The warts gene as a novel target of the Drosophila DRE/DREF transcription pathway

Abstract

The Hippo tumor suppressor pathway in Drosophila represses expression of DIAP1 and Cyclin E via inactivation of the transcription co-activator Yorkie, resulting in cell cycle arrest and induction of apoptosis. The warts (wts) gene is well known as a core kinase in this pathway, but its transcriptional regulation has yet to be clarified. In Drosophila, DREF binds to a target sequence named DRE (5'-TATCGATA) and regulates transcription of cell proliferation-related genes containing the DRE sequence in their promoter regions. Here we found half reduction of the wts gene dose to enhance the DREF-induced rough eye phenotype, suggesting a DREF genetic interaction with the Hippo pathway in vivo. Three DREs indentified in the wts gene promoter region exhibited strong promoter activity with a luciferase transient expression assay in Drosophila S2 cells, this decreasing under DREF-RNAi conditions. In addition, knockdown of DREF in S2 cells reduced the level of endogenous wts mRNA. Chromatin immunoprecipitation assays with anti-DREF antibody revealed that DREF binds specifically to the wts gene promoter region containing DREs in vivo. These results indicate that the DRE/DREF pathway is required for transcriptional regulation of the wts gene, indicating a novel link between the DRE/DREF and the Hippo pathways.

Keywords: DRE; DREF; Hippo pathway; transcription; tumor suppressor; warts.

Figure 1

The wts gene genetically interacts with DREF. Scanning electron micrographs of adult eyes. (A) GMR-GAL4/Y; UAS-DREF/+, (B) GMR-GAL4/Y; UAS -DREF/+; +/wts³-¹⁷. Lower panels indicate higher magnification images of the upper panels. Scale bars are for 12.5 μm and 50 μm, respectively.

Figure 2

The wts gene carries DRE and DRE-like sequences in the 5'-flanking region. The transcription initiation site is indicated by the arrow and designated as +1. The positions and nucleotide sequences of DRE1, DRE2 and DRE3 are shown.

Figure 3

DREs play essential roles in wts gene promoter activity in cultured cells. (A) Schematic features of the wts promoter-luciferase fusion plas-mid wts-WT-luc, and its base-substituted derivatives (wts-DRE1mut, wts-DRE2mut, wts-DRE3mut, wts-DRE1,2mut, wts-DRE1,3mut, wts-DRE2,3mut and wts-DREallmut) are illustrated. DRE is represented by an open box and mutated DRE by a closed box. Plasmids were transfected into S2 cells and luciferase activities measured at 48 h thereafter. Luciferase activity was normalized to Renilla luciferase activity and expressed relative to that of wts-WT-luc. The mean activities with standard deviations from three independent transfections are shown. (B) Three days after treatment with DREF dsRNA (DREF RNAi) or YFP dsRNA (YFP RNAi), S2 cells were transfected with 0.5 μg each of wts-WT-luc or wts-DREallmut-Luc. Promoter activities were measured at 48 h after trans-fection. The luciferase activity was normalized to Renilla luciferase activity and expressed relative to that of wts-WT-luc in non-RNA treated cells (Mock). (C) Western immunoblot analysis showing decreased DREF protein level in S2 cells after DREF dsRNA treatment. The upper panel shows data for the DREF band with anti-DREF polyclonal antibody. The lower panel shows findings for α-tubulin as a loading control.

Figure 4

DREF dsRNA treatment reduces endogenous wts mRNA levels in S2 cells. DREF mRNA and wts mRNA in DREF dsRNA-treated cells were measured by quantitative RT-PCR and compared with values for non-dsRNA treated cells (Mock). The mRNA for (β-tubulin was used as a negative control.

Figure 5

Binding of DREF to the DRE-containing genomic region of the wts gene. (A) Schematic of the 5'-flanking region of the wts gene. Arrowheads indicate positions of the primers used for real-time PCR reactions. (B) Crosslinked chromatin of S2 cells was immunoprecipitated with either anti-DREF IgG or the control rabbit IgG. Genomic regions containing wtsDRE1, 2 or wtsDRE3 were amplified by PCR, and compared with amplicons from immunoprecipitates with the control rabbit IgG.

Figure 6

Model of balanced cell proliferation via the DRE/DREF system. DRE/DREF may simultaneously function as both strong enhancer and mild suppressor of cell proliferation.

Figure 7

The wts gene genetically interacts with DREF. Scanning electron micrographs of adult eyes. (A) UAS-DREFIR/+; GMR-GAL4/+, (B) UAS-DREFIR/+; GMR-GAL4/+; + /wts³-¹⁷. Scale bars are for 50 μm in upper panels, and 12.5μm in lower panels.