Sequence-specific DNA binding by the alphaNAC coactivator is required for potentiation of c-Jun-dependent transcription of the osteocalcin gene

Abstract

Since the c-Jun coactivator alphaNAC 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 alphaNAC. We had also previously shown that alphaNAC can specifically bind DNA in vitro, but it remained unclear whether the DNA-binding function of alphaNAC is expressed in vivo or if it is required for coactivation. We have identified an alphaNAC binding site within the murine osteocalcin gene proximal promoter region and demonstrated that recombinant alphaNAC or alphaNAC from ROS17/2.8 nuclear extracts can specifically bind this element. Using transient transfection assays, we have shown that alphaNAC specifically potentiated the c-Jun-dependent transcription of the osteocalcin promoter and that this activity specifically required the DNA-binding domain of alphaNAC. Chromatin immunoprecipitation confirmed that alphaNAC occupies its binding site on the osteocalcin promoter in living osteoblastic cells expressing osteocalcin. Inhibition of the expression of endogenous alphaNAC 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 alphaNAC coactivating function, and we propose that alphaNAC is specifically targeted to the osteocalcin promoter through its DNA-binding activity as a means to achieve increased specificity in gene transcription.

FIG. 1.

αNAC specifically potentiates the transcription of the osteocalcin gene promoter by c-Jun. (A) COS-7 cells were transiently transfected with an osteocalcin-luciferase reporter and expression vectors for c-Jun, αNAC, or Runx2/Cbfa1, alone or in combinations. c-Jun induced the transcription of the reporter, and this induction was potentiated by αNAC. The coactivating function of αNAC was specific to c-Jun, as αNAC expression did not affect Runx2/Cbfa1-activated osteocalcin gene transcription. The expression level detected in cells transfected with the reporter construct alone was arbitrarily given a value of 1. Results are means ± standard errors of the means of three independent transfections performed in triplicate. *, P < 0.05; ***, P < 0.001. (B) Immunoblot probed with the anti-αNAC antibody that shows expression levels of endogenous and FLAG-tagged αNAC. The order of the numbered tracks corresponds to the order of the bars in panel A.

FIG. 2.

αNAC and c-Jun bind the osteocalcin proximal promoter. EMSAs using a probe from the murine osteocalcin gene and purified recombinant αNAC (A), nuclear extracts (NE) from ROS 17/2.8 cells (B), or recombinant c-Jun and recombinant αNAC (C). In panel A, a specific complex can be detected (αNAC arrow) that was “supershifted” in the presence of anti-αNAC antibodies (Ab). Preimmune serum (Serum) did not influence complex migration. The complex was competed with increasing amounts of the canonical αNAC binding site (1xNAC) but not with a mutated sequence (mut). DBD-deleted αNAC (Δ69-80) did not bind the probe (lane 10). Panel B shows that αNAC from ROS 17/2.8 osteoblastic cells bound the probe. The complex was specifically but not completely supershifted by the anti-αNAC antibody (Ab). In panel C, the probe used covered positions −103 to −30 of the osteocalcin promoter and encompassed the αNAC binding site and the OC box 1 (34). Recombinant c-Jun did not bind the probe by itself (lane 2), but a complex containing c-Jun could be detected (c-Jun arrow) when the probe was incubated with both c-Jun and αNAC (lanes 4 and 5).

FIG. 3.

Coactivation of c-Jun-dependent osteocalcin gene transcription requires the αNAC DBD. (A) Transient transfection assays were set up as described in the legend to Fig. 1. The recombinant αNACΔ69-80 protein, devoid of DNA-binding activity, did not potentiate the activity of c-Jun. **, P < 0.01; ***, P < 0.001. (B) The expression of the recombinant proteins was monitored by Western blot assay using the antibodies (Ab) listed above each panel. Probing with anti-TBP served as a loading control. Note that FLAG-αNACΔ69-80 migrates at the same position as endogenous αNAC in SDS-PAGE, so that the band detected by the anti-αNAC antibody (third panel, lanes 5 and 6) represents the combined signal of endogenous αNAC and FLAG-αNACΔ69-80.

FIG. 4.

Coactivation of c-Jun-dependent osteocalcin gene transcription requires the αNAC binding site on the promoter. Transient transfection assays were performed as described in the legend to Fig. 1. (A) Reporters used included the wild-type osteocalcin promoter driving luciferase (OCN-Luc) or a mutated osteocalcin promoter in which the αNAC binding site was deleted (OCN-Luc ΔBDG; the deletion covered from −32 to −60 relative to the transcription start site). (B) Reporters used included OCN-Luc and a mutated osteocalcin promoter in which the αNAC binding site was mutated by site-specific mutagenesis (OCN-Luc mut BDG; the engineered mutation was 5′-GCACgGgGTAG-3′). αNAC did not potentiate the activity of c-Jun when its binding site was mutated or deleted from the promoter. **, P < 0.01; ***, P < 0.001.

FIG. 5.

αNAC DNA-binding activity is specifically required for c-Jun-dependent osteocalcin gene transcription. Transient transfection assays were set up as described in the legend to Fig. 1. Two c-Jun responsive promoters were used as reporter constructs: the wild-type osteocalcin promoter (OCN-Luc) or the proximal 670 bp of the mmp-9 gene promoter (MMP9-Luc). Wild-type αNAC could potentiate the activity of c-Jun on both promoters, but the DNA-binding domain-deleted αNAC mutant (αNACΔ69-80) was active only on the MMP9-Luc template. **, P < 0.01; ***, P < 0.001.

FIG. 6.

αNAC binds the osteocalcin gene promoter in living cells expressing osteocalcin. Wild-type osteoblastic MC3T3-E1 cells or MC3T3-E1 cells stably transfected with pSI-NAC-Flag (A) or the Flag epitope-tagged DBD-deleted αNAC mutant, αNACΔ69-80 (D), were grown for 14 (panel A) or 21 (panels A and D) days postconfluence in the presence of ascorbic acid and beta-glycerophosphate. Immunoprecipitation assays were performed with formaldehyde-cross-linked chromatin and antibodies against Runx2/Cbfa1, αNAC, or the Flag epitope. Ethidium bromide-stained agarose gels of PCR products obtained with primers flanking the Runx2/Cbfa1 binding site (lanes 1 and 2) or the αNAC binding site (lanes 3 to 9) within the mouse osteocalcin gene promoter are shown. Input, amplification of DNA prior to immunoprecipitation; IgG, immunoglobulin G; I.P., immunoprecipitate; M, molecular size markers. (B) Immunoblot probed with the anti-αNAC antibody that shows expression levels of endogenous and FLAG-tagged αNAC proteins. Note that FLAG-αNACΔ69-80 migrates at the same position as endogenous αNAC in SDS-PAGE, so that the band detected by the anti-αNAC antibody (lane 3) represents the combined signal of endogenous αNAC and FLAG-αNACΔ69-80. (C) Northern blot showing OCN mRNA expression. 28S, ribosomal 28S RNA used to monitor loading.

FIG. 7.

Inhibition of endogenous αNAC expression by RNA interference affects osteocalcin promoter activity. MC3T3-E1 cells stably transfected with a luciferase reporter gene under the control of the 1.3-kb mouse osteocalcin promoter fragment (46) were transfected with a control, unrelated siRNA, or an siRNA directed against αNAC. (A) Expression of the endogenous αNAC or osteocalcin mRNAs was monitored by real-time PCR. ***, P < 0.001. (B) Expression of the endogenous αNAC protein was assessed by immunoblotting with the anti-αNAC antibody. Lane 1, treatment with the transfection reagent alone; lane 2, transfection with the control siRNA; lane 3, transfection with the αNAC siRNA. Staining of the membrane with Ponceau red (bottom panel) was used to monitor for even loading of each lane on the gel. (C) Expression of the reporter luciferase gene under the control of the osteocalcin promoter was monitored with a luminometer. The control bar represents expression measured in the presence of the transfection reagent alone. **, P < 0.01; Ctrl siRNA, control siRNA. Treatment of cells with the specific αNAC siRNA repressed both endogenous osteocalcin mRNA levels and the transcription of the osteocalcin promoter-controlled luciferase reporter.