Recent genome-wide association studies (GWAS) identified 518 significant loci associated with bone mineral density (BMD),including variants at the RUNX1 locus (rs13046645, rs2834676, and rs2834694). However, their reg...Recent genome-wide association studies (GWAS) identified 518 significant loci associated with bone mineral density (BMD),including variants at the RUNX1 locus (rs13046645, rs2834676, and rs2834694). However, their regulatory impact on RUNX1expression and bone formation remained unclear. This study utilized human induced pluripotent stem cells (iPSCs) differentiatedinto osteoblasts to investigate these variants’ regulatory roles. CRISPR/Cas9 was employed to generate mutant (Δ) iPSC lines lackingthese loci at the RUNX1 locus. Deletion lines (Δ1 and Δ2) were created in iPSCs to assess the effects of removing regions containingthese loci. Deletion lines exhibited enhanced osteogenic potential, with increased expression of osteogenic marker genes andAlizarin Red staining. Circularized chromosome conformation capture (4C-Seq) was utilized to analyze interactions between BMDassociatedloci and the RUNX1 promoter during osteogenesis. Analysis revealed altered chromatin interactions with multiple genepromoters including RUNX1 isoform, as well as SETD4, a histone methyltransferase, indicating their regulatory influence.Interestingly, both deletion lines notably stimulated the expression of the long isoform of RUNX1, with more modest effects on theshorter isoform. Consistent upregulation of SETD4 and other predicted targets within the Δ2 deletion suggested its removalremoved a regulatory hub constraining expression of multiple genes at this locus. In vivo experiments using a bone defect model inmice demonstrated increased bone regeneration with homozygous deletion of the Δ2 region. These findings indicate that BMDassociatedvariants within the RUNX1 locus regulate multiple effector genes involved in osteoblast commitment, providing valuableinsights into genetic regulation of bone density and potential therapeutic targets.展开更多
基金supported by NIH-R21 Award (R21AR071536) to HDfunds from Emory University。
文摘Recent genome-wide association studies (GWAS) identified 518 significant loci associated with bone mineral density (BMD),including variants at the RUNX1 locus (rs13046645, rs2834676, and rs2834694). However, their regulatory impact on RUNX1expression and bone formation remained unclear. This study utilized human induced pluripotent stem cells (iPSCs) differentiatedinto osteoblasts to investigate these variants’ regulatory roles. CRISPR/Cas9 was employed to generate mutant (Δ) iPSC lines lackingthese loci at the RUNX1 locus. Deletion lines (Δ1 and Δ2) were created in iPSCs to assess the effects of removing regions containingthese loci. Deletion lines exhibited enhanced osteogenic potential, with increased expression of osteogenic marker genes andAlizarin Red staining. Circularized chromosome conformation capture (4C-Seq) was utilized to analyze interactions between BMDassociatedloci and the RUNX1 promoter during osteogenesis. Analysis revealed altered chromatin interactions with multiple genepromoters including RUNX1 isoform, as well as SETD4, a histone methyltransferase, indicating their regulatory influence.Interestingly, both deletion lines notably stimulated the expression of the long isoform of RUNX1, with more modest effects on theshorter isoform. Consistent upregulation of SETD4 and other predicted targets within the Δ2 deletion suggested its removalremoved a regulatory hub constraining expression of multiple genes at this locus. In vivo experiments using a bone defect model inmice demonstrated increased bone regeneration with homozygous deletion of the Δ2 region. These findings indicate that BMDassociatedvariants within the RUNX1 locus regulate multiple effector genes involved in osteoblast commitment, providing valuableinsights into genetic regulation of bone density and potential therapeutic targets.
