Transcriptional regulation of the Drosophila caudal homeobox gene by DRE/DREF

Abstract

The caudal-related homeobox transcription factors are required for the normal development and differentiation of intestinal cells. Recent reports indicate that misregulation of homeotic gene expression is associated with gastrointestinal cancer in mammals. However, the molecular mechanisms that regulate expression of the caudal-related homeobox genes are poorly understood. In this study, we have identified a DNA replication-related element (DRE) in the 5' flanking region of the Drosophila caudal gene. Gel-mobility shift analysis reveals that three of the four DRE-related sequences in the caudal 5'-flanking region are recognized by the DRE-binding factor (DREF). Deletion and site-directed mutagenesis of these DRE sites results in a considerable reduction in caudal gene promoter activity. Analyses with transgenic flies carrying a caudal-lacZ fusion gene bearing wild-type or mutant DRE sites indicate that the DRE sites are required for caudal expression in vivo. These findings indicate that DRE/DREF is a key regulator of Drosophila caudal homeobox gene expression and suggest that DREs and DREF contribute to intestinal development by regulating caudal gene expression.

Figure 1

The nucleotide sequences and relative positions of DRE-related sequences in the 5′-flanking region of Drosophila caudal gene promoter. The four potential DRE sites in the Drosophila caudal promoter, termed cad-DRE1, cad-DRE2, cad-DRE3 and cad-DRE4, are shown. The maternal transcription initiation site is indicated by the arrowhead and designated +1. Lower case letters indicate mutated bases in the DRE sites with numbering relative to the maternal transcription initiation site. The zygotic transcription initiation site is indicated by +1(Z, −159).

Figure 2

Complex formation between putative DRE sites in the caudal promoter and GST–DREF1-125. Radiolabeled double-stranded cad-DRE oligonucleotides were incubated with GST–DREF1-125 fusion proteins in the presence of increasing amounts of competitor oligonucleotides. Lanes 1, 6, 11 and 16, GST only. Lanes 2, 7, 12 and 17, minus competitor. Lanes 3, 8, 13 and 18, unlabeled competitor oligonucleotides with the wt cad-DRE sequences. Lanes 4, 9, 14 and 19, unlabeled competitor oligonucleotides with the mutant having three base changes in cad-DRE sequences (mut). Lanes 5, 10, 15 and 20, unlabeled competitor oligonucleotides with the wt TBP-DRE1 sequences. TBP-DRE1, oligonucleotide containing DRE site 1 of the Drosophila TBP gene promoter.

Figure 3

Complex formation between putative DRE sites in the caudal promoter and Kc cell nuclear extracts factors. (A) Radiolabeled double-stranded cad-DRE oligonucleotides were incubated with Kc cell nuclear extracts in the absence or presence of unlabeled TBP-DRE1 wt competitor oligonucleotides. Radiolabeled cad-DRE1 (B) or cad-DRE3 (C) oligonucleotides were incubated with Kc cell nuclear extracts in the absence or presence (lanes 16 and 21) of anti-DREF monoclonal antibody No. 4 (mAb 4). Lanes 12 and 17, no extract added. Lanes 13 and 18, binding without competitor. Lanes 14 and 19, unlabeled competitor oligonucleotides with the wt cad-DRE sequences. Lanes 15 and 20, unlabeled competitor oligonucleotides with mutant having three base changes in cad-DRE sequences (mut).

Figure 4

Analysis of caudal promoter deletion constructs. (A) Schematic representation of caudal promoter deletion constructs containing progressively shorter sequences at the 5′-end generated by restriction enzyme digestion. The putative DRE sites and transcription start sites are shown. (B) Transfections were performed with Drosophila S2 cells and promoter activities measured as luciferase activities normalized to β-galactosidase activities 48 h after transfection. The mean activities ± SE from three independent transfections are shown.

Figure 5

Effects of DRE-related sequences on caudal gene promoter activity. (A) Schematic features of the promoter–luciferase fusion plasmids are illustrated. DRE-related sequences are indicated by open box and mutated DREs are marked by crossed box. (B) Transfections were performed with Drosophila S2 cells and promoter activities measured as luciferase activities normalized to β-galactosidase activities 48 h after transfection. The mean activities ± SE from three independent transfections are shown.

Figure 6

Effects of DRE-related sequences on caudal gene expression in vivo. (A) β-Galactosidase expression in the transgenic flies carrying cad–lacZ or cadDmut7–lacZ was detected by X-gal staining. The third instar larvae or adults were dissected and stained with 0.2% X-gal solution in the dark at 25°C overnight. Reduced expression of the cadDmut7–lacZ is apparent in the larval hindgut, salivary gland, adult hindgut, ovary and ejaculatory duct compared with cad–lacZ. (B) Quantitative β-galactosidase activities of the transgenic flies bearing cad–lacZ or cadDmut7–lacZ fusion gene. Crude extracts were prepared from third instar larvae or 5-day-old adult transgenic flies as described under Materials and Methods. The β-galactosidase activities are expressed as absorbance units at 574 nm/h/mg of protein. Average values obtained from three independent experiments with ± SE values are shown.

Figure 7

Expression of DREF and caudal in UAS-DREF/+;hs-GAL4/+ third instar larvae. Total RNA was prepared from the third instar larvae carrying hs-GAL4 and UAS-DREF after heat shock at 37°C for 45 min and incubated at 25°C for various time periods. RT–PCR was performed to determine DREF and caudal mRNA levels. The line carrying hs-GAL4/+ was used as a control and processed in the same way as that carrying UAS-DREF/+;hs-GAL4/+.

Figure 8

DREF overexpression stimulates the caudal gene promoter activity in vivo. (A) The β-galactosidase activities of third instar larvae from +/+;cad–lacZ/hs-GAL4 or UAS-DREF/+;cad–lacZ/hs-GAL4 lines. Crude extracts were prepared after 45 min of heat shock at 37°C and incubated at 25°C for an additional 8 h. Average values obtained from three independent experiments with ± SE values are shown. (B) DREF mRNA overexpression by heat shock induction. Total RNA was prepared from the third instar larvae carrying +/+;cad–lacZ/hs-GAL4 (a) or UAS-DREF/+;cad–lacZ/hs-GAL4 (b) following 45 min of heat shock at 37°C and incubation at 25°C for an additional 3 h, after which DREF mRNA levels were measured by RT–PCR.