Characterization of dFOXO binding sites upstream of the Insulin Receptor P2 promoter across the Drosophila phylogeny

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.

Fig 1. Constructions and analyses of transgenic flies.

(A) Schematic representation of the InR gene and its upstream region in Drosophila melanogaster with promoters P1, P2 and P3 coordinates from FlyBase (Release 6). The 1.3 kb fragment upstream of P2 is enlarged to show the location of the dFOXO footprints in this region. (B) Schematic representation of the 1.3 kb fragment upstream of the InR P2 promoter of wild type and mutated-insert transgenic lines. A discontinuous vertical line indicates the nucleotide position (-650) that divides the fragment into two halves. Grey and brown boxes indicate wild type and mutated footprints, respectively, with their width varying according to the extent of the footprint. (C) Expression level of the eGFP reporter gene in the different transgenic lines relative to that of the two endogenous genes. RRGE, relative reporter gene expression. * P<0.05; ** P<0.01; *** P<0.001.

Fig 2. dFOXO footprints upstream of the InR P2 promoter across the Drosophila phylogeny.

Schematic representation of the 1.3 kb fragment used to identify footprints in each of the five species studied (M, D. melanogaster; S, D. simulans; Y, D. yakuba; P, D. pseudoobscura; V, D. virilis). Grey boxes show the location of footprints with their width varying according to the extent of the footprint. Vertical black and red bars indicate the presence of DBE and FKH core sequences, respectively, detected in footprint areas. In species other than D. melanogaster, footprints that give reliable alignments are indicated with the same number than in D. melanogaster, with the remaining footprints in D. pseudoobscura and D. virilis indicated with the species initial and a correlative small letter. Marks (a) and (b) on the bp ruler correspond to the points used as limits to calculate the local densities of DBE and FKH motifs (-1042 and -650, respectively). Limit (b) was used for that purpose because of its former use in transgenic constructs (see Fig 1). Limit (a) was established at the midpoint between footprints 14 and 13 given that the latter footprint and footprints 12 and 11 are the only ones in D. melanogaster that do not harbor any core motifs.