Interpretation of X chromosome dose at Sex-lethal requires non-E-box sitesfor the basic helix-loop-helix proteins SISB and daughterless

For Drosophila melanogaster flies, sexual fate is determined by the X chrom osome number. The basic helix-loop-helix. protein product of the X-Linked s isterlessB (sisB or scute) gene is a key indicator of the X dose and functi ons 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 activ ate transcription. We examined SISB-Da binding at Sri by using footprinting and gel mobility shift assays and found that SISB-Da binds numerous cluste red sites in the establishment promoter Sxl(Pe). Surprisingly, most SISB-Da sites at Sxl(Pe) differ from the canonical CANNTG E-box motif. These nonca nonical sites have 6-bp CA(GIC)CCG and 7-bp CA(GIC)CTTG cores and exhibit a range of binding affinities. We show that the noncanonical sites can media te SISB-Da-activated transcription in cell culture. P-element transformatio n experiments show that these noncanonical sites are essential for Sxl(Pe) activity in embryos. Together with previous deletion analysis, the data sug gest that the number, affinity, and position of SISB-Da sites may all be im portant for the operation of the Sxl(Pe) switch. Comparisons with other dos e-sensitive promoters suggest that threshold responses to diverse biologica l signals have common molecular mechanisms, with important variations tailo red to suit particular functional requirements.