Our previous genome-wide association study identified 56 loci that were associated with bone mineral density (BMD) and fracture risk in men and women. One locus on chromosome 12q23.3 harboring the gene TMEM263 (Transmembrane protein 263) was strongly associated with hip BMD (p<9.6x10-10). TMEM263 encodes a multi-pass transmembrane protein of unknown family. To identify the role of this gene in bone biology, we tested the SNP (rs1053051) associated with hip BMD in the TMEM263 gene for allele-specific expression (ASE) differences using human femoral bone samples. We observed a unidirectional allelic imbalance in mRNA expression for this SNP, suggesting that variation in ASE in TMEM263 may contribute to variation in hip BMD. The mouse Tmem263 gene was highly expressed in skeletal tissue compared to non- skeletal tissues, and osteoblasts and osteocytes expressed 6-fold higher mRNA levels of Tmem263 compared to osteoclasts. Also, in an established osteoblast cell line, OB6, gene-silencing with shRNA specific for Tmem263 revealed decreased expression of genes important for bone formation and increased expression of genes related to bone resorption. We recently made osteoblast-specific Tmem263 KO mice. Preliminary data show that these mice display broken bones and significantly lower whole body aBMD, BMC and trabecular bone mass and compromised bone micro-architecture compared to WT mice. Therefore, we hypothesize that: 1) the Tmem263 gene plays an important role in the acquisition and maintenance of bone mass; 2) osteoblast- specific deletion of Tmem263 in mice will lead to reduced bone mass and strength compared to controls; 3) lower BMD and strength in KO mice will result from decreased proliferation, impaired differentiation and/or diminished activity of osteoblasts. We will test these hypotheses in global and osteoblast-specific Tmem263 knockout mice and Tmem263 overexpressing Tg mice. We will measure areal BMD of whole body, femur and spine by in-vivo DXA and determine volumetric BMD for cortical and trabecular bone in femur by in-vivo μCT. Bone strength will be tested in femur by 3-point bending. Further, we will determine the molecular and cellular mechanisms responsible for changes in bone phenotype by measuring serum markers of bone turnover, quantifying expression of genes important in bone formation and resorption, and performing static and dynamic bone histomorphometry. We will identify protein-protein interaction partners of Tmem263 using two complementary proteomics techniques. We will also use RNA sequencing in KO and overexpressing Tmem263 newborn pups to identify global gene expression changes. The proteomic and RNA sequencing studies will identify specific pathways and networks involved in Tmem’s role in bone health. In summary, we propose to use novel mouse models to understand the function of a unique gene product on bone and mineral metabolism. Successful completion of the proposed studies could open a new area of bone biology and ...