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.

Computational and functional characterization of four SNPs in the SOST locus associated with osteoporosis.

The SOST gene encodes sclerostin, a C-terminal cysteine knot-like domain containing key negative regulator of osteoblastic bone formation that inhibits LRP5/6-mediated canonical Wnt signaling. Numerous single nucleotide polymorphisms (SNPs) in the SOST locus are firmly associated with bone mineral density (BMD) and fracture in genome-wide association studies (GWAS) and candidate gene association studies. However, the validation and mechanistic elucidation of causal genetic variants, especially for SNPs located beyond the promoter-proximal region, remain largely unresolved. By employing computational and experimental approaches, here we identify four SNPs rs1230399, rs7220711, rs1107748 and rs75901553 as functional variants which display allelic variation in SOST gene expression. The osteoporosis associated SNP rs1230399 in the SOST distal upstream regulatory region shows FOXA1 binding activity with subsequent transinactivation in a T allele-specific manner. The BMD GWAS lead SNPs rs7220711 and rs1107748 both reside in the 52-kb regulatory element deletion 35-kb downstream of the SOST gene which leads to Van Buchem disease. The rs7220711-A has a higher affinity for the transcriptional repressors MAFF or MAFK homodimers than rs7220711-G, while rs1107748 confers C allele specific transcriptional enhancer activity via a CTCF binding element. The variant rs75901553 C>T located in a conserved site of the SOST 3' UTR abolishes a target binding site for miR-98-5p which is negatively responsive to parathyroid hormone or 17ß-estradiol in osteoblastic cell lines. Our findings uncover the biological consequences of four independent genetic variants in the SOST region and their important roles in SOST expression via diverse mechanisms, providing new insights into the genetics and molecular pathogenesis of osteoporosis.