Abstract
The insulin/TOR signal transduction pathway plays a critical role in determining such important traits as body and organ size, metabolic homeostasis and life span. Although this pathway is highly conserved across the animal kingdom, the affected traits can exhibit important differences even between closely related species. Evolutionary studies of regulatory regions require the reliable identification of transcription factor binding sites. Here we have focused on the Insulin Receptor (InR) expression from its P2 promoter in the Drosophila genus, which in D. melanogaster is up-regulated by hypophosphorylated Drosophila FOXO (dFOXO). We have finely characterized this transcription factor binding sites in vitro along the 1.3 kb region upstream of the InR P2 promoter in five Drosophila species. Moreover, we have tested the effect of mutations in the characterized dFOXO sites of D. melanogaster in transgenic flies. The number of experimentally established binding sites varies across the 1.3 kb region of any particular species, and their distribution also differs among species. In D. melanogaster, InR expression from P2 is differentially affected by dFOXO binding sites at the proximal and distal halves of the species 1.3 kb fragment. The observed uneven distribution of binding sites across this fragment might underlie their differential contribution to regulate InR transcription.
Highlights
The insulin receptor (INR) of the insulin/TOR signal transduction pathway is one of the key sensors of nutrient availability that plays an important role in the control of cellular proliferation, cell size determination, and the response to nutrient availability in metazoans
DNA footprinting of the 1.3 kb fragment upstream of this promoter was performed using several overlapping fragments (300–500 bp long) covering this region. Each strand of these fragments was FAM labeled and subsequently incubated with either Drosophila FOXO (dFOXO) or BSA. dFOXO binding sites are protected from DNase I cleavage, which results in clusters of protected residues in each FAM labeled strand that can be identified by a DNA analyzer machine
Automated footprinting of the 1.3 kb region upstream of P2 revealed that dFOXO produces 18 footprints in this region, which include a total of 444 bp
Summary
The insulin receptor (INR) of the insulin/TOR signal transduction pathway is one of the key sensors of nutrient availability that plays an important role in the control of cellular proliferation, cell size determination, and the response to nutrient availability in metazoans. This pathway is critical for determining body and organ size [1,2]—via regulation of growth and proliferation [3]—as well as metabolic homeostasis and life span [4,5] in Drosophila, Caenorhabditis and mammals. DFOXO binding sites upstream of InR P2 promoter in Drosophila and analysis, decision to publish, or preparation of the manuscript
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