Ectopic expression of DREF induces DNA synthesis, apoptosis, and unusual morphogenesis in the Drosophila eye imaginal disc: possible interaction with Polycomb and trithorax group proteins - PubMed (original) (raw)

Ectopic expression of DREF induces DNA synthesis, apoptosis, and unusual morphogenesis in the Drosophila eye imaginal disc: possible interaction with Polycomb and trithorax group proteins

F Hirose et al. Mol Cell Biol. 2001 Nov.

Abstract

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory element DRE (5'-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs' chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, we established transgenic flies in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induced ectopic DNA synthesis in the cells behind the morphogenetic furrow, which are normally postmitotic, and abolished photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype was suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, we found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppressed and that reduction of Distal-less enhanced the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators.

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Figures

FIG. 1

Immunostaining of eye imaginal discs with anti-DREF antibodies. (b to d) Eye imaginal discs from wild-type third-instar larva were stained with rabbit anti-DREF polyclonal antibody (b), mouse anti-DREF monoclonal antibody 2 (c), and mouse anti-DREF monoclonal antibody 4 (d). (a) Negative control without primary antibody. P, posterior; A, anterior; MF, morphogenetic furrow. The time of color development for alkaline phosphatase is 1 h.

FIG. 2

Ectopic expression of DREF protein with the GMR promoter. Immunostaining of eye imaginal discs with the anti-DREF antibody is shown. (a) GMR-GAL4/+; +/+, (b) GMR-GAL4/+; UAS-DREF/+. The time of color development for alkaline phosphatase is 5 min.

FIG. 3

Scanning and transmission electron micrographs of adult compound eyes. (a and b) Wild-type eye. (c and d) GMR-GAL4/+;+/+. (e and f) +/+. UAS-DREF/+. (g and h) GMR-GAL4/+; UAS-DREF/+. (i and j) +/+; GMR-DREF#22/GMR-DREF#22. These flies were developed at 28°C. The rough eye phenotype is evident in panels g and h.

FIG. 4

Patterns of BrdU incorporation in eye imaginal discs. (a to c) GMR-GAL4/+; +/+. (d to f) GMR-GAL4/+; UAS-DREF/+. The eye disc was stained with an anti-BrdU antibody. MF and arrows indicate the position of the MF. Panels b and e show high-magnification basal views of the same focal planes as panels a and d, respectively. Panels c and f show BrdU signals at the apical focal planes of a and d, respectively.

FIG. 5

Immunostaining of eye imaginal discs with an anti-β-galactosidase antibody. Wild-type (a and c) or GMR-GAL4; UAS-DREF transgenic (b and d) flies were crossed with enhancer trap lines carrying D120 (inserted in scabrous) (A), BB02 (B), X63 (inserted in rhomboid) (C), AE127 (inserted in seven-up) (D), or H214 (inserted in klingon) (E) specifically expressing the β-galactosidase marker in photoreceptor cells (R) of early R8, late R8, R2/R5/R8, R3/R4/R1/R6, and R7, respectively, and F1 larvae were immunohistochemically stained with the anti-β-galactosidase antibody.

FIG. 6

Detection of apoptotic cells in eye imaginal discs. The TUNEL method was used with (a and b) and without (c) terminal deoxynucleotidyltransferase. (a) GMR-GAL4/+;+/+. (b and c) GMR-GAL4/+; UAS-DREF/+. MF and arrows indicate the position of the MF.

FIG. 7

Suppression of the DREF-induced rough eye phenotype by expression of DIAP1 and p35. (A) Scanning electron micrographs of adult compound eyes. (a and d) GMR-GAL4/+; UAS-DREF/+;+/+. (b and e) GMR-GAL4/+; UAS-DREF/+; UAS-DIAP1/+. (c and f) GMR-GAL4/+; UAS-DREF/+; GMR-p35/+. (B) Horizontal sections of adult Drosophila eyes stained with hematoxylin-eosin. (a) Canton S. (b) GMR-GAL4/+; UAS-DREF/; +/+. (c) GMR-GAL4/+; UAS-DREF/+; GMR-p35/+. These flies were developed at 28°C.

FIG. 8

A half-dose reduction of the dE2F gene suppresses the DREF rough eye phenotype. (A) Overexpression of DREF activates lacZ expression under the control of the dE2F gene promoter. Female flies expressing DREF (GMR-GAL4/GMR-GAL4; UAS-DREF/UAS-DREF;+/+) or female flies expressing GAL4 only (GMR-GAL4/GMR-GAL4; +/+; +/+) were crossed with dE2F729 male flies. F1 third-instar larvae developed at 28°C were dissected, and eye imaginal discs were used for detection of lacZ. In dE2F729 flies, the P element-carrying lacZ gene is inserted 48 nucleotides upstream of the initiator methionine of the dE2F gene in the same orientation as the dE2F gene; therefore, lacZ expression directed by the dE2F gene promoter can be detected in eye imaginal discs from dE2F729/+ larvae. (a) GMR-GAL4; +/+; dE2F729/+ eye disc stained with anti-β-galactosidase antibody. (b) GMR-GAL4; UAS-DREF/+; dE2F729/+ eye disc stained with anti-β-galactosidase antibody. (B) Scanning electron micrographs of adult compound eyes. Female flies expressing DREF (GMR-GAL4/GMR-GAL4; UAS-DREF/UAS-DREF; +/+) were crossed with dE2F729, dE2F7172, or dE2F91 male flies (w/Y; +/+; _dE2F_−/TM3), and F1 progeny developing at 28°C without TM3 balancer were used for analysis of eye phenotype. (a) GMR-GAL4/+; UAS-DREF/+; +/+. (b) GMR-GAL4/+; UAS-DREF/+; dE2F91/+. (c) GMR-GAL4/+; UAS-DREF/+; dE2F729/+. (d) GMR-GAL4/+; UAS-DREF/+; dE2F7172/+. In dE2F7172 flies, the P element is inserted 33 nucleotides downstream of the initiator methionine. The E2F91 allele contained a C-to-T transition at nucleotide +91 that converted Gln-31 of dE2F to a stop codon.

FIG. 9

Scanning electron micrographs of adult compound eyes. Female flies expressing DREF (GMR-GAL4/GMR-GAL4; UAS-DREF/UAS-DREF; +/+) were crossed with brm2/TM6B, osa2/TM6B, mor1/TM6B, or Dll5/SM5 male flies, and F1 progeny developing at 28°C without balancer chromosome were used for analysis of eye phenotype. (a and b) GMR-GAL4/+; UAS-DREF/+; +/+. (c and d) GMR-GAL4/+; UAS-DREF/+; brm2/+. (e and f) GMR-GAL4/+; UAS-DREF/+; osa2/+. (g and h) GMR-GAL4/+; UAS-DREF/+; mor1/+. (i and j) GMR-GAL4/+; UAS-DREF/Dll5; +/+.

FIG. 10

Summary of the deficiencies screened. The numbered divisions and lettered subdivisions of salivary gland polytene chromosomes are marked. The boxes indicate the influence of each deficiency on the DREF-induced rough eye phenotype: black lines, no effect; shaded box, enhancement; open box, suppression. The deletions for dominant enhancers and suppressor of both DREF- and p53-induced rough eye phenotype are indicated by italics.

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Ectopic expression of DREF induces DNA synthesis, apoptosis, and unusual morphogenesis in the Drosophila eye imaginal disc: possible interaction with Polycomb and trithorax group proteins - PubMed (original) (raw)