Interpretation of X Chromosome Dose atSex-lethalRequires Non-E-Box Sites for the Basic Helix-Loop-Helix Proteins SISB and Daughterless

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Abstract
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 atSxl by using footprinting and gel mobility shift assays and found that SISB-Da binds numerous clustered sites in the establishment promoter SxlPe. Surprisingly, most SISB-Da sites at SxlPe 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 SxlPeactivity 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 SxlPeswitch. 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.