Ectopic Expression of DREF Induces DNA Synthesis, Apoptosis, and Unusual Morphogenesis in the Drosophila Eye Imaginal Disc: Possible Interaction with Polycomband trithorax Group Proteins

Abstract

, 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.The promoters of Drosophila genes related to DNA replication, such as those for the 180-kDa catalytic subunit and 73-kDa subunit polypeptide of DNA polymerase ␣ and proliferating-cell nuclear antigen (PCNA), contain a common 8-bp palindromic sequence (5Ј-TATCGATA), named the DNA replication-related element (DRE) (21) in addition to E2F recognition sites (13, 54). The DRE requirement for promoter activation has been confirmed in both cultured cells and transgenic flies (22, 51, 52). Studies using the latter have shown that DRE is required for the function of the PCNA promoter throughout development except in adult females. We found a specific DRE-binding factor (DREF) consisting of an 80-kDa polypeptide homodimer, and molecular cloning of its cDNA has led to confirmation that DREF is a trans activator for DRE-containing genes (22).Recently, we found that the N-terminal fragment of the DREF polypeptide containing a region responsible for DRE binding and dimer formation acts as dominant negative effector against DREF (23). Expression of a dominant-negative form of DREF in cells of salivary glands and eye imaginal discs using the GAL4-upstream activation site (UAS...

Role of DREF in transcriptional regulation of the Drosophila p53 gene

“…Activation of endogenous dmp53 gene expression in eye imaginal disks from flies overexpressing DREF To address whether overexpression of DREF can activate dmp53 gene expression in vivo, we used the fly line GMR-GAL4/ þ ; UAS-DREF/ þ ; þ for immunostaining of the eye imaginal disks in which DREF is overexpressed in the region posterior to the morphogenetic furrow (Hirose et al, 2001). As reported earlier, overexpression was confirmed by immunostaining with anti-DREF antibodies (Figure 7g).…”

“…Half reduction of the dmp53 gene dose suppresses the DREF-induced rough eye phenotype Overexpression of DREF-induced ectopic DNA synthesis and apoptosis and inhibited photoreceptor cell differentiation in eye imaginal disks, so that adult flies showed a mild rough eye phenotype (Figures 2a and b), although they are known to be viable and fertile (Hirose et al, 2001). Therefore, these flies are useful genetic tool for screening mutants for genes whose products affect phenotype induced by DREF overexpression Nakamura et al, 2008;Ida et al, 2009).…”

“…This line was generated by homologous recombination and is homozygous viable (Sogame et al, 2003). The UAS-DREF line was described earlier (Hirose et al, 2001) as well as the transgenic fly line carrying GMR-GAL4 on the X chromosome (Takahashi et al, 1999). All other stocks used in this study were obtained from the Bloomington, IN, USA stock center.…”

“…Ectopic expression of the dominant-negative form of DREF using the GAL4-UAS targeted expression system causes inhibition of both endoreplication in larval salivary gland cells and mitotic DNA replication in eye disk cells . Ectopic expression of full-length DREF in eye imaginal disks induces ectopic DNA synthesis and apoptosis in otherwise post-mitotic cells, and inhibits photoreceptor cell differentiation that results in a severe rough eye phenotype (Hirose et al, 2001). RNA interference (RNAi)-mediated knockdown of DREF in growing tissues has also provided direct evidence that the factor is necessary for cell cycle and cell growth control (Yoshida et al, 2004;Hyun et al, 2005).…”

Human DNA Replication-related Element Binding Factor (hDREF) Self-association via hATC Domain Is Necessary for Its Nuclear Accumulation and DNA Binding

“…In vivo and in vitro studies from our and other groups have shown that dDREF positively regulates processes involved not only in DNA replication but also cell growth and differentiation (5,6). It has been reported that dDREF is a component of a transcription initiation complex containing Drosophila TATA-binding protein-related factor 2 (dTRF2), so that it might direct dTRF2 complexes to dDRE-containing genes (7).…”