Bone Abstracts

Epigenetic mechanisms regulating osteoblast differentiation are still inadequately described. In a genome wide transcriptional profiling of MC3T3-E1 osteoblastic cell line, we identified RCOR2 as a significantly upregulated gene during a differentiation time-course from proliferative to mature osteoblasts. Similar expression profile of RCOR2 was found in mouse calvarial osteoblasts. RCOR2 belongs to CoREST/RCOR family of proteins that regulate action of lysine-specific histone demethylase 1 (LSD1). LSD1 controls gene expression, for example, by demethylating histone H3K4me1/2, leading generally to gene repression. Lentiviral shRNA-mediated silencing of RCOR2 expression in MC3T3-E1 cells led to impaired osteoblastic differentiation shown by decreased ALP staining and number of mineralized bone nodules, and reduced expression of osteoblast-related genes. LSD1 is abundantly expressed in MC3T3-E1 cells and we showed that RCOR2 and LSD1 co-immunoprecipitated in HEK293T cells. However, we did not find differences in global H3K4me1/2 levels between shRCOR2 and control cells. To identify specific genes and pathways associated with RCOR2 action, we performed RNA-seq analysis of MC3T3-E1-shRCOR2 cells at the early stages of differentiation. GO pathway analysis showed upregulation of genes related to cellular metabolism and embryonic morphogenesis in shRCOR2 cells, suggesting RCOR2 to repress these pathways at the onset of differentiation. Using our genome-wide H3K4me1 ChIP-seq map of MC3T3-E1 cells, we identified putative enhancer regions in selected shRCOR2-affected genes and tested them for H3K4me1 occupancy in shRCOR2 and control cells by ChIP. We observed alterations in H3K4me1 levels relative to total histone H3 occupancy in RCOR2 knockdown cells, suggesting changes in LSD1 activity at specific genomic sites. In conclusion, RCOR2 is an important new player in regulating the transition from proliferative to committed osteoblastic phenotype. Our data suggest that RCOR2 could function at least in part via regulating histone methylation and provide important new information on the epigenetic regulation of chromatin landscape during osteoblast differentiation.