Highly Cooperative Homodimerization Is a Conserved Property of Neural POU Proteins

Abstract

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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