Identification of the Drosophila eIF4A gene as a target of the DREF transcription factor

The DNA replication-related element-binding factor (DREF) regulates cell proliferation-related gene expression in Drosophila. We have carried out a genetic screening, taking advantage of the rough eye phenotype of transgenic flies that express full-length DREF in the eye imaginal discs and identified the eukaryotic initiation factor 4A (eIF4A) gene as a dominant suppressor of the DREF-induced rough eye phenotype. The eIF4A gene was here found to carry three DRE sequences, DRE1 (-40 to -47), DRE2 (-48 to -55), and DRE3 (-267 to -274) in its promoter region, these all being important for the eIF4A gene promoter activity in cultured Drosophila Kc cells and in living flies. Knockdown of DREF in Drosophila S2 cells decreased the eIF4A mRNA level and the eIF4A gene promoter activity. Furthermore, specific binding of DREF to genomic regions containing DRE sequences was demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. Band mobility shift assays using Kc cell nuclear extracts revealed that DREF could bind to DRE1 and DRE3 sequences in the eIF4A gene promoter in vitro, but not to the DRE2 sequence. The results suggest that the eIF4A gene is under the control of the DREF pathway and DREF is therefore involved in the regulation of protein synthesis.     

Regulation of the Drosophila p38b gene by transcription factor DREF in the adult midgut

The Drosophila midgut is an excellent model for evaluation of gene networks that regulate adult stem cell proliferation and differentiation. The Drosophila p38b (D-p38b) gene has been shown to be involved in intestinal stem cell (ISC) proliferation and differentiation in the adult midgut. Here, we report that D-p38b gene expression is regulated by DREF (DNA replication-related element binding factor) in the adult midgut. We have identified a DRE in the 5′-flanking region of the D-p38b gene and showed that DREF could bind to this DRE via a gel mobility shift assay and a ChIP assay. Base-substitution mutations of the D-p38b promoter DRE and analyses of transformants carrying D-p38b-lacZ or D-p38b-DREmut-lacZ indicated that this DRE is required for the activity of the D-p38b gene promoter. Furthermore, by using the GAL4-UAS system, we showed that DREF regulates the activity of the D-p38b gene promoter in adult ISCs and progenitors. In addition, the D-p38b knockdown phenotypes in the midgut were rescued by DREF overexpression, suggesting a functional link between these two factors. Our results suggest that the D-p38b gene is regulated by the DREF pathway and that DREF is involved in the regulation of proliferation and differentiation of Drosophila ISCs and progenitors.     

Characterization of the novel gene BjDREB1B encoding a DRE-binding transcription factor from Brassica juncea L

A novel DREB (dehydration responsive element binding protein) gene, designated BjDREB1B, was isolated from Brassica juncea L. BjDREB1B contains a conserved EREBP/AP2 domain and was classified into the A-1 subgroup of the DREB subfamily based on phylogenetic tree analysis. RT-PCR showed that BjDREB1B was induced by abiotic stresses and exogenous phytohormones, such as drought, salt, low temperature, heavy metals, abscisic acid, and salicylic acid. Gel shift assay revealed that BjDREB1B specifically bound to the DRE element in vitro. Yeast one-hybrid assay showed that full-length BjDREB1B or its C-terminal region functioned effectively as a trans-activator. Furthermore, overexpression of BjDREB1B in tobacco up-regulated the expression of NtERD10B, and BjDREB1B transgenic plants accumulated higher levels of proline than control plants under normal and saline conditions, together showing that BjDREB1B plays important roles in improving plant tolerance to drought and salinity.     

A major bristle QTL from a selected population of Drosophila uncovers the zinc-finger transcription factor poils-au-dos, a repressor of achaete-scute

Traditional screens aiming at identifying genes regulating development have relied on mutagenesis. Here, we describe a new gene involved in bristle development, identified through the use of natural variation and selection. Drosophila melanogaster bears a pattern of 11 macrochaetes per heminotum. From a population initially sampled in Marrakech, a strain was selected for an increased number of thoracic macrochaetes. Using recombination and single nucleotide polymorphisms, the factor responsible was mapped to a single locus on the third chromosome, poils au dos, that encodes a zinc-finger-ZAD protein. The original, as well as new, presumed null, alleles of poils au dos, is associated with ectopic achaete-scute expression that results in the additional bristles. This suggests a possible role for Poils au dos as a repressor of achaete and scute. Ectopic expression appears to be independent of the activity of known cis-regulatory enhancer sequences at the achaete-scute complex that mediate activation at specific sites on the notum. The target sequences for Poils au dos activity were mapped to a 14 kb region around scute. In addition, we show that pad interacts synergistically with the repressor hairy and with Dpp signaling in posterior and anterior regions of the notum, respectively.