We report the characterization of clone 1.9.2, a gene expressed in mineralizing osteoblasts. Remarkably, clone 1.9.2 is the murine homolog of the alpha chain of the nascent polypeptide-associated complex (␣-NAC). Based on sequence similarities between ␣-NAC/1.9.2 and transcriptional regulatory proteins and the fact that the heterodimerization partner of ␣-NAC was identified as the transcription factor BTF3b (B. Wiedmann, H. Sakai, T. A. Davis, and M. Wiedmann, Nature 370:434-440, 1994), we investigated a putative role for ␣-NAC/ 1.9.2 in transcriptional control. The ␣-NAC/1.9.2 protein potentiated by 10-fold the activity of the chimeric activator GAL4/VP-16 in vivo. The potentiation was shown to be mediated at the level of gene transcription, because ␣-NAC/1.9.2 increased GAL4/VP-16-mediated mRNA synthesis without affecting the half-life of the GAL4/VP-16 fusion protein. Moreover, the interaction of ␣-NAC/1.9.2 with a transcriptionally defective mutant of GAL4/VP-16 was severely compromised. Specific protein-protein interactions between ␣-NAC/1.9.2 and GAL4/VP-16 were demonstrated by gel retardation, affinity chromatography, and protein blotting assays, while interactions with TATA box-binding protein (TBP) were detected by immunoprecipitation, affinity chromatography, and protein blotting assays. Based on these interactions that define the coactivator class of proteins, we conclude that the ␣-NAC/1.9.2 gene product functions as a transcriptional coactivator.

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Tyrosine hydroxylase (TH) is the rate-limiting enzyme of dopamine and (nor)adrenaline biosynthesis. Regulation of its gene expression is complex and different regulatory mechanisms appear to be operative in various neuronal lineages. Pitx3, a homeodomain-containing transcription factor, has been cloned from neuronal tissues and, in the CNS, mouse Pitx3 is exclusively expressed in midbrain dopaminergic (MesDA) neurons from embryonic day 11 (E11). TH appears in these neurons at E11.5, consistent with a putative role of Pitx3 in TH transcription. We show that Pitx3 activates the TH promoter through direct interaction with a single high-af®nity binding site within the promoter and that this site is suf®cient for Pitx3 responsiveness. In contrast, we did not observe an effect of Nurr1, an orphan nuclear receptor essential for normal development of MesDA neurons, on TH promoter activity. Pitx3 activation of TH promoter activity appears to be cell-dependent suggesting that Pitx3 action may be modulated by other(s) regulatory mechanism(s) and factor(s).

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