Suthon Sarocha, Perkins Rachel S, Lin Jianjian, Crockarell John R, Miranda-Carboni Gustavo A, Krum Susan A
Department of Orthopaedic Surgery and Biomedical Engineering, University of Tennessee Health Science Center, Memphis, TN, USA.
Campbell Clinic, Memphis, TN, USA.
Hum Genet. 2022 Dec;141(12):1849-1861. doi: 10.1007/s00439-022-02463-8. Epub 2022 Jun 9.
Osteoporosis is a serious public health problem that affects 200 million people worldwide. Genome-wide association studies have revealed the association between several single nucleotide polymorphisms (SNPs) near WNT/β-catenin signaling genes and bone mineral density (BMD). The activation of β-catenin by WNT ligands is required for osteoblast differentiation. SNP rs9921222 is an intronic variant of AXIN1 (a scaffold protein in the destruction complex that regulates β-catenin signaling) that correlates with BMD. However, the biological mechanism of SNP rs9921222 has never been reported. Here, we show that the genotype of SNP rs9921222 correlates with the expression of AXIN1 in human osteoblasts. RNA and genomic DNA were analyzed from primary osteoblasts from 111 different individuals. Homozygous TT at rs9921222 correlates with a higher expression of AXIN1 than homozygous CC. Regional association analysis showed that rs9921222 is in high linkage disequilibrium (LD) with SNP rs10794639. In silico transcription factor analysis predicted that rs9921222 is within a GATA4 motif and rs10794639 is adjacent to an estrogen receptor alpha (ERα) motif. Mechanistically, GATA4 and ERα bind at SNPs rs9921222 and rs10794639 as detected by ChIP-qPCR. Luciferase assays demonstrate that rs9921222 is the causal SNP to alter ERα and GATA4 binding. GATA4 promoted the expression, and in contrast, ERα suppressed the expression of AXIN1 via the histone deacetylase complex member SIN3A. Functionally, the level of AXIN1 negatively correlates with the level of transcriptionally active β-catenin. In summary, we have discovered a molecular mechanism of the SNP rs9921222 to regulate AXIN1 through GATA4 and ERα binding in human osteoblasts.
骨质疏松症是一个严重的公共卫生问题，全球有2亿人受其影响。全基因组关联研究揭示了WNT/β-连环蛋白信号基因附近的几个单核苷酸多态性（SNP）与骨密度（BMD）之间的关联。WNT配体对β-连环蛋白的激活是成骨细胞分化所必需的。SNP rs9921222是AXIN1（一种在调节β-连环蛋白信号的破坏复合物中的支架蛋白）的内含子变体，与骨密度相关。然而，SNP rs9921222的生物学机制从未被报道过。在此，我们表明SNP rs9921222的基因型与人类成骨细胞中AXIN1的表达相关。对来自111个不同个体的原代成骨细胞的RNA和基因组DNA进行了分析。rs9921222处的纯合子TT与AXIN1的表达高于纯合子CC相关。区域关联分析表明，rs9921222与SNP rs10794639处于高度连锁不平衡（LD）状态。计算机转录因子分析预测，rs9921222位于一个GATA4基序内，rs10794639与雌激素受体α（ERα）基序相邻。从机制上讲，通过染色质免疫沉淀定量PCR（ChIP-qPCR）检测发现，GATA4和ERα在SNP rs9921222和rs10794639处结合。荧光素酶测定表明，rs9921222是改变ERα和GATA4结合的因果SNP。GATA4促进了表达，相反，ERα通过组蛋白去乙酰化酶复合物成员SIN3A抑制了AXIN1的表达。在功能上，AXIN1的水平与转录活性β-连环蛋白的水平呈负相关。总之，我们发现了SNP rs9921222在人类成骨细胞中通过GATA4和ERα结合来调节AXIN1的分子机制。
