Sequence-Specific DNA Binding by the αNAC Coactivator Is Required for Potentiation of c-Jun-Dependent Transcription of the Osteocalcin Gene

Abstract

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.

The PTH-Gαs-Protein Kinase A Cascade Controls αNAC Localization To Regulate Bone Mass

“…Ocn remains the best-characterized transcriptional target of ␣NAC to date (16), although in vitro studies and mutant mouse models have hinted at other relevant promoters (17,34,54). The regulatory pathway characterized in this study broadens the set of putative targets regulated by ␣NAC.…”

“…Nuclear ␣NAC binds to and regulates transcription from the Ocn promoter (16,34). Since PTH induces ␣NAC nuclear translocation, we examined the effect of PTH or cAMP analog treatment on recruitment of wild-type or S99A ␣NAC proteins at the Ocn gene.…”

“…CREB then stimulates the transcription of members of genes coding for the AP-1 family of basic domain- leucine zipper transcription factors, c-Fos and c-Jun (38)(39)(40). While the importance of AP-1 proteins in mediating the anabolic PTH response remains to be determined, it is noteworthy that ␣NAC interacts with the N-terminal activation domain of c-JUN to regulate transcription (16,20,21,41). Interaction with c-JUN involves residues 89 to 129 within ␣NAC (41), a region containing 5 ␤-sheets (42).…”

“…Nuclear ␣NAC potentiates Osteocalcin (Ocn) gene transcription by stabilizing the binding of a c-JUN dimer to its AP-1 binding site within the Ocn gene promoter (20,21). It has further been shown that ␣NAC can specifically bind DNA and that this DNA-binding capacity is required to potentiate AP-1-mediated Ocn gene expression in osteoblasts (16). To determine the physiological role of nuclear ␣NAC, we engineered a knock-in mouse model with a serine-to-alanine mutation at position 43 (S43A).…”

“…One of the molecules affecting maximal Ocn expression is the transcriptional coregulator ␣NAC (nascent-polypeptide-associated complex and coregulator alpha) (16,17). ␣NAC shuttles between the cytoplasm and the nucleus, and one of the signals that induce its nuclear accumulation is phosphorylation by integrinlinked kinase (ILK) (18) at residue Ser43 (19).…”