Identification of Amino Acid Residues in theCaenorhabditis elegansPOU Protein UNC-86 That Mediate UNC-86–MEC-3–DNA Ternary Complex Formation

Abstract

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...

Caenorhabditis elegans aristaless/Arx gene alr-1 restricts variable gene expression

“…Earlier studies have shown the cooperative role of the MEC-3 and UNC-86 transcription factors in defining the terminal fate of the TRNs and in maintaining the expression of MEC-3 (13)(14)(15)(16)(17)(18)). Here we demonstrate that, due to stochastic fluctuations, MEC-3 autoregulation is insufficient to maintain TRN differentiation.…”

“…UNC-86 is needed not only to produce touch cell lineages but also to initiate transcription from the mec-3 gene (12). The maintained expression of mec-3 and the subsequent expression of TRN characteristics require the combinatorial action of MEC-3 and UNC-86; MEC-3 acts (as a coactivator with UNC-86) in an autoregulatory loop to maintain its own expression and to induce the expression of TRN-specific genes (13)(14)(15)(16)(17)(18).…”

Genetic Models of Mechanotransduction: The NematodeCaenorhabditis elegans

“…unc-86 is expressed throughout the touch cell life, suggesting that it may function in maintenance as well as in lineage specification (27,122,191). MEC-3 and UNC-86 associate to form heterodimers that bind to well-defined sites in the mec-3 and other touch cell-specific gene promoters (such as mec-4 and mec-7) to stimulate gene expression (252,344,345,447,449,474,475). The MEC-3 LIM homeodomain and the UNC-86 POU homeodomain are sufficient to mediate interaction in vitro, but more of each protein is needed for heterodimer formation on DNA in vivo (474,475).…”

Specification ofDrosophilamotoneuron identity by the combinatorial action of POU and LIM-HD factors

“…In C. elegans touch receptor neurons, the LIM-HD factor, MEC-3 and the POU protein UNC-86, physically interact to control specification (Rockelein et al, 2000;Xue et al, 1993). In the pituitary, the LIM domain of Lhx3 (P-Lim) specifically interacts with the Pit1 POU domain and is required for synergistic interactions with Pit1 (Bach et al, 1995).…”

“…Furthermore, LIM-HD and POU members have been shown to function together to direct cell differentiation in both vertebrates and invertebrates. For example, UNC-86 and the LIM-HD factor MEC-3 interact genetically and physically to regulate tough sensory neuron differentiation in C. elegans (Rockelein et al, 2000;Xue et al, 1993).…”