Targeted expression of the DNA binding domain of DRE-binding factor, a Drosophila transcription factor, attenuates DNA replication of the salivary gland and eye imaginal disc

Abstract

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory elements consisting of an 8-bp palindromic DNA replication-related element (DRE) sequence (5'-TATCGATA). The specific DRE-binding factor (DREF), a homodimer of the polypeptide with 709 amino acid residues, is a positive trans-acting factor for transcription of DRE-containing genes. Both DRE binding and dimer formation are associated with residues 16 to 115 of the N-terminal region. We have established transgenic flies expressing the full-length DREF polypeptide or its N-terminal fragment (amino acid residues 1 to 125) under the control of the heat shock promoter, the salivary gland-specific promoter, or the eye imaginal disc-specific promoter. Heat shock induction of the N-terminal fragment during embryonic, larval, or pupal stages caused greater than 50% lethality. This lethality was overcome by coexpression of the full-length DREF. In salivary glands of the transgenic larvae expressing the N-terminal fragment, this fragment formed a homodimer and a heterodimer with the endogenous DREF. Ectopic expression of the N-terminal fragment in salivary gland cells reduced the contents of mRNAs for the 180-kDa subunit of DNA polymerase alpha and for dE2F and the extent of DNA endoreplication. Ectopic expression of the N-terminal fragment in the eye imaginal discs significantly reduced DNA replication in cells at the second mitotic wave. The lines of evidence suggest that the N-terminal fragment can impede the endogenous DREF function in a dominant negative manner and that DREF is required for normal DNA replication in both mitotic cell cycle and endo cycle.

FIG. 1

Western immunoblotting and gel mobility shift assay to detect endogenous DREF and ectopically expressed DREF_1–125. (A) Extracts were prepared from embryos of Canton S (CS) flies (lane 2) and from transgenic embryos carrying hs-GAL4 and UAS-DREF_1–125 without heat shock (HS) (lane 3), at 2 h after HS (lane 4), at 4 h after HS (lane 5), and at 6 h after HS (lane 6), and 20-μg aliquots of proteins were analyzed by Western immunoblotting with anti-DREF MAb 1. The arrow indicates signals for the endogenous DREF polypeptide. Signals for DREF_1–125 are indicated with an asterisk. Lane 1, size markers. (B) Extracts were prepared from salivary glands from third-instar larvae carrying Sg-GAL4 and UAS-DREF_1–125. Endogenous DREF and the N-terminal fragment were immunoprecipitated by using protein G-Sepharose beads with control IgG (lane 2) or anti-DREF MAb 1 (lane 3) and then analyzed by immunoblotting with anti-DREF MAb 1 (lanes 2 and 3). Samples for each lane contained 100 μg of protein. The arrow indicates signals for the endogenous DREF polypeptide. Signals for DREF_1–125 are indicated with an asterisk. (C) Radiolabeled double-stranded DRE-P oligonucleotides were incubated with salivary gland extracts in the presence or absence of competitor oligonucleotide (11, 13). Lane 1, salivary gland extract of transgenic larvae carrying only Sg-GAL4; lanes 2 to 11, salivary gland extracts of transgenic larvae carrying Sg-GAL4 and UAS-DREF_1–125. (D) Extracts were prepared from salivary glands from third-instar larvae carrying Sg-GAL4 and UAS-DREF_1–125. Aliquots (4 μl) were preincubated with control antibody (C) (lane 1), anti-DREF MAb 1 (lane 2), or anti-DREF MAb 4 (lane 3) and then mixed with radiolabeled double-stranded DRE-P oligonucleotides.

FIG. 2

Lethality in transgenic flies expressing DREF_1–125. Eggs were counted and animals at various developmental stages were administered a single heat shock for 45 min at 37°C. The numbers of animals developing into adults were counted. The values shown were normalized for the rate of maturation into adults without heat treatment.

FIG. 3

Melanotic tumors after heat shock induction of DREF_1–125. (A) Melanotic tumor (arrow) observed in a second-instar larva at 24 h after heat shock. (B) Melanotic tumors (arrows) observed in a third-instar larva at 24 h after heat shock. Note that more than half of larvae had died by that time.

FIG. 4

Phenotypes of salivary glands expressing DREF_1–125. Transcripts of the rp49 gene (A and B), the DNA polymerase α 180-kDa subunit gene (C and D), and the dE2F gene (E and F) in salivary glands were detected by in situ hybridization. Salivary glands from third-instar larvae at 60 h after hatching were hybridized with antisense DIG-labeled RNA probes. Staining was detected with alkaline phosphatase. (G and H) DAPI staining of the salivary glands. (I and J) DAPI staining of imaginal ring cells of the same salivary glands as in panels G and H, respectively. (A, C, E, G, and I) Control fly carrying Sg-GAL4 alone; (B, D, F, H, and J) transgenic fly carrying Sg-GAL4 and UAS-DREF_1–125. Magnifications: for panels A through F, ×153; for panels G and H, ×307; and for panels I and J, ×383.

FIG. 5

Ectopic expression of DREF_1–125-reduced endoreplication. Larvae at 36 h after hatching were dissected in Drosophila Ringer’s solution, labeled with BrdU at 25°C for 30 min in Grace’s medium, and stained with anti-BrdU. (A) Control larva carrying Sg-GAL4 alone; (B) larva carrying Sg-GAL4 and UAS-DREF_1–125; (C) salivary glands from a control larva carrying Sg-GAL4 alone; (D) salivary glands from a larva carrying Sg-GAL4 and UAS-DREF_1–125. sg, salivary gland; ir, imaginal ring.

FIG. 6

Ectopic expression of DREF_1–125 inhibits DNA replication of cells in the second mitotic wave. Shown are results for immunostaining of eye imaginal discs with anti-DREF MAb 1. (A) GMR-GAL4/+; +. (B) GMR-GAL4/+; UAS-DREF_1–125/+. Patterns of BrdU incorporation in eye imaginal discs are apparent. (C) GMR-GAL4/+; +. (D) GMR-GAL4/+; UAS-DREF_1–125/+. The eye discs from a third-instar larva were stained with an anti-BrdU antibody. Arrows indicate the position of the morphogenetic furrow (MF). The anterior (A) of the discs is on the left. P, posterior.