Inserting the Ftz homeodomain into engrailed creates a dominant transcriptional repressor that specifically turns off Ftz target genes in vivo.
The Engrailed homeodomain protein is an 'active' or dominant transcriptional repressor in cultured cells. In contrast, the Fushi Tarazu homeodomain protein is an activator, both in cultured cells and in Drosophila embryos, where it activates several known target genes, including its own gene. This auto-activation has been shown to depend on targeting to a fushi tarazu enhancer by the Fushi Tarazu homeodomain. We combined Fushi Tarazu targeting and Engrailed active repression in a chimeric regulator, EFE. When EFE is ubiquitously expressed, it overrides endogenous Fushi Tarazu and causes a fushi tarazu mutant phenotype. Normal Fushi Tarazu target genes are affected as they are in fushi tarazu mutants. One such target gene is repressed by EFE even where Fushi Tarazu is not expressed, suggesting that the repression is active. This is confirmed by showing that the in vivo activity of EFE depends on a domain that is required for active repression in culture. A derivative that lacks this domain, while it cannot repress the endogenous fushi tarazu gene, can still reduce the activity of the fushi tarazu autoregulatory enhancer, suggesting that it competes with endogenous Fushi Tarazu for binding sites in vivo. However, this passive repression is much less effective than active repression.
John
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Department of Microbiology and Immunology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia PA 19107, USA.
Smith
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Jaynes
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D000818:Animals; D004330:Drosophila / Q000196:embryology / Q000235:genetics*; D029721:Drosophila Proteins; D004742:Enhancer Elements, Genetic; D051717:Fushi Tarazu Transcription Factors; D018507:Gene Expression Regulation, Developmental; D018014:Gene Transfer Techniques; D005801:Genes, Homeobox; D018398:Homeodomain Proteins / Q000235:genetics*; D007301:Insect Hormones / Q000235:genetics*; D009024:Morphogenesis / Q000235:genetics; D010641:Phenotype; D011994:Recombinant Proteins / Q000494:pharmacology; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
D029721:Drosophila Proteins; C063459:En protein, Drosophila; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D007301:Insect Hormones; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C497169:ftz protein, Drosophila