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The Brn-3a POU transcription factor is associated with survival and the differentiation of sensory neuronal cells during development. Brn-3a mediates its effects either by the direct regulation of target genes or indirectly upon interaction with proteins such as p53. Brn-3a differentially regulates p53-mediated gene expression and modifies its effect on cell fate. Here we show that, like Bax, Brn-3a antagonizes p53-mediated transcription of another proapoptotic target, Noxa, significantly reducing transactivation of the Noxa promoter by p53. This effect requires the p53 binding site, and electrophoretic mobility shift assay studies suggest that Brn-3a is associated with p53 when it is bound to its site in the Noxa promoter. The wild type but not the mutant promoter can be immunoprecipitated with Brn-3a in chromatin immunoprecipitation assays. Thus, Brn-3a may act by preventing the recruitment of cofactors required for p53 to transactivate this promoter. The co-expression of Brn-3a and p53 results in decreased endogenous Noxa protein in the neuronal cell line, ND7, suggesting a direct functional effect of this interaction. Moreover, there is a significant elevation of both proapoptotic Bax and Noxa proteins in sensory neuronal tissue taken from Brn-3a-/- embryos during development, compared with wild type controls. Striking changes occurred at embryonic day 14.5, a time that precedes a significant loss of specific neurons in the mutant embryos, but not at embryonic day 16.5 when Brn-3a-expressing cells are already lost by apoptosis. Therefore, the lack of antagonism by Brn-3a on activation of proapoptotic p53 target genes may contribute to the increased apoptosis seen in the Brn-3a-/- embryos. These results support a crucial role for Brn-3a in determining the pathway taken by p53 when co-expressed during development and thus in controlling the fate of these cells.

The Brn-3a POU family transcription factor has previously been shown to activate the human papilloma virus type 16 (HPV-16) promoter driving the expression of the E6- and E7-transforming proteins. Moreover, Brn-3a is overexpressed approximately 300-fold in cervical biopsies from women with cervical intra-epithelial neoplasia type 3 (CIN3) compared with normal cervical material. To test the role of Brn-3a in cervical neoplasia we have manipulated its expression in cervical carcinoma-derived cell lines with or without endogenous HPV genes. In HPV-expressing cells, reduction in Brn-3a expression specifically reduces HPV gene expression, growth rate, saturation density and anchorage-independent growth, whereas these effects are not observed when Brn-3a expression is reduced in cervical cells lacking HPV genomes. Together with our previous observations, these findings indicate a critical role for Brn-3a in regulating HPV gene expression and thereby in controlling the growth/transformation of cervical cells.

POU-domain proteins have been shown to play important roles in the development of the nervous, endocrine, and immune systems. However, the distinctive DNA recognition properties of the six major POU subclasses have not been well defined. Here, we have used random oligonucleotide selection and competitive binding assays to determine the optimal DNA recognition elements for the POU-III and POU-VI protein classes, represented by Brn-2 and Brn-5, respectively. The optimal Brn-5 consensus binding sequence GCATAA(T/A)TTAT strongly resembles that previously determined for the POU-IV (Brn-3) class, whereas Brn-2 exhibits highest affinity for non-octamer sites of the form ATG(A/C)AT(A/T)0-2ATTNAT and for octamer sites that contain a full associated heptamer sequence. Brn-2, Brn-3.0, and their invertebrate homologues all exhibit highly cooperative homodimerization on the Brn-2 consensus sequence, demonstrating that cooperative dimerization is a general property of these neural POU proteins. However, modified sites to which Brn-2 binds only as a monomer mediate the transcriptional effects of Brn-2 better than the consensus sequence, demonstrating that dimerization on these sites diminishes the transactivation ability of the protein. Together with the findings of our prior studies these data greatly facilitate the identification of functional POU recognition elements in the regulatory regions of neural genes.

The POU domain transcription factor Brn-3a is able to stimulate neurite outgrowth when overexpressed in the neuronal ND7 cell line, whereas the closely related Brn-3b factor does not have this effect. We show that Brn-3a overexpression also enhances the expression of the three neurofilament genes at both the mRNA and protein levels, whereas Brn-3b overexpression has no effect. In addition Brn-3a activates the three neurofilament gene promoters in co-transfection assays in both neuronal and non-neuronal cells. As observed for enhanced neurite outgrowth, the stimulation of neurofilament gene expression and activation of the neurofilament gene promoters is observed with the isolated POU domain of Brn-3a. A single amino acid change in the POU homeodomain of Brn-3a to the equivalent amino acid in Brn-3b abolishes its ability to activate the neurofilament promoters, whereas the reciprocal change converts Brn-3b to an activator of these promoters.

The differentiation of the ND7 neuronal cell line to a nondividing phenotype bearing numerous neurite processes is accompanied by a dramatic increase in the levels of the activating POU family transcription factor Brn-3a and a corresponding fall in the levels of the closely related inhibitory factor Brn-3b. We have previously shown that the artificial overexpression of Brn-3a in these cells can induce neurite outgrowth and the activation of genes encoding synaptic vesicle proteins in the absence of a differentiation-inducing stimulus. Here we show that overexpression of Brn-3b can reduce process outgrowth and synaptic vesicle gene expression following exposure to a stimulus which would normally induce differentiation. These inhibitory effects are abolished by altering a single amino acid in the POU homeodomain of Brn-3b to its equivalent in Brn-3a. The converse mutation in Brn-3a allows it to inhibit process outgrowth in response to a differentiation-inducing stimulus. Hence a single amino acid difference results in these closely related factors having opposite effects and allows the balance between them to regulate differentiation.