Brn3a is a POU-domain transcription factor expressed in peripheral sensory neurons and in specific interneurons of the caudal CNS. Sensory expression of Brn3a is regulated by a specific upstream enhancer, the activity of which is greatly increased in Brn3a knockout mice, implying that Brn3a negatively regulates its own expression. Brn3a binds to highly conserved sites within this enhancer, and alteration of these sites abolishes Brn3a regulation of reporter transgenes. Furthermore, endogenous Brn3a expression levels in the sensory ganglia of Brn3a +/+ and Brn3a +/-mice are similar, demonstrating that autoregulation can compensate for the loss of one allele by increasing transcription of the remaining gene copy. Conversely, transgenic overexpression of Brn3a in the trigeminal ganglion suppresses the expression of the endogenous gene. These findings demonstrate that the Brn3a locus functions as a self-regulating unit to maintain a constant expression level of this key regulator of neural development.

The POU-IV or Brn-3 class of transcription factors exhibit conserved structure, DNA-binding properties, and expression in specific subclasses of neurons across widely diverged species. In the mouse CNS, Brn-3.0 expression characterizes specific neurons from neurogenesis through the life of the cell. This irreversible activation of expression suggests positive autoregulation. To search for cis-acting elements that could mediate autoregulation we used a novel method, complex stability screening, which we applied to rapidly identify functional Brn-3.0 recognition sites within a large genomic region encompassing the mouse brn-3.0 locus. This method is based on the observation that the kinetic stability of Brn-3.0 complexes with specific DNA sequences, as measured by their dissociation half-lives, is highly correlated with the ability of those sequences to mediate transcriptional activation by Brn-3.0. The principal Brn-3.0 autoregulatory region lies ϳ5 kb upstream from the Brn-3.0 transcription start site and contains multiple Brn-3.0-binding sites that strongly resemble the optimal binding site for this protein class. This region also mediates transactivation by the closely related protein Brn-3.2, suggesting a regulatory cascade of POU proteins in specific neurons in which Brn-3.2 expression precedes Brn-3.0. Key words: Brn-3; POU-domain; autoregulation; transcription factor; homeodomain; dissociation kinetics; retina; habenula; inferior olive; tectumThe development of the vertebrate brain requires the differentiation of a large number of specific types of neurons from a relatively undifferentiated cell layer, the neural plate. Many developing neurons and their precursors express characteristic DNA-binding transcription factors. The expression domains of these factors overlap in unique combinations in specific regions of the neural tube and in groups of differentiating neurons, suggesting that development of each neuronal type is guided by a transcriptional code. However, little is known about how these factors act at the molecular level to determine and maintain specific neuronal phenotypes.Many of the transcription factors that are expressed in specific groups of neurons belong to the POU, Pax, and LIM families of variant homeodomain proteins. Some of these factors appear early in development in regions of the dividing neuroepithelium, whereas others are restricted to terminally differentiating neurons. Factors of the POU-IV or Brn-3 class are expressed as specific sets of C NS and peripheral sensory neurons exit the cell cycle (Fedtsova and T urner, 1995;Artinger et al., 1998), and they have been shown to be necessary for the normal development of the retina, auditory system, cranial sensory ganglia, and certain CNS nuclei (Ryan and Rosenfeld, 1997).The expression patterns of most of the neuron-specific transcription factors have been characterized only in the developing brain, and in many cases little information is available about their expression in the adult nervous system. Here we demonstrate that expressi...

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 -2 ATTNAT 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 factors are a class of homeodomain proteins that interact with DNA via a two-part binding domain, consisting of a POU homeodomain and a POUspecific domain (1). POU proteins have been identified as developmental regulators in diverse invertebrate species, and in mammals they have important roles in the development of the immune, endocrine, and nervous systems (2). Based entirely on structural homology, the POU-domain factors have been divided into six distinct subclasses. In vertebrates, proteins of the POU-III, POU-IV, and POU-VI classes are expressed predominantly in the nervous system.The POU-III class proteins Brn-1, Brn-2, and Brn-4 are expressed in specific hypothalamic nuclei and also in the neocortex (3), whereas SCIP/Tst-1/Oct-6 has a unique role in differentiation of myelinating glia and is also expressed in the neocortex (4). Expression of the POU-IV class proteins Brn-3.0, Brn-3.1, and Brn-3.2 is confined almost entirely to the caudal CNS 1 and sensory system (5). Brn-5 (also known as emb and CNS-1) is significantly diverged from neural POU proteins of the POU-III and POU-IV classes and has been assigned to a separate structural category (POU-VI). Brn-5 is expressed widely in the developing mammalian CNS and has a somewhat lamina-specific pattern in the mature cerebral cortex (6 -8). It is not currently known whether these proteins play similar regulatory roles in their respective brain regions or instead have distinct transcriptional functions conferred by different DNA binding properties between POU classes or by other unique features of the individual factors. We have previo...

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