Identification of amino acid residues in the Caenorhabditis elegans POU protein UNC-86 that mediate UNC-86-MEC-3-DNA ternary complex formation.
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.
Röckelein
I
I
Genzentrum, Ludwig-Maximilians-Universität, D-81377 Munich, Germany.
Röhrig
S
S
Donhauser
R
R
Eimer
S
S
Baumeister
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D018509:DNA, Helminth / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D019077:Helix-Turn-Helix Motifs; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051863:Host Cell Factor C1; D008969:Molecular Sequence Data; D009154:Mutation; D050811:Octamer Transcription Factor-1; D050809:POU Domain Factors; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D029742:Caenorhabditis elegans Proteins; D018797:Cell Cycle Proteins; D018509:DNA, Helminth; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D051863:Host Cell Factor C1; C099938:MEC3 protein, S cerevisiae; D050811:Octamer Transcription Factor-1; D050809:POU Domain Factors; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C497691:hcf-1 protein, C elegans; C081539:unc-86 protein, C elegans