A binding site for multiple transcriptional activators in the fushi tarazu proximal enhancer is essential for gene expression in vivo.
The Drosophila homeobox gene fushi tarazu (ftz) is expressed in a highly dynamic striped pattern in early embryos. A key regulatory element that controls the ftz pattern is the ftz proximal enhancer, which mediates positive autoregulation via multiple binding sites for the Ftz protein. In addition, the enhancer is necessary for stripe establishment prior to the onset of autoregulation. We previously identified nine binding sites for multiple Drosophila nuclear proteins in a core 323-bp region of the enhancer. Three of these nine sites interact with the same cohort of nuclear proteins in vitro. We showed previously that the nuclear receptor Ftz-F1 interacts with this repeated module. Here we purified additional proteins interacting with this module from Drosophila nuclear extracts. Peptide sequences of the zinc finger protein Ttk and the transcription factor Adf-1 were obtained. While Ttk is thought to be a repressor of ftz stripes, we have shown that both Adf-1 and Ftz-F1 activate transcription in a binding site-dependent fashion. These two proteins are expressed ubiquitously at the time ftz is expressed in stripes, suggesting that either may activate striped expression alone or in combination with the Ftz protein. The roles of the nine nuclear factor binding sites were tested in vivo, by site-directed mutagenesis of individual and multiple sites. The three Ftz-F1-Adf-1-Ttk binding sites were found to be functionally redundant and essential for stripe expression in transgenic embryos. Thus, a biochemical analysis identified cis-acting regulatory modules that are required for gene expression in vivo. The finding of repeated binding sites for multiple nuclear proteins underscores the high degree of redundancy built into embryonic gene regulatory networks.
Han
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Brookdale Center for Developmental and Molecular Biology, Mt. Sinai School of Medicine, New York, New York 10029, USA.
Yu
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Su
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Kohanski
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Pick
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D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D004330:Drosophila; D029721:Drosophila Proteins; D004742:Enhancer Elements, Genetic; D051717:Fushi Tarazu Transcription Factors; D015870:Gene Expression; D018398:Homeodomain Proteins / Q000378:metabolism*; D019476:Insect Proteins / Q000378:metabolism; D007763:Lac Operon; D008969:Molecular Sequence Data; D017354:Point Mutation; D018160:Receptors, Cytoplasmic and Nuclear; D011988:Receptors, Thyroid Hormone / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D054339:Steroidogenic Factor 1; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D016335:Zinc Fingers
C072263:Adf1 protein, Drosophila; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D019476:Insect Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D054339:Steroidogenic Factor 1; D015534:Trans-Activators; D014157:Transcription Factors; C497169:ftz protein, Drosophila; C499712:nuclear hormone receptor FTZ-F1, Drosophila