PROJECT SUMMARY Osteoporosis is the most common aging-related metabolic bone disease mainly characterized by low bone mineral density (BMD) and deteriorated bone quality/strength. Peripheral blood monocytes (PBMs) can not only act as precursors of osteoclasts but also produce cytokines important for osteoclast differentiation and function, and thus represent major systemic cells for bone metabolism. DNA methylation as an important epigenetic regulator of gene expression may have significant and potential sex-specific effects in the etiology of complex diseases. However, the significance of global DNA methylation profiles underlying osteoporosis risk is largely unknown, particularly in males who suffer significantly higher morbidity and mortality rates upon osteoporotic fractures than females. Our General Hypothesis is that altered DNA methylation profiles in PBMs and the associated changes in gene expression and osteoclastogenesis contribute to peak BMD and bone quality/strength variation in males. Our Goals/Expectations are to 1) identify and characterize, at the epigenome-wide level, differentially methylated regions (DMRs) in PBMs associated with osteoporosis risk in Caucasian males; 2) ascertain the DNA methylation mediated epigenetic mechanisms of osteoporosis, that is, how the DMRs regulate the expression of the coding/non- coding target genes and subsequent osteoclastogenesis. We will accomplish the following Specific Aims: 1) Identification/validation of DMRs significantly associated with peak BMD and bone quality/strength (QCT and FEA) in Caucasian males. We will perform systematic epigenome- wide and regional focused comparative DNA methylation profiling studies in PBMs of 100 discordant Caucasian males (?Discovery cohort?) at peak bone mass aged 20-30, including half with extremely high BMDs and the other half with extremely low BMDs, and validate the top most significant DMRs in both of the ?Discovery cohort? and an independent ?Replication cohort? of 100 Caucasian males discordant for peak BMDs. 2) Determination of the sex- and ethnic-generality/specificity of the significant DMRs. The replicated DMRs will be tested in three independent samples, including a) 160 Caucasian females (through a DNA methylation study for female osteoporosis, R01AR059781), b) 100 African American males, and c) 160 Chinese males. 3) In-depth molecular investigation of the functional roles of the validated DMRs in regulating coding/non-coding gene expression and osteoclastogenesis. We will identify potential DMR cis-regulated coding/non-coding genes by integrative analyses of DNA methylation data, transcriptomic data (Proj 2), and DNA sequence data (Proj 1) in the same set of PBMs from the same 100 Caucasian males in the Discovery Cohort, and conduct in vitro cell-based functional assays to assess how DNA methylation at these DMRs regulates target gene expression and osteoclastogenesis. This novel project holds a great promise of award to generate breakthroughs in the osteoporosis research field. The results will give new insights into the epigenetic mechanisms underlying osteoporosis. The knowledge gained may ultimately lead to novel approaches to better prevention and treatment of osteoporosis.