The POU homeodomain protein UNC-86 and the LIM homeodomain protein MEC-3 are essential for the differentiation of the six mechanoreceptor neurons in the nematode Caenorhabditis elegans. Previous studies have indicated that UNC-86 and MEC-3 bind cooperatively to at least three sites in the mec-3 promoter and synergistically activate transcription. However, the molecular details of the interactions of UNC-86 with MEC-3 and DNA have not been investigated so far. Here we used a yeast system to identify the functional domains in UNC-86 required for transcriptional activation and to characterize the interaction of UNC-86 with MEC-3 in vivo. Our results suggest that transcriptional activation is mediated by the amino terminus of UNC-86, whereas amino acids in the POU domain mediate DNA binding and interaction with MEC-3. By random mutagenesis, we identified mutations that only affect the DNA binding properties of UNC-86, as well as mutations that prevent coactivation by MEC-3. We demonstrated that both the POU-specific domain and the homeodomain of UNC-86, as well as DNA bases adjacent to the proposed UNC-86 binding site, are involved in the formation of a transcriptionally active complex with MEC-3. These data suggest that some residues involved in the contact of UNC-86 with MEC-3 also contribute to the interaction of the functionally nonrelated POU protein Oct-1 with Oca-B, whereas other positions have different roles.POU domain transcription factors are characterized by their bipartite DNA binding domain, consisting of a helix-turn-helix POU-specific domain (POU S ) and an adjacent POU homeodomain (POU HD ) (for reviews, see references 15 and 28). Both protein domains contact DNA and are necessary for high-affinity DNA binding (15). POU class IV is comprised of the mammalian Brn-3-encoding genes, the Drosophila I-POU/ acj6 gene, and the Caenorhabditis elegans unc-86 gene (28). These are all expressed exclusively in the nervous system, but their expression is not limited to one specific neuronal cell type. In vitro studies have shown that POU class IV proteins bind very similar DNA sequences (12, 24). Therefore, it has been proposed that any differences in target gene activation are due to modulatory protein interactions (28). Most of the data for determination of the promoter specificity of POU proteins are derived from the nonrelated human POU class III protein Oct-1 (13, 41).POU homeobox gene unc-86 is expressed in 57 neurons in adult C. elegans. These neurons comprise one-fifth of the animal's nervous system and represent 27 different functional classes (10). Consequently, null alleles of unc-86 result in several behavioral defects affecting mechanosensation, egg laying, chemosensation, and thermosensation and cause an uncoordinated phenotype (5,10,18,26,37). The diversity of neuron types that require UNC-86 raises the possibility that in different cell types, UNC-86 targets the promoters of different genes. This is supported by the fact that in at least two unc-86 mutants, only a subset of the unc-86-mediated be...

The Caenorhabditis elegans POU protein UNC-86 specifies the HSN motor neurons, which are required for egg-laying, and six mechanosensory neurons. To investigate how UNC-86 controls neuronal specification, we characterized two unc-86 mutants that do not respond to touch but show wild-type egg-laying behavior. Residues P145 and L195, which are altered by these mutations, are located in the POU-specific domain and abolish the physical interaction of UNC-86 with the LIM homeodomain protein, MEC-3. This results in a failure to maintain mec-3 expression and in loss of expression of the mechanosensory neuron-specific gene, mec-2. unc-86-dependent expression of genes in other neurons is not impaired. We conclude that distinct residues in the POU domain of UNC-86 are involved in modulating UNC-86 activity during its specification of different neurons. A structural model of the UNC-86 POU domain, including base pairs and amino acid residues required for MEC-3 interaction, revealed that P145 and L195 are part of a hydrophobic pocket which is similar to the OCA-B-binding domain of the mammalian POU protein, Oct-1.