Multiple Functional Variants at 13q14 Risk Locus for Osteoporosis Regulate RANKL Expression Through Long-Range Super-Enhancer

Abstract

RANKL is a key regulator involved in bone metabolism, and a drug target for osteoporosis. The clinical diagnosis and assessment of osteoporosis are mainly based on bone mineral density (BMD). Previous powerful genomewide association studies (GWASs) have identified multiple intergenic single-nucleotide polymorphisms (SNPs) located over 100 kb upstream of RANKL and 65 kb downstream of AKAP11 at 13q14.11 for osteoporosis. Whether these SNPs exert their roles on osteoporosis through RANKL is unknown. In this study, we conducted integrative analyses combining expression quantitative trait locus (eQTL), genomic chromatin interaction (high-throughput chromosome conformation capture [Hi-C]), epigenetic annotation, and a series of functional assays. The eQTL analysis identified six potential functional SNPs (rs9533090, rs9594738, r8001611, rs9533094, rs9533095, and rs9594759) exclusively correlated with RANKL gene expression (p < 0.001) at 13q14.11. Co-localization analyses suggested that eQTL signal for RANKL and BMD-GWAS signal shared the same causal variants. Hi-C analysis and functional annotation further validated that the first five osteoporosis SNPs are located in a super-enhancer region to regulate the expression of RANKL via long-range chromosomal interaction. Particularly, dual-luciferase assay showed that the region harboring rs9533090 in the super-enhancer has the strongest enhancer activity, and rs9533090 is an allele-specific regulatory SNP. Furthermore, deletion of the region harboring rs9533090 using CRISPR/Cas9 genome editing significantly reduced RANKL expression in both mRNA level and protein level. Finally, we found that the rs9533090-C robustly recruits transcription factor NFIC, which efficiently elevates the enhancer activity and increases the RANKL expression. In summary, we provided a feasible method to identify regulatory noncoding SNPs to distally regulate their target gene underlying the pathogenesis of osteoporosis by using bioinformatics data analyses and experimental validation. Our findings would be a potential and promising therapeutic target for precision medicine in osteoporosis. © 2018 American Society for Bone and Mineral Research.

lncRNA Neat1 Stimulates Osteoclastogenesis Via Sponging miR-7

“…Recent studies shown that noncoding GWAS SNPs regulate distal target genes via long‐range looping formation. ( 8,23,41,42 ) Similarly, we first demonstrated that NEAT1 could be regulated by one distal noncoding SNP (rs12789028) associated with eBMD through long‐range chromatin interaction (113 kb) in an allele‐specific manner. In consistent with the GWAS association analyses, our functional assays showed that the reported osteoporosis risk allele of rs12789028‐G augments NEAT1 expression compared with rs12789028‐A, whose increased expression further decreases bone mass by promoting osteoclastogenesis.…”

VDR and TNFSF11 polymorphisms are associated with osteoporosis in Thai patients

“…It is possible that base pair change at these SNPs may affect the binding of the transcription factor, resulting in the regulation of RANKL expression. This particularly applies to rs9533090, where deletion of the region harboring this SNP could result in the reduction of RANKL expression (34). The SNP rs9533090 can be C or T; however, allele C has greater ability to recruit the transcription factor nuclear factor I C compared with allele T, and may thus more efficiently elevate enhancer activity and increase RANKL expression (33); therefore, it is plausible to consider an association between rs9533090 and osteoporosis.…”

Functions and Clinical Significance of Super-Enhancers in Bone-Related Diseases

“…Diseases-related cells acquire SEs through various mechanisms, including (i)mutations and genomic alterations like deletions, duplications, translocations, insertions, inversions (Mansour et al, 2014;Oldridge et al, 2015;Zhang et al, 2019); (ii) single-nucleotide polymorphisms (SNPs) (Zhu et al, 2018); (iii) chromosomal rearrangements (Affer et al, 2014;Gröschel et al, 2014;Demchenko et al, 2016); and (iv) 3D genome structural changes (Furlong and Levine, 2018). Once the SEs originated through the above mechanisms, resulting in the dysregulation of nearby SEs-target genes, which ultimately accelerates the deterioration of diseases.…”

“…Osteoporosis has become a global health concern. Zhu et al (2018) found a SE-associated gene-RANKL-was critical for the progress of osteoporosis, by analyzing the ChIPseq Data from ENCODE. Some significant genome-wide association studies (GWAS) have shown that single nucleotide polymorphisms (SNPs) near RANKL are associated with osteoporosis on chromosome 13q14.11 (Zhang et al, 2014;Kuosmanen et al, 2016).…”

“…Some significant genome-wide association studies (GWAS) have shown that single nucleotide polymorphisms (SNPs) near RANKL are associated with osteoporosis on chromosome 13q14.11 (Zhang et al, 2014;Kuosmanen et al, 2016). Zhu et al (2018) identified five BMD-associated SNPs (rs9533094, rs9533090, rs9594738, rs8001611, and rs9533095) that may be related to osteoporosis in the SEs region via bioinformatics data analysis and experimental validation. RANKL is a key regulator involved in bone remodeling and is critical for osteoclast differentiation, activation, survival, and enhancement of bone resorption (Lacey et al, 1998;Hsu et al, 1999;Jimi et al, 1999;Sobacchi et al, 2007).…”