αNAC interacts with histone deacetylase corepressors to control Myogenin and Osteocalcin gene expression

In the nucleus of differentiated osteoblasts, the DNA-binding αNAC protein acts as a transcriptional coactivator of the Osteocalcin gene. Chromatin immunoprecipitation-microarray assays (ChIP-chip) showed that αNAC binds the Osteocalcin promoter but also identified the Myogenin promoter as an αNAC target. Here, we confirm these array data using quantitative ChIP and further detected that αNAC binds to these promoters in myoblasts. Since these genes are differentially regulated during osteoblastogenesis or myogenesis, these results suggest cell- and promoter-context specific functions for αNAC. We hypothesized that αNAC dynamically recruits corepressors to inhibit Myogenin expression in cells committing to the osteoblastic lineage or to inhibit Osteocalcin transcription in differentiating myoblasts. Using co-immunoprecipitation assays, we detected complexes between αNAC and the corepressors HDAC1 and HDAC3, in myoblasts and osteoblasts. Sequential ChIP confirmed HDAC1 recruitment by αNAC at the Osteocalcin and Myogenin promoters. Interaction with the corepressors was detectable in pre-osteoblasts and in myoblasts but disappeared as the cells differentiate. Treatment with an HDAC inhibitor caused de-repression of Osteocalcin expression in myoblasts. Overexpression of αNAC in myoblasts inhibits expression of Myogenin and differentiation. However, overexpression of an N-terminus truncated αNAC mutant allowed myoblasts to express Myogenin and differentiate, and this mutant did not interact with HDAC1 or HDAC3. This study identified an additional DNA-binding target and novel protein-protein interactions for αNAC. We propose that αNAC plays a role in regulating gene transcription during mesenchymal cell differentiation by differentially recruiting corepressors at target promoters.