Regulation of human ZNF687, a gene associated with Paget's disease of bone

Mutations in Zinc finger 687 (ZNF687) were associated with Paget's disease of bone (PDB), a disease characterized by increased bone resorption and excessive bone formation. It was suggested that ZNF687 plays a role in bone differentiation and development. However, the mechanisms involved in ZNF687 regulation remain unknown. This study aimed to obtain novel knowledge regarding ZNF687 transcriptional and epigenetic regulation. Through in silico analysis, we hypothesized three ZNF687 promoter regions located upstream exon 1 A, 1B, and 1 C and denominated promoter regions 1, 2, and 3, respectively. Their functionality was confirmed by luciferase activity assays and positive/negative regulatory regions were identified using promoter deletions constructs. In silico analysis revealed a high density of CpG islands in these promoter regions and in vitro methylation suppressed promoters' activity. Using bioinformatic approaches, bone-associated transcription factor binding sites containing CpG dinucleotides were identified, including those for NFκB, PU.1, DLX5, and SOX9. By co-transfection in HEK293 and hFOB cells, we found that DLX5 specifically activated ZNF687 promoter region 1, and its methylation impaired DLX5-driven promoter stimulation. NFκB repressed and activated promoter regions 1 and 2, respectively, and these activities were affected by methylation. PU.1 induced ZNF687 promoter region 1 which was affected by methylation. SOX9 differentially regulated ZNF687 promoters in HEK293 and hFOB cells that were impaired after methylation. In conclusion, this study provides novel insights into ZNF687 regulation by demonstrating that NFκB, PU.1, DLX5, and SOX9 are regulators of ZNF687 promoters, and DNA methylation influences their activity. The contribution of the dysregulation of these mechanisms in PDB should be further elucidated.