The use of the polymerase chain reaction in conjunction with degenerate oligonucleotides has allowed the isolation of cDNA clones derived from each of the POU family transcription factors expressed in the proliferating ND7 neuronal cell line. In addition to the previously characterized Oct-1, Oct-2 and Brn-3 factors, ND7 cells have been shown by this means to express a novel POU factor. This factor is closely related to Brn-3 but differs at seven amino acids in the POU domain, one of which is located in a region which is critical for protein-protein interactions between different POU proteins. Like Brn-3, the novel factor is expressed at high levels in embryonic brain and declines in abundance during neuronal development. In contrast to Brn-3 however, it is absent in mature sensory neurons and its expression declines during the in vitro differentiation of ND7 cells to a non-dividing phenotype whilst Brn-3 expression increases. The significance of these distinct but overlapping expression patterns is discussed in terms of the possible role of these two factors in regulating neuronal gene expression.

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SNAP-25 is a presynaptic nerve terminal protein which is also essential for the process of neurite outgrowth in vivo and in vitro. However the processes regulating its expression have not been characterized previously. We show that the gene encoding this protein, SNAP, is strongly activated by the Brn-3a POU (Pit-Oct-Unc) family transcription factor. Expression of both Brn-3a and SNAP-25 increases when ND7 neuronal cells are induced to extend neurite processes by serum removal. Inhibition of Brn-3a expression in these cells inhibits SNAP-25 expression and abolishes the neurite outgrowth that normally occurs in response to serum removal. These results identify Brn-3a as the first transcription factor having a role in process outgrowth in neuronal cells acting, at least in part, via the activation of SNAP-25 gene expression.

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The POU (Pit-Oct-Unc) family transcription factor Brn-3a contains two distinct activation domains, one at the N terminus of the molecule and one at the C terminus coincident with the DNA binding domain. These different activation domains have been shown previously to differ in their ability to activate an artificial test promoter containing a Brn-3a binding site and the naturally occurring ␣-internexin gene promoter. Here we identify the target site for Brn-3a in the ␣-internexin gene promoter and show that it can confer responsiveness to Brn-3a on a heterologous promoter. One of the single-stranded DNA sequences derived from either this novel Brn-3a binding site or from the previously characterized site in the test promoter are shown to bind Brn-3a preferentially compared with the complementary single strand or the corresponding double-stranded sequence. The pattern of responsiveness of these two sequences when cloned upstream of the same test promoter and co-transfected with constructs encoding various portions of Brn-3a indicates that the activity of the two Brn-3a activation domains is dependent upon differences in the context of the target sequence in each promoter rather than on differences in the target sequence itself.The POU (Pit-Oct-Unc) family of transcription factors play a critical role in the regulation of gene expression in neuronal cells acting by binding to sequences related to the consensus octamer motif ATGCAAAT in the promoters of their target genes (for review, see Verrijzer and van der Vliet (1993) and Wegner et al. (1993)). The Brn-3 family of mammalian POU factors are of particular interest as the mammalian factors most closely related to the nematode POU protein Unc-86 whose inactivation results in the failure to develop specific neuronal cell types particularly sensory neurons (Desai et al., 1988;Finney et al., 1988). Three mammalian Brn-3 factors encoded by distinct genes exist (Theil et al., 1993 and are known as Brn-3a (also known as Brn-3.0: Gerrero et al., 1993;Lillycrop et al., 1992), Brn-3b (also known as Brn-3.2: Lillycrop et al., 1992;Turner et al., 1994), and Brn-3c (also known as Brn-3