Project Summary Worldwide osteoporosis affects over 200 million people annually, particularly postmenopausal women who suffer ~9 million fractures or one fracture every 3 seconds. It is a disease in which bone density and strength is diminished to a point where the skeleton cannot adequately perform its support functions, increasing the risk of fractures and contributing to substantial morbidity and mortality in the elderly. In addition, other bone thinning disorders such as osteopenia, osteogenesis imperfect and renal osteodystrophy are prevalent, creating an urgent need for effective therapies that promote bone health. Mutations along the WNT signaling pathways have been shown to play key roles in bone metabolism, triggering tremendous interest in determining and possibly exploiting the role of WNT signaling in bone as a new therapeutic approach for the treatment of osteoporosis, other diseases with LBM and increased bone fragility. Our research program has generated important data interconnecting Mef2C and Sost as critical components of WNT signaling pathway in osteoblasts/osteocytes. Genetic analysis of Mef2C, ECR5 and Sost has established these proteins and Sost's regulatory element as key mediators of bone homeostasis. In this proposal we intended to pinpoint precisely what molecular functions are associated with which cell type(s) in bone to dissect out cell-type specific contributions of Mef2C and Sost to distinct functions during bone metabolism. Particular focus will be given in Aim 1 to Mef2C role in osteoclasts; using a combination of different Cre-recombinase transgenic mice we will delete Mef2C in osteoclasts and osteoblasts to determine: a) if Mef2C has dual roles in bone by activating genes that promote bone resorption in osteoclasts and genes that inhibit bone formation in osteoblasts; b) if Mef2C KO causes high bone mass by mechanism independent of Sost. In Aim 2 we will determine whether Mef2C directly or indirectly controls the transcription of energy metabolism genes, in osteoclasts. Through a combination of RNAseq, ChIPseq, enhancer validation, siRNA and overexpression of Mef2C we will determine whether (1) Ppargc1? and ppargc1? are direct transcriptional targets of Mef2C in osteoclasts; (2) Mef2C physically interact with Ppargc1? and/or ppargc1? to bind to similar DNA elements in osteoclasts, and whether (3) 162 energy metabolism genes down-regulated in Mef2CcKO; Ctsk-Cre mice are direct transcriptional targets of Mef2C. In Aim 3 we will identify putative Mef2C osteoclast and osteoblast enhancers, and validate them in vitro, in cell line models. Validated enhancers in combination with their transcriptional target genes will be used to build transcriptional networks that are modulated during bone metabolism. Our overarching goal is to understand how Mef2C contributes to bone metabolism by regulating osteoclast and osteoblast gene expression and to identify the molecules involved in this process; this work could ultimately lead to the discovery of new candidate molecules that could be therapeutically targeted to improve human bone health.