USF3 modulates osteoporosis risk by targeting WNT16, RANKL, RUNX2, and two GWAS lead SNPs rs2908007 and rs4531631.

Osteoporotic fractures cause major morbidity and mortality in the aging population. Genome-wide association studies (GWAS) have identified USF3 as the novel susceptibility gene of osteoporosis. However, the functional role in bone metabolism and the target gene of the basic helix-loop-helix transcription factor USF3 are unclear. Here, we show that USF3 enhances osteoblast differentiation and suppresses osteoclastogenesis in cultured human osteoblast-like U-2OS cells. Mechanistic studies revealed that transcription factor USF3 antagonistically interacts with anti-osteogenic TWIST1/TCF12 heterodimer in the WNT16 and RUNX2 promoter, and counteracts CREB1 and JUN/FOS in the RANKL promoter. Importantly, the osteoporosis GWAS variant g.1744A>G (rs2908007A>G) located in the WNT16 promoter confers G-allele-specific transcriptional modulation by USF3, TWIST1/TCF12 and TBX5/TBX15, and USF3 transactivates the osteoclastogenesis suppressor WNT16 promoter activity and antagonizes the repression of WNT16 by TWIST1 and TCF12. The risk G allele of osteoporosis GWAS variant g.3260A>G (rs4531631A>G) in the RANKL promoter facilitates the binding of CREB1 and JUN/FOS and enhances transactivation of the osteoclastogenesis contributor RANKL that is inhibited by USF3. Our findings uncovered the functional mechanisms of osteoporosis novel GWAS-associated gene USF3 and lead single nucleotide polymorphisms rs2908007 and rs4531631 in the regulation of bone formation and resorption.