ABSTRACT
A screen for modifiers of Dpp adult phenotypes led to the identification of the Drosophila homolog of the Sno oncogene (dSno). The dSno locus is large, transcriptionally complex and contains a recent retrotransposon insertion that may be essential for dSno function, an intriguing possibility from the perspective of developmental evolution. dSno is highly transcribed in the embryonic central nervous system and transcripts are most abundant in third instar larvae. dSno mutant larvae have proliferation defects in the optic lobe of the brain very similar to those seen in baboon (Activin type I receptor) and dSmad2 mutants. This suggests that dSno is a mediator of Baboon signaling. dSno binds to Medea and Medea/dSno complexes have enhanced affinity for dSmad2. Alternatively, Medea/dSno complexes have reduced affinity for Mad such that, in the presence of dSno, Dpp signaling is antagonized. We propose that dSno functions as a switch in optic lobe development, shunting Medea from the Dpp pathway to the Activin pathway to ensure proper proliferation. Pathway switching in target cells is a previously unreported mechanism for regulating TGFbeta signaling and a novel function for Sno/Ski family proteins.
Subject(s)
Brain/metabolism , Drosophila Proteins , Drosophila/physiology , Insect Proteins/metabolism , Signal Transduction , Amino Acid Sequence , Amino Acid Substitution , Animals , Asparagine/metabolism , Base Sequence , Drosophila/embryology , Drosophila/genetics , Embryo, Nonmammalian , Gene Deletion , Immunoprecipitation , Insect Proteins/genetics , Molecular Sequence Data , Phylogeny , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary , Sequence Homology, Amino AcidABSTRACT
The Kruppel-like factor Klf4 is implicated in tumorigenesis and maintaining stem cell pluripotency, and Klf4 can both activate and repress gene expression. We show that the Pbx1 and Meis2 homeodomain proteins interact with Klf4 and can be recruited to DNA elements comprising a Klf4 site or GC box, with adjacent Meis and Pbx sites. Meis2d and Pbx1a activate expression of p15(Ink4a) and E-cadherin, dependent on the Meis2d transcriptional activation domain. In HepG2 cells, reducing expression of endogenous Meis2 or Pbx1 decreases p15 gene expression and increases the number of cells entering S phase. Although DNA binding by all three proteins contributes to full cooperative activation, the sequence requirements for binding by Meis2 and Pbx1 are variable. In the E-cadherin promoter, a Pbx-like site is required for full activation, whereas in the p15 promoter, the Klf4 site appears to play the major role. Through a bioinformatics search we identified additional genes with conserved binding sites for Klf4, Meis2, and Pbx1 and show that at least some of these genes can be activated cooperatively by Klf4 and Meis2/Pbx1. We suggest a model in which genes with Klf4 sites can be cooperatively activated by Meis2/Pbx1 and Klf4, dependent primarily on recruitment by Klf4. This provides a mechanism to modulate transcriptional regulation by the multifunctional Klf4 transcription factor.
Subject(s)
Cadherins/biosynthesis , Cyclin-Dependent Kinase Inhibitor p15/biosynthesis , DNA-Binding Proteins/metabolism , Homeodomain Proteins/metabolism , Kruppel-Like Transcription Factors/metabolism , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Animals , Base Composition , COS Cells , Cadherins/genetics , Cell Cycle/physiology , Cell Line, Tumor , Chlorocebus aethiops , Chromatin Immunoprecipitation , Cyclin-Dependent Kinase Inhibitor p15/genetics , DNA/genetics , DNA/metabolism , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Gene Expression , Gene Expression Regulation , HeLa Cells , Hep G2 Cells , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/genetics , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/biosynthesis , Kruppel-Like Transcription Factors/genetics , Mice , Pre-B-Cell Leukemia Transcription Factor 1 , Promoter Regions, Genetic , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , RNA Interference , RNA, Small Interfering , Regulatory Sequences, Nucleic Acid , Transcription Factors/biosynthesis , Transcription Factors/geneticsABSTRACT
Myeloid ecotropic insertion site (Meis)2 is a homeodomain protein containing a conserved homothorax (Hth) domain that is present in all Meis and Prep family proteins and in the Drosophila Hth protein. The Hth domain mediates interaction with Pbx homeodomain proteins, allowing for efficient DNA binding. Here we show that, like Meis1, Meis2 has a strong C-terminal transcriptional activation domain, which is required for full activation of transcription by homeodomain protein complexes composed of Meis2 and Pbx1. We also show that the activity of the activation domain is inhibited by the Hth domain, and that this autoinhibition can be partially relieved by the interaction of Pbx1 with the Hth domain of Meis2. Targeting of the Hth domain to DNA suggests that it is not a portable trans-acting repression domain. However, the Hth domain can inhibit a linked activation domain, and this inhibition is not limited to the Meis2 activation domain. Database searching reveals that the Meis3.2 splice variant, which is found in several vertebrate species, disrupts the Hth domain by removing 17 codons from the 5'-end of exon 6. We show that the equivalent deletion in Meis2 derepresses the C-terminal activation domain and weakens interaction with Pbx1. This work suggests that the transcriptional activity of all members of the Meis/Prep Hth protein family is subject to autoinhibition by their Hth domains, and that the Meis3.2 splice variant encodes a protein that bypasses this autoinhibitory effect.
Subject(s)
Homeodomain Proteins/genetics , Transcription Factors/genetics , Alternative Splicing , Amino Acid Sequence , Animals , Cell Line, Tumor , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster , Humans , Mice , Molecular Sequence Data , Pre-B-Cell Leukemia Transcription Factor 1 , Protein Structure, Tertiary , Proto-Oncogene Proteins/genetics , Sequence Alignment , Transcriptional ActivationABSTRACT
The Sno oncogene (Snoo or dSno in Drosophila) is a highly conserved protein and a well-established antagonist of Transforming Growth Factor-beta signaling in overexpression assays. However, analyses of Sno mutants in flies and mice have proven enigmatic in revealing developmental roles for Sno proteins. Thus, to identify developmental roles for dSno we first reconciled conflicting data on the lethality of dSno mutations. Then we conducted analyses of wing development in dSno loss of function genotypes. These studies revealed ectopic margin bristles and ectopic campaniform sensilla in the anterior compartment of the wing blade suggesting that dSno functions to antagonize Wingless (Wg) signaling. A subsequent series of gain of function analyses yielded the opposite phenotype (loss of bristles and sensilla) and further suggested that dSno antagonizes Wg signal transduction in target cells. To date Sno family proteins have not been reported to influence the Wg pathway during development in any species. Overall our data suggest that dSno functions as a tissue-specific component of the Wg signaling pathway with modest antagonistic activity under normal conditions but capable of blocking significant levels of extraneous Wg, a role that may be conserved in vertebrates.