFP42-TET1-mediated DNA methylation.

BACKGROUND: Human genetic analyses have identified numerous single-nucleotide polymorphism (SNP) loci in noncoding regions associated with obesity-related traits; however, the functional contributions of such SNP loci to obesity are largely unknown. The noncoding variant rs713586, with its risk allele C, is linked to two candidate genes, DNAJC27 and ADCY3, potentially implicated in obesity. However, whether rs713586 primary targets ADCY3 or DNAJC27 gene to regulate body weight and what molecular mechanisms underlie this process remain unclear. METHODS: We conducted bioinformatics analyses using BMI data from the UK biobank and GIANT consortium, and prioritised functional variants on chromosome 2 linked to ADCY3 gene for experimental validation. The variant rs713586 was identified as a functional regulator of ADCY3 and DNAJC27 expression. We investigated the molecular mechanisms by which rs713586 participates in obesity through epigenetic regulation. Dual-luciferase reporter assay and genome-editing in cell lines were conducted to assess the impacts of the rs713586-C risk allele or a proximal enhancer (Enh) on ADCY3 and DNAJC27 promoter activity and expression levels. CRISPR/Cas9-mediated knockout of Dnajc27 was performed in mice to evaluate its role in obesity. Mechanistic studies examined the interactions between the rs713586-T or -C alleles and the transcription factor ZFP42. Additionally, we assessed the DNA methylation patterns within the Enh and promoter regions of ADCY3 to evaluate their impact on ADCY3 expression. FINDINGS: First, the rs713586-C risk allele significantly reduced the promoter activity of ADCY3 and DNAJC27 and thus reduced their expression levels. However, Dnajc27 knockout mice did not develop obesity, thereby excluding DNAJC27 as the target gene through which rs713586 mediates obesity. Further, we demonstrate that the rs713586-C allele impaired ZFP42 binding, leading to decreased TET1 recruitment and increased DNA methylation in the Enh and promoter regions of ADCY3, ultimately suppressing its expression. Given that ADCY3 is a well-established gene involved in obesity, we conclude that the rs713586-C risk allele may associated with obesity susceptibility, concomitant with downregulated ADCY3 expression. INTERPRETATION: Our findings establish the rs713586-ZFP42-TET1-ADCY3 epigenetic regulatory axis, providing insights into the mechanism of rs713586-mediated obesity pathogenesis. FUNDING: National Natural Science Foundation of China and Natural Science Foundation of Hebei Province of China (32470645, 32070567, 32202840), and Priority-Funded Postdoctoral Research Project, Zhejiang Province (ZJ2025118). Full funding details are provided in the Acknowledgements.