The Nk-2 box of the Drosophila homeodomain protein, Vnd, contributes to its repression activity in a Groucho-dependent manner.
The transcription factor, Vnd, is a dual regulator that specifies ventral neuroblast identity in Drosophila by both repressing and activating target genes. Vnd and its homologues have a conserved amino acid sequence, the Nk-2 box or Nk specific domain, as well a conserved DNA-binding homeodomain and an EhI-type Groucho interaction domain. However, the function of the conserved Nk-2 box has not been fully defined. To explore its function, we deleted the Nk-2 box and compared the regulatory activity of mutant Vnd in transgenic over-expression assays to that of the wild-type protein. We were unable to assign regulatory activity to the Nk-2 box using an over-expression assay, because the mutant protein activated expression of endogenous Vnd, masking a requirement for the Nk-2 box. However, in transgenic rescue assays, Vnd lacking the Nk-2 box repressed ind expression at 30% lower levels than the wild-type protein. Moreover, in transient transfection assays using Gal4 DNA-binding domain-Vnd chimeras, the repression activity of Vnd lacking the Nk-2 box was compromised. Because Vnd represses target gene expression in conjunction with Groucho, we asked whether the Nk-2 box affects Vnd's ability to interact with this co-repressor. Vnd lacking the Nk-2 box binds Groucho 30% less efficiently than wild-type Vnd in co-immunoprecipitations. These data suggest that the Nk-2 box contributes to the repression activity of Vnd by stabilizing its interaction with the co-repressor, Groucho.
Uhler
Jay
J
Pathology Department, University of Michigan, Ann Arbor, MI 48109-0646, USA.
Zhang
Huanqing
H
Syu
Li-Jyun
LJ
Mellerick
Dervla M
DM
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D005801:Genes, Homeobox; D017344:Genes, Insect; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009420:Nervous System / Q000196:embryology / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D051792:Basic Helix-Loop-Helix Proteins; D018076:DNA, Complementary; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C091023:gro protein, Drosophila; C094797:vnd protein, Drosophila