Identification of Amino Acid Residues in the Caenorhabditis elegans POU 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.

ACKNOWLEDGMENTS

We thank W. Hammerschmidt for the modified pGBT9 vector and the yeast strain RH1533, M. Chalfie for pTU47, and R. Grosschedl and the members of the Kolanus, Meisterernst, and Baumeister labs for helpful suggestions and comments on the manuscript.

This work was supported by a grant from the Deutsche Forschungsgemeinschaft to R.B.