Since the c-Jun coactivator ␣NAC was initially identified in a differential screen for genes expressed in differentiated osteoblasts, we examined whether the osteocalcin gene, a specific marker of terminal osteoblastic differentiation, could be a natural target for the coactivating function of ␣NAC. We had also previously shown that ␣NAC can specifically bind DNA in vitro, but it remained unclear whether the DNA-binding function of ␣NAC is expressed in vivo or if it is required for coactivation. We have identified an ␣NAC binding site within the murine osteocalcin gene proximal promoter region and demonstrated that recombinant ␣NAC or ␣NAC from ROS17/2.8 nuclear extracts can specifically bind this element. Using transient transfection assays, we have shown that ␣NAC specifically potentiated the c-Jun-dependent transcription of the osteocalcin promoter and that this activity specifically required the DNA-binding domain of ␣NAC. Chromatin immunoprecipitation confirmed that ␣NAC occupies its binding site on the osteocalcin promoter in living osteoblastic cells expressing osteocalcin. Inhibition of the expression of endogenous ␣NAC in osteoblastic cells by use of RNA interference provoked a decrease in osteocalcin gene transcription. Our results show that the osteocalcin gene is a target for the ␣NAC coactivating function, and we propose that ␣NAC is specifically targeted to the osteocalcin promoter through its DNA-binding activity as a means to achieve increased specificity in gene transcription.
HES6 is a novel member of the family of basic helix–loop–helix mammalian homologues of Drosophila Hairy and Enhancer of split. We have analyzed the biochemical and functional roles of HES6 in myoblasts. HES6 interacted with the corepressor transducin-like Enhancer of split 1 in yeast and mammalian cells through its WRPW COOH-terminal motif. HES6 repressed transcription from an N box–containing template and also when tethered to DNA through the GAL4 DNA binding domain. On N box–containing promoters, HES6 cooperated with HES1 to achieve maximal repression. An HES6–VP16 activation domain fusion protein activated the N box–containing reporter, confirming that HES6 bound the N box in muscle cells. The expression of HES6 was induced when myoblasts fused to become differentiated myotubes. Constitutive expression of HES6 in myoblasts inhibited expression of MyoR, a repressor of myogenesis, and induced differentiation, as evidenced by fusion into myotubes and expression of the muscle marker myosin heavy chain. Reciprocally, blocking endogenous HES6 function by using a WRPW-deleted dominant negative HES6 mutant led to increased expression of MyoR and completely blocked the muscle development program. Our results show that HES6 is an important regulator of myogenesis and suggest that MyoR is a target for HES6-dependent transcriptional repression.
c-Jun is an immediate-early gene whose degradation by the proteasome pathway is required for an efficient transactivation. In this report, we demonstrated that the c-Jun coactivator, nascent polypeptide associated complex and coactivator alpha (alphaNAC) was also a target for degradation by the 26S proteasome. The proteasome inhibitor lactacystin increased the metabolic stability of alphaNAC in vivo, and lactacystin, MG-132, or epoxomicin treatment of cells induced nuclear translocation of alphaNAC. We have shown that the ubiquitous kinase glycogen synthase kinase 3beta (GSK3beta) directly phosphorylated alphaNAC in vitro and in vivo. Inhibition of the endogenous GSKappa3beta activity resulted in the stabilization of this coactivator in vivo. We identified the phosphoacceptor site in the C-terminal end of the coactivator, on position threonine 159. We demonstrated that the inhibition of GSK3beta activity by treatment of cells with the inhibitor 5-iodo-indirubin-3'-monoxime, as well as with a dominant-negative GSK3beta mutant, induced the accumulation of alphaNAC in the nuclei of cells. Mutation of the GSK3beta phosphoacceptor site on alphaNAC induced a significant increase of its coactivation potency. We conclude that GSK3beta-dependent phosphorylation of alphaNAC was the signal that directed the protein to the proteasome. The accumulation of alphaNAC caused by the inhibition of the proteasome pathway or the activity of GSK3beta contributes to its nuclear translocation and impacts on its coactivating function.
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