Interpretation of X chromosome dose at Sex-lethal requires non-E-box sites for the basic helix-loop-helix proteins SISB and daughterless.
For Drosophila melanogaster flies, sexual fate is determined by the X chromosome number. The basic helix-loop-helix protein product of the X-linked sisterlessB (sisB or scute) gene is a key indicator of the X dose and functions to activate the switch gene Sex-lethal (Sxl) in female (XX), but not in male (XY), embryos. Zygotically expressed sisB and maternal daughterless (da) proteins are known to form heterodimers that bind E-box sites and activate transcription. We examined SISB-Da binding at Sxl by using footprinting and gel mobility shift assays and found that SISB-Da binds numerous clustered sites in the establishment promoter Sxl(Pe). Surprisingly, most SISB-Da sites at Sxl(Pe) differ from the canonical CANNTG E-box motif. These noncanonical sites have 6-bp CA(G/C)CCG and 7-bp CA(G/C)CTTG cores and exhibit a range of binding affinities. We show that the noncanonical sites can mediate SISB-Da-activated transcription in cell culture. P-element transformation experiments show that these noncanonical sites are essential for Sxl(Pe) activity in embryos. Together with previous deletion analysis, the data suggest that the number, affinity, and position of SISB-Da sites may all be important for the operation of the Sxl(Pe) switch. Comparisons with other dose-sensitive promoters suggest that threshold responses to diverse biological signals have common molecular mechanisms, with important variations tailored to suit particular functional requirements.
Department of Biological Sciences, Columbia University, New York, New York 10027.
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D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D002478:Cells, Cultured; D017124:Conserved Sequence; D018983:DNA Footprinting; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000378:metabolism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics*; D019143:Evolution, Molecular; D005260:Female; D017353:Gene Deletion; D018257:Helix-Loop-Helix Motifs; D008297:Male; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000235:genetics*; D019849:Sex Determination Processes; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D019076:Transgenes; D014960:X Chromosome; D001616:beta-Galactosidase / Q000378:metabolism
D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D029721:Drosophila Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; D001616:beta-Galactosidase; C090080:Da protein, Drosophila; C067777:Sxl protein, Drosophila; C089197:sc protein, Drosophila; C000604451:sna protein, Drosophila