ABSTRACT
Heparan sulfate proteoglycans play a vital role in signaling of various growth factors in both Drosophila and vertebrates. In Drosophila, mutations in the tout velu (ttv) gene, a homolog of the mammalian EXT1 tumor suppressor gene, leads to abrogation of glycosaminoglycan (GAG) biosynthesis. This impairs distribution and signaling activities of various morphogens such as Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp). Mutations in members of the exostosin (EXT) gene family lead to hereditary multiple exostosis in humans leading to bone outgrowths and tumors. In this study, we provide genetic and biochemical evidence that the human EXT1 (hEXT1) gene is conserved through species and can functionally complement the ttv mutation in Drosophila. The hEXT1 gene was able to rescue a ttv null mutant to adulthood and restore GAG biosynthesis.
Subject(s)
Drosophila Proteins/genetics , Drosophila/genetics , Evolution, Molecular , Membrane Proteins/genetics , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/physiology , Animals , Animals, Genetically Modified , Cells, Cultured , Drosophila/embryology , Drosophila Proteins/metabolism , Embryo, Nonmammalian , Female , Genes, Tumor Suppressor/physiology , Heparin/analogs & derivatives , Heparin/biosynthesis , Humans , Male , Membrane Proteins/metabolism , N-Acetylglucosaminyltransferases/metabolism , Protein Binding , Proteoglycans/biosynthesis , Sequence Homology , Tissue DistributionABSTRACT
DAF-16, a forkhead transcription factor, is a key regulator of longevity, metabolism and dauer diapause in Caenorhabditis elegans. The precise mechanism by which DAF-16 regulates multiple functions, however, is poorly understood. Here, we used chromatin immunoprecipitation (ChIP) to identify direct targets of DAF-16. We cloned 103 target sequences containing consensus DAF-16 binding sites and selected 33 targets for further analysis. Expression of most of these genes is regulated in a DAF-16-dependent manner, and inactivation of more than half of these genes significantly altered DAF-16-dependent functions, including life span, fat storage and dauer formation. Our results show that the ChIP-based cloning strategy leads to greater enrichment for DAF-16 target genes than previous screening strategies. We also demonstrate that DAF-16 is recruited to multiple promoters to coordinate regulation of its downstream targets. The large number of target genes discovered provides insight into how DAF-16 controls diverse biological functions.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Longevity/physiology , Transcription Factors/metabolism , Alleles , Animals , Caenorhabditis elegans/physiology , Chromatin Immunoprecipitation , Forkhead Transcription Factors , Gene Expression Regulation , Genes, Helminth , PhenotypeABSTRACT
TATA-box-binding protein (TBP) is a highly conserved RNA polymerase II general transcription factor that binds to the core promoter and initiates assembly of the preinitiation complex. Two proteins with high homology to TBP have been found: TBP-related factor 1 (TRF1), described only in Drosophila melanogaster, and TRF2, which is broadly distributed in metazoans. Here, we report the identification and characterization of an additional TBP-related factor, TRF3. TRF3 is virtually identical to TBP in the C-terminal core domain, including all residues involved in DNA binding and interaction with other general transcription factors. Like other TBP family members, the N-terminal region of TRF3 is divergent. The TRF3 gene is present and expressed in vertebrates, from fish through humans, but absent from the genomes of the urochordate Ciona intestinalis and the lower eukaryotes D. melanogaster and Caenorhabditis elegans. TRF3 is a nuclear protein that is present in all human and mouse tissues and cell lines examined. Despite the highly homologous TBP-like C-terminal core domain, gel filtration analysis indicates that the native molecular weight of TRF3 is substantially less than that of TFIID. Interestingly, after mitosis, reimport of TRF3 into the nucleus occurs subsequent to TBP and other basal transcription factors. In summary, TRF3 is a highly conserved vertebrate-specific TRF whose phylogenetic conservation, expression pattern, and other properties are distinct from those of TBP and all other TRFs.
Subject(s)
TATA Box Binding Protein-Like Proteins/metabolism , TATA Box Binding Protein-Like Proteins/physiology , Transcription Factors/metabolism , Transcription Factors/physiology , Amino Acid Sequence , Animals , Caenorhabditis elegans/metabolism , Cell Line , Cell Nucleus/metabolism , Chromatography, Gel , Ciona intestinalis/metabolism , Computational Biology , DNA/chemistry , Drosophila melanogaster/metabolism , Fluorescent Antibody Technique, Indirect , HeLa Cells , Humans , Mice , Mitosis , Molecular Sequence Data , Nuclear Proteins , Phylogeny , Protein Biosynthesis , Protein Structure, Tertiary , RNA Polymerase II/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , TATA Box Binding Protein-Like Proteins/chemistry , Tissue Distribution , Transcription Factor TFIID/chemistry , Transcription Factors/chemistry , Transcription, GeneticABSTRACT
The general transcription factor TFIID is composed of the TATA box binding protein (TBP) and multiple TBP-associated factors (TAFs). In yeast, promoters can be grouped into two classes based on the involvement of TAFs. TAF-dependent (TAF(dep)) promoters require TAFs for transcription, and TBP and TAFs are present at comparable levels on these promoters. TAF-independent (TAF(ind)) promoters do not require TAFs for activity, and TAFs are either absent or present at levels far below those of TBP on these promoters. Here, we demonstrate that the upstream activating sequence (UAS) mediates the selective recruitment of TAFs to TAF(dep) promoters. A TAF(ind) UAS fails to recruit TAFs and to direct efficient transcription when inserted upstream of a TAF(dep) core promoter. This transcriptional defect can be overcome by a potent activator, indicating that a strong activation domain can compensate for the absence of TAFs on a TAF(dep) core promoter. Our results reveal a requirement for compatibility between the UAS and core promoter and thus help explain previous reports that only certain yeast UAS-core promoter combinations and mammalian enhancer-promoter combinations are efficiently transcribed. The differential recruitment of TAFs by UASs provides strong evidence for the proposal that in vivo TAFs are the targets of some, but not all, activators.
