Specific antibodies were prepared against Drosophila DNA polymerase epsilon and DREF, a regulatory factor for DNA replication-related genes. Using these antibodies together with those for DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), expression patterns and sub-cellular distributions of these proteins were studied during Drosophila development. DNA polymerase alpha, epsilon and PCNA proteins were maternally stored in unfertilized eggs and maintained at high levels during embryogenesis. With distinct nuclear localization, proteins were observed in embryos at interphase stages throughout the 13 nuclear division cycles, suggesting that they all participate in rapid nuclear DNA replication during these cycles. In contrast, maternal storage of a DREF protein was relatively low and its level increased throughout embryogenesis. Strong nuclear staining with the anti-DREF antibody was not observed until the nuclear division cycle 8. Immunostaining of various larval tissues from transgenic flies carrying the PCNA gene promoter-lacZ fusion gene revealed co-expression of DREF, PCNA and lacZ, suggesting that DREF regulates the expression of PCNA gene in these tissues. In addition, a relatively high level of DREF was detected in adult males as well as females. Since DNA polymerase alpha, epsilon and PCNA are hardly detectable in adult males, DREF very likely regulates genes other than those closely linked to DNA replication in adult males (Yamaguchi, 1995a).
In situ hybridization studies show that Dref transcripts are detected in neural precursor cells of the CNS (including NBs and GMCs) and in SOPs the PNS. No transcripts are detected in post-mitotic neurons (Brody, 2002).
The distribution of endogenous DREF was examined in wild-type eye imaginal discs. Anti-DREF polyclonal antibody, monoclonal antibody 2 or monoclonal antibody 4 all strongly stain cells in front of the MF, when the cells are entering the G1 phase of the second mitotic cycle. In addition, an elevated level of DREF was observed in the cells just posterior to the MF, corresponding to the S-phase zone in the second mitotic wave. Relatively low levels of DREF are present in the postmitotic cells undergoing differentiation. The expression pattern is very similar to that of dE2F. The results suggest that DREF is involved in differentiation processes in addition to cell proliferation (Hirose, 2001).
Glass protein is a transcription factor that is expressed in all cells in and posterior to the MF. The pGMR vector contains a multimer of Glass-binding sites that are sufficient to drive the expression of coding regions placed downstream of these binding sites. To investigate the consequence of the ectopic expression of DREF, a transgenic line was established bearing pGMR-GAL4 crossed with a transgenic fly carrying DREF cDNAs under the control of a GAL4-binding sequence (pUAS-DREF1-709). Immunostaining using anti-DREF polyclonal antibody revealed elevated levels of DREF expressed in all cells in and posterior to the MF of the third-instar larvae of the progeny (Hirose, 2001).
The eyes of adults carrying one copy of pGMR-GAL4 and one copy of pUAS-DREF1-709 are severely rough in appearance, and most bristles are missing. In addition, the organized array of each ommatidium is destroyed and some ommatidia are fused each other. Examination of retinal sections revealed the number of reduced ommatidia and the number and shape of the abnormal photoreceptor cells. Pigment cells were found to be missing in all ommatidia, and some remaining tissues were vacuolated in transgenic flies carrying one copy of pGMR-GAL4 and one copy of pUAS-DREF1-709. To confirm that ectopic expression of DREF-directly affects eye development, transgenic flies were established carrying pGMR-DREF1-709. Flies carrying pGMR-DREF1-709 did not exhibit an apparent rough eye phenotype on inspection by scanning electron microscopy; however, the abnormal photoreceptor cells and missing pigment cells were seen in sections of eyes from transgenic flies carrying pGMR-DREF1-709, indicating that abnormality of eye development is indeed induced by overexpression of DREF polypeptide and not by overexpression of GAL4 protein. Furthermore, the eye phenotype in these flies suggests that DREF overexpression affects eye development without causing the apparent rough eye under scanning electron microscopy (Hirose, 2001).
To assess whether expression of DREF in late G1 can drive cells destined to become postmitotic cells into the S phase, imaginal discs were pulse-labeled with BrdU for 30 min in vitro and stained with an anti-BrdU antibody. No significant difference between control and DREF-expressing discs was observed in the region anterior to the MF, where the GMR promoter was inactive. In control discs, a 30-min pulse of BrdU incorporation led to labeling of cells in one or two columns posterior to the MF, while in imaginal discs of transgenic flies expressing DREF, the equivalent of five columns were labeled. Therefore, overexpression of DREF either expands the S phase or induces an extra S phase. In addition, some cells in the region more posterior to the MF, where cells are committed to the neuronal fate and normally differentiate into specific cells such as photoreceptors, were labeled with BrdU. Most of the nuclei of cells with BrdU incorporated occupied a more apical position in the imaginal discs than did nuclei in the synchronous S-phase cells posterior to the MF and in uncommitted cells, suggesting that DREF overexpression caused ectopic S phase in some cells that are normally postmitotic (Hirose, 2001).
It has been reported that overexpression of dE2F in eye imaginal disc causes ectopic S phases in differentiation-uncommitted reservoir cells, although committed cells in the eye disc are relatively resistant to the effects of ectopic E2F expression. However, overexpression of DREF appears to result in the occurrence of an extra round of S phase in the committed cells. Therefore, the effects of DREF expression on photoreceptor specifications were examined in detail. In wild-type discs, developmentally uncommitted cells are sequentially recruited into clusters that comprise ommatidial precursors. Cluster formation is first observed within the MF, where cells are in G1. Cells either leave the cell cycle and differentiate or undergo a final synchronous round of cell division. Overt ommatidial organization starts in the MF when cells are grouped into equally spaced concentric aggregates, which convert into preclusters. Photoreceptor cells have been found to be generated in a stereotyped order: R8 is generated first, with movement posterior from the MF, then cells are added pairwise (R2 and R5, R3 and R4, and R1 and R6), and R7 is the last photoreceptor to be added to each cluster. Several enhancer trap lines expressing a nucleus-localized form of Escherichia coli β-galactosidase depend on the specific enhancer-promoter located nearby the P-element. They were used here to determine the identities of each photoreceptor. Seven enhancer trap lines were used, D120 (inserted in scabrous), BB02, ro156, X63 (inserted in rhomboid), P82, AE127 (inserted in seven-up), and H214 (inserted in klingon), specifically expressing the β-galactosidase marker in photoreceptor cells (R) of early R8, late R8, late R8, R2/R5/R8, R3/R4/R7, R3/R4/R1/R6, and R7, respectively. The imaginal discs from F1 larva from mating of enhancer trap lines and DREF-expressing transgenic flies were immunohistochemically stained with the anti-β-galactosidase antibody. In ommatidia of DREF-expressing animals, nuclei of early R8, late R8, and R2/R5/R8 demonstrated a similar staining pattern to nuclei of control ommatidia. With AE127, the ommatidia of DREF-expressing progeny were found to contain R3 and R4 nuclei but did not contain R1 or R6 nuclei. In addition, signals for R7 cells were not detected in imaginal discs expressing DREF. The results indicate that expression of DREF inhibits the differentiation of R1, R6, and R7 photoreceptor cells (Hirose, 2001).
Failure of normal cell cycle progression and disturbance of differentiation processes are known to cause apoptosis. For example, it has been reported that overexpression of dE2F and dDP in eye imaginal discs using a GMR promoter induces apoptosis and that this counterbalances cells that enter an abnormal S phase. DREF expression leads to ectopic DNA synthesis and inhibition of differentiation. Therefore, whether overexpression of DREF can induce apoptosis in eye imaginal disc cells was investigated. In wild-type discs of third-instar larvae, there were very few apoptotic cells. In contrast, staining of eye imaginal discs from transgenic flies expressing DREF revealed apoptotic cells to be significantly increased in the region posterior to the MF. Apoptosis seemed to begin in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation into R1/R6 might induce apoptosis (Hirose, 2001).
The rough eye phenotype was suppressed when the transgenic line expressing DREF was crossed with those expressing DIAP1 (Drosophila homologue of the baculovirus inhibitor of apoptosis 1), DIAP2, or baculovirus p35 protein. Horizontal sections of eyes of adult flies also showed that the DREF-induced eye degeneration was at least partially suppressed by expression of p35 proteins. The result indicates that the rough eye phenotype resulted at least partially from ectopic induction of apoptosis (Hirose, 2001).
The transgenic lines expressing DREF in the eye imaginal discs exhibit a rough eye phenotype but normal viability and fertility. Therefore, they can be used as a genetic screen to identify mutations that enhance or suppress the rough eye phenotype. A pilot experiment was performed to test their validity for this purpose by using dE2F mutant flies. dE2F is one of the genes under regulation by DREF. Expression of DREF in the eye imaginal discs activates the dE2F gene promoter, which can be monitored by measuring the expression of β-galactosidase in the eye imaginal disc of a dE2F mutant, dE2F729, in which the lacZ gene had been inserted near the translation initiation site of the dE2F gene in the same orientation as the dE2F gene. It was therefore predicted that a half-dose reduction of dE2F would affect the rough eye phenotype caused by DREF overexpression. Consistent with this prediction, the rough eye phenotype was suppressed when a half-dose reduction of the dE2F was achieved by crossing GMR-GAL4/UAS-DREF flies for loss-of-function mutant flies of dE2F (dE2F91, dE2F729, and dE2F7172) (Hirose, 2001).
Recently, Hart (1999) proposed a novel function of DREF as a modulator of boundary element activity. Therefore, DREF might activate the transcription of DRE-containing genes through interaction with proteins involved in modification of chromatin structure (for example, establishment, maintenance, or cancellation of the chromatin boundary). The Polycomb group proteins are required to preserve the transcriptionally silenced state, whereas the trithorax group genes are needed to perpetuate the transcriptionally active state. The function of these factors is not limited to homeotic gene regulation; rather, they are involved in the control of diverse developmental processes. Several observations suggest that they change the chromatin structure, establishing a configuration that is either permissive or nonpermissive for transcription. The effects of mutations in Polycomb or trithorax group genes on the rough eye phenotype induced by expression of DREF was examined. A half-dose reduction of the trithorax group genes brahma (brm), osa, and moira (mor) significantly suppressed the rough eye phenotype. Mutation of enhancer of Polycomb [E(Pc)] weakly suppressed the rough eye phenotype. Genetic crossing of the DREF-expressing strain with Dll9, a hypomorphic allele, resulted in a weakly enhanced rough eye phenotype and crossing with Dll5 resulted in a severe small eye phenotype. Mutations in other trithoax and Polycomb group genes tested, including Polycomb (Pc), Polycomb-like (Pcl), suppressor of zesta [Su(z)], Posterior sex combs (Psc), multiple wing hair (mwh), super sex comb (sxc), trithorax (trx), and kohtalo (kto), had no effect on the GMR-GAL4; UAS-DREF phenotype, suggesting that the effect is specific to only certain members of the trithorax class of genes (Hirose, 2001).
brm is the Drosophila homologue of the yeast SWI2/SNF2 gene, and the BRM complex containing OSA and MOR is an essential coactivator for the trithorax group protein Zeste. Therefore, suppression of the DREF-induced rough eye phenotype by a half-dose reduction of some members of the BRM complex genes suggests that it may contribute to the regulation of DREF activity (Hirose, 2001).
A collection of Drosophila deficiency stocks was used to cross with the transgenic flies expressing DREF, and the eye morphology of their F1 progeny was compared with that of F1 progeny between transgenic flies and Canton S. A total of 132 deficient lines were examined, permitting a screen of approximately 61% of the euchromatic region. To determine whether these deletions specifically modify the rough eye phenotype induced by DREF overexpression, crosses were made with transgenic flies having a rough eye phenotype induced by overexpression of human p53. Expression of p53 inhibited cell cycle progression in the S-phase zone behind the MF and induced extensive apoptosis, but photoreceptor cell differentiation appeared to be normal. Apoptosis is a phenomenon induced in common by p53 and DREF overexpression. Therefore, if a dominant enhancer (or suppressor) of the p53-induced rough eye phenotype also enhances (or suppresses) the DREF-induced rough eye phenotype, the deletion might contain a gene involved in the apoptotic pathway. In fact, nine deletions were identified for dominant enhancers (21D2-3;21F2-22A1, 34F4;35D7, 37B2-12;38D2-5, 37C2-5;38B2-38C1, 41A, 43E6;44B6, 48A;48B, 57B4;58B, and 87B11-13;87E8-11) and one for a dominant suppressor (75B3-6;75C) of both DREF- and p53-induced rough eyes. Furthermore dominant modifiers specific to the DREF function could be determined. Ten lines specifically suppressed the rough eye phenotype induced by DREF expression, and 11 lines demonstrated specific enhancement. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators (Hirose, 2001).
DNA replication-related element binding factor (DREF) has been suggested to be involved in regulation of DNA replication- and proliferation-related genes in Drosophila. While the effects on the mutation in the DNA replication-related element (DRE) in cultured cells have been studied extensively, the consequences of elevating wild-type DREF activity in developing tissues have hitherto remained unclear. DREF was over-expressed in the wing imaginal disc using a GAL4-UAS targeted expression system in Drosophila. Over-expression of DREF induced a notching wing phenotype, which was associated with ectopic apoptosis. A half reduction of the reaper, head involution defective and grim gene dose suppressed this DREF-induced notching wing phenotype. Furthermore, this was also the case with co-expression of baculovirus P35, a caspase inhibitor. In addition, over-expression of the 32 kDa boundary element-associated factor (BEAF-32), thought to compete against DREF for common binding sites in genomic regions, rescued the DREF-induced notching wing phenotype, while a half reduction of the genomic region, including the BEAF-32 gene, exerted enhancing effects. This is the first evidence for a genetic interaction between DREF and BEAF-32. It is concluded that the DREF-induced notching wing phenotype is caused by induction of apoptosis in the Drosophila wing imaginal disc (Yoshida, 2001).
Based on overexpression studies and target gene analyses, the transcription factor DNA replication-related element factor (DREF) has been proposed to regulate growth and replication in Drosophila. This study presents loss-of-function experiments to analyze the contribution of DREF to these processes. RNA interference-mediated extinction of DREF function in vivo demonstrates a requirement for the protein for normal progression through the cell cycle and consequently for growth of imaginal discs and the derived adult organs. DREF regulates the expression of genes that are required for the transition of imaginal disc cells through S phase. In conditions of suppressed apoptosis, DREF activation can cause overgrowth of developing organs. These data establish DREF as a global regulator of transcriptional programs that mediate cell proliferation and organ growth during animal development (Hyun, 2005).
The lack of loss-of-function alleles for Dref has hampered the functional characterization of this gene and the analysis of its contribution to normal tissue growth. To overcome this limitation, transgenic fly lines were generated in which an inverted repeat of the Dref transcript can be expressed under the control of a yeast Gal4 upstream activating sequence (UAS DREFRNAi). In these flies, DREF function can be ablated by RNAi in a spatially and temporally controlled manner using the Gal4/UAS system. Two independent RNAi lines were analyzed by semiquantitative reverse transcriptase PCR (RT-PCR). When RNAi expression was directed ubiquitously under the control of T80Gal4, both RNAi lines showed significantly decreased levels of endogenous Dref mRNA, with the construct inserted on the X chromosome causing a stronger suppression than the one residing on the third chromosome. The stronger X-linked RNAi construct was used in mos experiments described in this study (Hyun, 2005).
The efficiency of spatially restricted DREF knockdown was studied using in situ hybridization with a Dref-specific probe that does not overlap with the double-stranded RNA construct. In the third instar larval wing imaginal disc of wild-type animals, Dref mRNA is uniformly distributed. However, when DREFRNAi was expressed in the posterior compartment using the engrailed Gal4 (enGal4) driver, Dref mRNA levels were markedly decreased in this region (Hyun, 2005).
Overexpression of DREF in the posterior compartment of the wing resulted in developmental defects. Similarly, loss of DREF function, brought about by DREFRNAi expression at high levels (from the X-linked transgene), severely disrupted normal wing development. These two effects neutralized each other, and the wing developed normally when DREFRNAi was coexpressed with DREF. This result indicates that the phenotype elicited by the RNAi construct was caused by a specific decrease of Dref mRNA. Consistent results were observed in the eye, where DREFRNAi could suppress the aberrant eye phenotype elicited by DREF overexpression (Hyun, 2005).
To test whether DREF is required for normal organ growth, the consequences were studied of DREF knockdown in the developing wing and eye. In the fly, the effect of transgenes on tissue growth can be conveniently assessed by overexpressing them under the control of enGal4 in the posterior compartment of the wing. In such a setting, the size of the anterior compartment serves as an internal wild-type control. A Drosophila line carrying the moderately expressing DREFRNAi transgene on the third chromosome was analyzed to assess wing disc growth in a DREF loss-of-function situation. Using this allele, the massive growth and developmental defects observed in wings in which DREF function was ablated more dramatically was avoided. Such conditions of limited knockdown of DREF expression did not affect patterning, but growth of the posterior compartment was significantly reduced, indicating that wild-type levels of DREF are critically required for normal tissue growth. The observed reduction in wing size correlates with a smaller cell size in the posterior compartment, as revealed by a higher density of trichomes in the area of DREFRNAi expression. At higher levels of DREFRNAi expression, more severe phenotypes manifested themselves, possibly including patterning defects. This is consistent with recent reports that implicate DREF in mitogen-associated protein kinase-dependent vein differentiation (Yoshida, 2004). Whether such aberrant patterning phenotypes are a primary consequence of DREF deficiency or an indirect effect of growth defects cannot be judged based on the evidence presently available (Hyun, 2005).
The expression pattern of Dref in the developing Drosophila eye imaginal disc is consistent with its proposed predominant function in cell proliferation. Dref mRNA is expressed at high levels in the dividing and growing cells of the eye imaginal disc, which are located anterior to the morphogenetic furrow (MF). The MF consists of cells that have arrested in G1 phase of the cell cycle in a coordinated fashion. Posterior to the MF, some cells become determined and differentiate into photoreceptors, while others undergo one more cell division and are thus part of the 'second mitotic wave.' DREF expression is low posterior to the second mitotic wave, suggesting that it is not required for normal photoreceptor differentiation. Consistent with this notion, expression of DREFRNAi in differentiating cells of the eye did not interfere with normal eye development. However, when expressed in the whole-eye imaginal disc, including areas of active cell proliferation, DREFRNAi induced drastically aberrant phenotypes. These ranged from small, rough eyes to the complete loss of the organ. It is concluded that DREF function is required for normal growth and cell proliferation in the eye but does not contribute significantly to the patterning and differentiation processes that shape the eye after cell proliferation has ceased (Hyun, 2005).
To observe the effect of DREF in cell proliferation directly, random EGFP-marked clones of cells expressing DREFRNAi were generated in third instar wing imaginal discs. Such clones were significantly smaller and less abundant than control clones expressing only EGFP. Furthermore, clones that were generated at earlier stages of larval development almost never survived through the third instar larval stage, while control clones were found abundantly. These results suggest that cell clones expressing DREFRNAi had a growth disadvantage and were eliminated in the course of wing disc growth (Hyun, 2005).
To investigate whether the requirement of DREF for organ growth might reflect a function in cell cycle regulation, the cell cycle profile wad assessed of wing imaginal disc cells in which DREF was knocked down. To this end, DREFRNAi expression was induced ubiquitously in third instar larvae using the TS-Gal80 TARGET system. At various time points after DREFRNAi induction, the cell cycle distribution was analyzed of dissociated wing disc cells using fluorescence-activated cell sorter (FACS) analysis. While the cell cycle profile did not change in control cells, among cells expressing DREFRNAi, the cell population residing in the G2 phase of the cell cycle was progressively lost over a time course of 16 h. This result suggests that DREF function is required for cells to progress through late G1 phase or S phase efficiently (Hyun, 2005).
Cell size as measured by forward light scatter supports the notion that the predominant consequence of DREF abrogation in mitotic cells is a defect in cell cycle regulation rather than growth. Two genotypes wee used to assess the effect of DREF suppression on cell size. First, DREFRNAi was expressed along with a GFP marker under the control of enGal4 in the posterior part of wing imaginal discs. GFP-positive G1/S cells of this genotype were larger than control cells in which GFP was expressed alone. The size of the anterior cells, in which enGal4 is not active, was not affected. To rule out possible differentiation or developmental effects of long-term DREF suppression as a cause for the change in the size of the G1/S cell population, wing imaginal disc cells were also examined in which DREFRNAi was expressed for just 16 h using the inducible TARGET system. Again, cells in which DREF activity was thus suppressed shifted to a larger size compared to control cells. These findings suggest that the inefficient progression into or through S phase is not a result of cells not reaching a critical size threshold. It rather seems that cells lacking DREF activity accumulate in G1 and continue to grow to a bigger size than cells in a wild-type disc. A requirement for DREF for cell cycle regulation is also supported by the identity of its target genes (Hyun, 2005).
A study was performed to see whether DREF-regulated gene expression might account for the cell cycle effects described above. This possibility is supported by the prevalence of DRE sites in the 5' region of genes involved in cell growth and proliferation. To analyze potential DREF-inducible changes in expression of such potential DREF targets, either wild-type DREF or DREFRNAi was ubiquitously expressed in larvae and semiquantitative RT-PCR analysis was performed. Consistent with previous observations, overexpression of wild-type DREF increased mRNA levels of genes that are known to promote G1-S transition and that are required for S phase, including cyclin E, cyclin A, dE2F1, myb, the DNA polymerase alpha gene, and PCNA. The same genes were down-regulated in loss-of-function conditions for DREF. These studies identified the orc2 gene (origin recognition complex subunit 2) as a novel DREF target. Its 5' promoter region was found to bear three putative DREF binding sites (48 bp before the start codon), and RT-PCR results showed that overexpression of DREF increased orc2 transcript levels and loss of DREF reduced them in vivo. These results demonstrate that DREF is sufficient and required to induce the expression of genes involved in S-phase progression in vivo and suggest that the absence of the DREF-induced gene expression program is the cause for the reduced size of DREFRNAi-expressing tissues. To test whether DREF would specifically be required for S phase or might also affect other stages of the cell cycle, the RNA levels of cyclin B and string (the Drosophila homolog of cdc25), as representative regulators of the G2-M transition, were measured in DREF gain- and loss-of-function conditions. As opposed to the effect on S-phase genes, gain of DREF function did not result in up-regulation of these mitotic genes, indicating that their expression is not controlled by the transcription factor. This is consistent with the absence of recognizable DREF binding motifs in the respective promoter regions. cyclin B and string expression levels were modestly reduced in the DREF knockdown background. This effect is most likely indirect and explained by the smaller fraction of cells that reach the G2-M phase in conditions of reduced DREF activity (Hyun, 2005).
Not all tested genes preceded by putative DREF binding sites were induced in response to DREF expression in vivo. For example, the helicase gene hlc carries three DREs in its upstream region (37 bp before its start site), but RT-PCR data showed no changes in its mRNA levels with either DREF or DREFRNAi overexpression. This finding suggests that for some genes DREF may not be sufficient, and additional inputs might be required for their transcriptional activation. Alternatively, it is possible that not all computationally identified DREF binding sites in the promoter regions are functional in the cells tested. It seems clear that DREF overexpression does not cause an indiscriminate and global activation of transcription (Hyun, 2005).
The results indicate that DREF directs a gene expression program that should promote cell proliferation in developing imaginal discs and increase organ size. Accordingly, gain-of-function situations for DREF may be expected to result in tissue overgrowth. It has been difficult to directly test this hypothesis, since DREF overexpression results in an increase in cell cycle markers in the developing disc that is accompanied by widespread apoptosis. The resulting adult organ thus typically does not show overgrowth. It was reasoned that higher than wild-type levels of DREF activity might cause problems during replication and result in cell death that is initiated by common cell cycle checkpoints and developmental safeguards (Hyun, 2005).
Whether a DREF-driven growth program would become apparent in conditions of suppressed apoptosis was examined. Thus, genetic interaction experiments were conducted by crossing flies in which DREF was overexpressed in cells of the developing eye imaginal disc with flies carrying a homozygous viable loss-of-function allele of the proapoptotic gene head involution defective, hid(W1). In agreement with the hypothesis, eyes expressing DREF in hid mutant backgrounds grew larger than eyes in control animals. In addition to increased eye circumference, DREF-overexpressing eyes frequently displayed bulged-out areas of overgrowth when both copies of wild-type hid were eliminated. These data demonstrate that, consistent with its molecular targets and its effects on cells, DREF overexpression is sufficient to promote tissue growth during larval development (Hyun, 2005).
The DNA replication-related element binding factor (DREF) has been suggested as being involved in regulation of DNA replication- and proliferation-related genes in Drosophila. Recently, by searching the Drosophila genome database, DRE-like sequences were also found in the 5'-flanking regions of many genes with other functions. In addition, immunostaining of polytene chromosomes with an anti-DREF monoclonal antibody revealed that DREF can bind to a hundred regions of polytene chromosomes, suggesting regulation of multiple genes and multiple roles in vivo. When DREF protein or inverted repeat RNA of the DREF gene were over-expressed in wing imaginal discs using the GAL4-UAS targeted expression system in Drosophila, the results were veins of increased width and a loss of veins, respectively. With DREF over-expression, Rolled, a Drosophila MAPK homologue, was ectopically activated. Furthermore, half reduction of the D-raf gene dose suppressed this DREF-induced vein of increased width phenotype. In addition, when DREF transcripts were reduced by introducing double-stranded RNA of the DREF gene into S2 cells, the D-raf gene promoter activity was diminished to 4%. These data indicate that DREF is involved in regulation of vein formation through the activation of EGFR signalling in the Drosophila wing imaginal discs (Yoshida, 2004).
Brody, T., Stivers, C., Nagle, J. and Odenwald, W. F. (2002). Identification of novel Drosophila neural precursor genes using a differential embryonic head cDNA screen. Mech. Dev. 113(1): 41-59. 11900973
Choi, T., et al. (2000). The DNA replication-related element (DRE)-DRE-binding factor (DREF) system may be involved in the expression of the Drosophila melanogaster TBP gene. FEBS Lett. 483(1):71-7. 11033359
Choi, T. Y., et al. (2004). Redox regulation of DNA binding activity of DREF (DNA replication-related element binding factor) in Drosophila. Biochem. J. 378(Pt 3): 833-8. 14651474
Choi, Y.-J., Choi, T.-Y., Yamaguchi, M., Matsukage, A., Kim, Y.-S., Yoo, M.-A. (2004). Transcriptional regulation of the Drosophila caudal homeobox gene by DRE/DREF. Nucleic Acids Res 32: 3734-3742. 15254275
Hart, C. M., Zhao, K. and Laemmli, U. K. (1997). The scs' boundary element: characterization of boundary element-associated factors. Mol. Cell. Biol. 17(2): 999-1009. 9001253
Hart, C. M., Cuvier, O. and Laemmli, U. K. (1999). Evidence for an antagonistic relationship between the boundary element-associated factor BEAF and the transcription factor DREF. Chromosoma 108(6): 375-83. 10591997
Hayashi, Y., et al. (1997). Identification of CFDD (common regulatory factor for DNA replication and DREF genes) and role of its binding site in regulation of the proliferating cell nuclear antigen gene promoter. J. Biol. Chem. 272(36):22848-58. 9278447
Hayashi, Y., Kato, M., Seto, H. and Yamaguchi, M. (2006). Drosophila distal-less negatively regulates dDREF by inhibiting its DNA binding activity. Biochim. Biophys. Acta. 1759(7): 359-66. 16949685
Hirose, F., et al. (1993). Novel 8-base-pair sequence (Drosophila DNA replication-related element) and specific binding factor involved in the expression of Drosophila genes for DNA polymerase and proliferating cell nuclear antigen. J. Biol. Chem. 268: 2092-2099. 8093616
Hirose, F., Yamaguchi, M. and Matsukage, A. (1994). Repression of regulatory factor for Drosophila DNA replication-related gene promoters by zerknüllt homeodomain protein. J. Biol. Chem. 269: 2937-42. 7905482
Hirose, F., et al. (1996). Isolation and characterization of cDNA for DREF, a promoter-activating factor for Drosophila DNA replication-related genes. J. Biol. Chem. 271: 3930-3937. 8632015
Hirose, F., Yamaguchi, M. and Matsukage, A. (1999). 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. Mol. Cell. Biol. 19(9): 6020-8. 10454549
Hirose, F., Ohshima, N., Shiraki, M., Inoue, Y. H., Taguchi, O., Nishi, Y. Matsukage, A. and Yamaguchi, M. (2001). 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. Mol. Cell. Biol. 21(21): 7231-42. 11585906
Hirose, F., Ohshima, N., Kwon, E.-J., Yoshida, H., Yamaguchi, M. (2002). Drosophila Mi-2 negatively regulates dDREF by inhibiting its DNA-binding activity. Mol. Cell. Biol. 22: 5182-5193. 12077345
Hochheimer, A., et al. (2002). TRF2 associates with DREF and directs promoter-selective gene expression in Drosophila. Nature 420(6914): 439-45. 12459787
Hyun, J., Jasper, H., Bohmann, D. (2005). DREF is required for efficient growth and cell cycle progression in Drosophila imaginal discs. Mol. Cell. Biol. 25: 5590-5598. 15964814
Jasper, H., et al. (2002). A genomic switch at the transition from cell proliferation to terminal differentiation in the Drosophila eye. Dev. Cell 3(4): 511-21. 12408803
Jin, J. S., et al. (2004). A DNA replication-related element downstream from the initiation site of Drosophila selenophosphate synthetase 2 gene is essential for its transcription. Nucleic Acids Res. 32(8): 2482-93. 15121905
Kwon, E., et al. (2003). Transcription control of a gene for Drosophila transcription factor, DREF by DRE and cis-elements conserved between Drosophila melanogaster and virilis. Gene 309(2): 101-16. 12758126
Kwon, E., et al. (2004). Armadillo/Pangolin regulates PCNA and DREF promoter activities. Biochim. Biophys. Acta 1679(3): 256-62. 15358517
Lefai, E., Fernandez-Moreno, M. A., Alahari, A., Kaguni, L. S. and Garesse, R. (2000). Differential regulation of the catalytic and accessory subunit genes of Drosophila mitochondrial DNA polymerase. J. Biol. Chem. 275(42): 33123-33. 10930405
Martin-Lanneree, S., et al. (2006). Characterization of the Drosophila myeloid leukemia factor. Genes Cells. 11(12): 1317-1335. 17121541
Masson, P., Lundgren, J. and Young, P. (2003). Drosophila proteasome regulator REGgamma: transcriptional activation by DNA replication-related factor DREF and evidence for a role in cell cycle progression. J. Mol. Biol. 327(5): 1001-12. 12662925
Matsukage, A., et al. (1995). The DRE sequence TATCGATA, a putative promoter-activating element for Drosophila melanogaster cell-proliferation-related genes. Gene 166: 233-236. 8543167
Ohno, K., et al. (1996). Transcriptional regulation of the Drosophila CycA gene by the DNA replication-related element (DRE) and DRE binding factor (DREF). Nucleic Acids Res. 24(20): 3942-3946.
Ohno, K., et al. (2000). Characterization of a Drosophila homologue of the human myelodysplasia/myeloid leukemia factor (MLF). Gene 260(1-2): 133-43. 11137299
Ohshima, N., Takahashi, M., Hirose, F. (2003). Identification of a Human Homologue of the DREF Transcription Factor with a Potential Role in Regulation of the Histone H1 Gene. J. Biol. Chem. 278: 22928-22938. 12663651
Okudaira, K., et al. (2005). Transcriptional regulation of the Drosophila orc2 gene by the DREF pathway. Biochim. Biophys. Acta 1732(1-3): 23-30. 16343659
Papai, G., et al. (2005). Intimate relationship between the genes of two transcriptional coactivators, ADA2a and PIMT, of Drosophila. Gene 348: 13-23. 15777699
Park, S. Y., Kim, Y.-S., Yang, D.-J., Yoo, M.-A. (2004). Transcriptional regulation of the Drosophila catalase gene by the DRE/DREF system. Nucleic Acids Res 32: 1318-1324. 14982956
Phuong Thao, D. T., Ida, H., Yoshida, H. and Yamaguchi, M. (2006). Identification of the Drosophila skpA gene as a novel target of the transcription factor DREF. Exp. Cell Res. 312(18): 3641-50. 16962096
Ruiz De Mena, I., Lefai, E., Garesse, R. and Kaguni, L. S. (2000). Regulation of mitochondrial single-stranded DNA-binding protein gene expression links nuclear and mitochondrial DNA replication in Drosophila. J. Biol. Chem. 275(18): 13628-36. 10788480
Ryu, J. R., et al. (1997). Transcriptional regulation of the Drosophila-raf proto-oncogene by the DNA replication-related element (DRE)/DRE-binding factor (DREF) system. Nucleic Acids Res. 25(4): 794-799.
Sawado, T., et al. (1998). The DNA replication-related element (DRE)/DRE-binding factor system is a transcriptional regulator of the Drosophila E2F gene. J. Biol. Chem. 273(40): 26042-51. 9748283
Seto, H., Hayashi, Y., Kwon, E., Taguchi, O. and Yamaguchi, M. (2006). Antagonistic regulation of the Drosophila PCNA gene promoter by DREF and Cut. Genes Cells. 11(5): 499-512. 16629902
Sharkov, N. V., Ramsay, G. and Katzen, A. L. (2002). The DNA replication-related element-binding factor (DREF) is a transcriptional regulator of the Drosophila myb gene. Gene 297(1-2): 209-19. 12384302
Takahashi, Y., et al. (1996). DNA replication-related elements cooperate to enhance promoter activity of the drosophila DNA polymerase alpha 73-kDa subunit gene. J. Biol. Chem. 271(24): 14541-7. 8662923
Takahashi, Y., Hirose, F., Matsukage, A. and Yamaguchi, M. (1999). Identification of three conserved regions in the DREF transcription factors from Drosophila melanogaster and Drosophila virilis. Nucleic Acids Res. 27(2): 510-6. 9862973
Takata, K., et al. (2001). Drosophila mitochondrial transcription factor A: characterization of its cDNA and expression pattern during development. Biochem. Biophys. Res. Commun. 287(2): 474-83. 11554753
Takata, K., Ishikawa, G., Hirose, F. and Sakaguchi, K. (2002). Drosophila damage-specific DNA-binding protein 1 (D-DDB1) is controlled by the DRE/DREF system. Nucleic Acids Res. 30(17): 3795-808. 12202765
Yamaguchi, M., et al. (1995a). Expression patterns of DNA replication enzymes and the regulatory factor DREF during Drosophila development analyzed with specific antibodies. Biol. Cell 85(2-3): 147-55. 8785516
Yamaguchi, M., et al., (1995b). A nucleotide sequence essential for the function of DRE, a common promoter element for Drosophila DNA replication-related genes. J. Biol. Chem. 270: 15808-15814. 7797583
Yamaguchi, M., Hirose, F. and Matsukage, A. (1996). Roles of multiple promoter elements of the proliferating cell nuclear antigen gene during Drosophila development. Genes Cells 1: 47-58. 9078366
Thao, D. T. P., et al. (2006). Identification of the Drosophila skpA gene as a novel target of the transcription factor DREF. Exp. Cell Res. 312: 3641-3650. Medline abstract: 16962096
Yoshida, H., et al. (2001). Over-expression of DREF in the Drosophila wing imaginal disc induces apoptosis and a notching wing phenotype. Genes Cells 6(10): 877-86. 11683916
Yoshida, H., Kwon, E., Hirose, F., Otsuki, K., Yamada, M., Yamaguchi, M. (2004). DREF is required for EGFR signalling during Drosophila wing vein development. Genes Cells 9: 935-944. 15461664
date revised: 25 May 2007
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