DREF has been revealed to be an important transcription factor for activating promoters of cell proliferation and differentiation related genes. The amino acid sequences of DREF are conserved in evolutionary separate Drosophila species, Drosophila melanogaster (Dm) and Drosophila virilis (Dv) in three regions. In the present study, evidence was obtained that there are several highly conserved regions in the 5' flanking region between the DmDREF and DvDREF genes. Band mobility shift assays using oligonucleotides corresponding to these conserved regions revealed that specific trans-acting factors can bind to at least three regions -554 to -543 (5'-TTTGTTCTTGCG), -81 to -70 (5'-GCCCACGTGGCT) and +225 to +234 (5'-GCAATCAGTG). Using a transient luciferase expression assay, the region -554 to -543 was shown to function as a negative regulatory element for DmDREF promoter activity, while the regions -77 to -70 (5'-ACGTGGCT) and +225 to +236 (5'-GCAATCAGTGTT) function as positive regulatory elements. Expression of the homeodomain protein Zerknullt (Zen) has been shown to repress PCNA gene transcription, by reducing the DNA binding activity of DREF. This study shows that Zen downregulates DREF gene promoter activity through action on the region between +241 and +254 (5'-AGAATACTCAACA). In addition, the DmDREF promoter contains five DREs. Using a double stranded RNA-mediated interference method, evidence was generated that expression of DmDREF could be auto-regulated by DREF through the third DRE located at +211 to +218. In living flies results were obtained consistent with those obtained in vitro and in cultured cells. The study thus indicates that DmDREF is effectively regulated via highly conserved regions between the DmDREF and DvDREF promoters, suggesting the existence of common regulatory factors, and that DmDREF can be positively regulated by itself via the third DRE located in its most highly conserved region (Kwon, 2003).
Armadillo and Pangolin (dTCF), downstream effectors of the Wingless (Wg) signal transduction pathway, activate transcription of the important DNA replication-related genes encoding Drosophila PCNA and DREF. By transient luciferase expression assays and band mobility shift assays, it has been demonstrated that the PCNA gene is a direct target gene for the Armadillo/Pangolin complex. Using a GAL4-UAS system, stimulation of the PCNA gene by Armadillo/Pangolin was confirmed in adult females. From the published reports of an inhibitory role, it was expected that Drosophila CREB-binding protein (dCBP) would interfere with activation. However, effects were only observed with the DREF but not the PCNA gene. In the latter case, as in mammals, dCBP can potentiate Armadillo-mediated activation. These results suggest that first, PCNA and DREF genes are targets of the Armadillo/Pangolin complex and second, dCBP modulates Wg signaling in a gene-specific manner (Kwon, 2004).
The Drosophila DNA polymerase alpha gene is repressed by Zerknüllt. The expression of zen results in reduction of the abundance of mRNA, both DNA polymerase alpha and PCNA. A positive cis-acting element found in both DNA polymerase alpha and PCNA genes is responsible for repression by ZEN protein or downstream of ZEN action. The nuclear extract of tissue culture cells transfected by a zen-expressing plasmid contains lesser amounts of a DNA replication-binding factor (DREF) than that of untransfected or mutant zen-transfected cells (Hirose, 1994).
Antibodies against DREF specifically inhibit the transcription of the DNA polymerase alpha promoter in vitro. Overproduction of DREF protein overcomes the repression of the proliferating cell nuclear antigen gene promoter by the zerknüllt gene product. DREF is a trans-activating factor for DNA replication-related genes. DREF polypeptide is present in nuclei after the eighth nuclear division cycle, suggesting that nuclear accumulation of DREF is important for the coordinate zygotic expression of DNA replication-related genes carrying DRE sequences (Hirose, 1996).
The Drosophila gene for cyclin A is expressed in dividing cells throughout development. This expression pattern is similar to that of genes related to DNA replication, suggesting involvement of some common control mechanism(s). In the upstream region (-71 to -64 with respect to the transcription initiation site) of the CycA gene, a sequence was found that is identical to the DNA replication-related element (DRE; 5'-TATCGATA), which is important for high level expression of replication-related genes such as those encoding DNA polymerase alpha and proliferating cell nuclear antigen. Deletion or base substitution mutations result in an extensive reduction in Cyclin A expression. Monoclonal antibodies against DRE binding factor (DREF) diminish or supershift the complex of the DREF and the DRE-containing fragment. The results indicate that the Drosophila CycA gene is under the control of a DRE/DREF system, as are DNA replication-related genes (Ohno, 1996).
An analysis was carried out on the promoter region of the Drosophila DNA polymerase alpha 73-kDa subunit gene and the factor(s) activating the promoter. Transcription initiation sites were newly identified in the region downstream of the previously determined sites. Full promoter activity resides within the region from -285 to +129 base pairs with respect to the newly determined major site. Within this region, three sequences were found identical or similar to the DNA replication-related element (DRE), 5'-TATCGATA, which is known as a common promoter-activating element for the Drosophila DNA polymerase alpha 180-kDa subunit gene and the proliferating cell nuclear antigen gene. These sites were located at positions -77 to -70 (DREalpha-I), -44 to -37 (DREalpha-II), and +3 to +10 (DREalpha-III). Footprinting analysis using the recombinant DRE-binding factor (DREF) or Kc cell nuclear extract demonstrated that DREF can bind to all three DRE-related sites. Introduction of mutation in even one of the three DRE-related sequences caused extensive reductions of the promoter activity and also the DREF-binding activity of the promoter-containing fragment. The results indicate that the three DREF-binding sites cooperate to enhance promoter activity of the DNA polymerase alpha 73-kDa subunit gene (Takahashi, 1996).
Promoter regions of the Drosophila proliferating cell nuclear antigen (PCNA) gene and the DNA polymerase alpha 180-kDa catalytic subunit gene contain a common 8 base pair (bp) promoter element, 5'-TATCGATA (DRE, Drosophila DNA replication-related element). Various base substitutions and internal deletions were generated in and around DRE (nucleotide positions -93 to -100 with respect to the transcription initiation site) of the PCNA gene in vitro and their effects were subsequently examined on the binding to DREF (DRE-binding factor) and PCNA gene promote activity in cultured Drosophila Kc cells as well as in living flies. Gel mobility shift assays using nuclear extracts of Kc cells with and without competitor DNA fragments carrying the mutations indicated that the 10-bp sequence from positions -91 to -100 is essential for complex formation with DREF. Transient expression assays of chloramphenicol acetyl-transferase (CAT) in Kc cells transfected with PCNA promoter-CAT fusion genes carrying the mutations revealed that the 8-bp sequence from -93 to -100 is essential for activation of the promoter in Kc cells. Examination of lacZ expression from PCNA promoter-lacZ fusion genes carrying the mutations, introduced into flies by germ-line transformation, revealed that the 8-bp sequence is also important for DRE function during development. However, two exceptional mutations were obtained in the 8-bp sequence that did not or only marginally affected the PCNA gene promoter activity in transgenic flies. Both of these mutations effectively reduced the promoter activity in CAT transient expression assay in Kc cells and the binding to DREF in vitro. Therefore, the 8-bp sequence requirement for DRE function appears to be less stringent in living flies than in the cultured cell or in vitro cases (Yamaguchi, 1995b).
The expression of genes involved in DNA replication is closely correlated with the proliferating state of cells and is repressed with the progression of differentiation during development. Promoter regions of the Drosophila proliferating cell nuclear antigen (PCNA) gene and the DNA polymerase alpha gene contain a common 8-base pair promoter element (DRE: DNA replication-related element). The examination of a common expression mechanism for DNA replication-related genes, which is regulated positively by growth signals and negatively by differentiation signals would be of interest. PCNA-LacZ fusion genes were generated in which the 5'-flanking sequence of the PCNA gene has been mutated. An examination of the expression of these fusion genes, introduced into flies by germ-line transformation, led to the identification of another distinct regulatory element, URE (upstream regulatory element), within the region from -168 to -119 with respect to the transcription initiation site. During embryogenesis, the region containing the DRE sequence (-108 to -91) greatly stimulated the PCNA gene minimal promoter (-86 to +130), when it was placed upstream of the promoter in both normal and reverse orientations. Addition of the URE sequence further stimulated the promoter activity twofold. During larval stages, both DRE and URE were indispensable to the promoter activity, since neither of the sequences alone activated the minimal promoter. Demonstration of beta-galactosidase activity indicated URE plays an essential role in various larval tissues such as salivary gland and imaginal disc. While the minimal promoter region alone directed maternal expression of lacZ in ovaries of adult females, both DRE and URE further stimulated promoter activity. These results show several elements of the PCNA gene promoter play roles during Drosophila development (Yamaguchi, 1996).
The Drosophila proliferating cell nuclear antigen (PCNA) gene promoter contains at least three transcriptional regulatory elements, the URE (upstream regulatory element), DRE (DNA replication-related element), and E2F recognition sites. In nuclear extracts of Drosophila Kc cells, a novel protein factor(s), CFDD (common regulatory factor for DNA replication and DREF genes) was identified that appeared to recognize two unique nucleotide sequences (5'-CGATA and 5'-CAATCA) and bind to three sites in the PCNA gene promoter. These sites were located at positions -84 to -77 (site 1), -100 to -93 (site 2) and -134 to -127 (site 3) with respect to the transcription initiation sites. Sites 2 and 3 overlapped with DRE and URE, respectively, and the 5'-CGATA matched with the reported recognition sequence of BEAF-32 (boundary element-associated factor of 32 kDa). Detailed analyses of CFDD recognition sequences and experiments with specific antibodies to DREF (DRE-binding factor) and BEAF-32 suggest that CFDD is different from DREF, UREF (URE-binding factor) and BEAF-32. A UV cross-linking experiment revealed that polypeptides of approximately 76 kDa in the nuclear extract interact directly with the CFDD site 1 sequence. Transient expression assays of chloramphenicol acetyltransferase (CAT) in Kc cells transfected with PCNA promoter-CAT fusion genes carrying mutations in CFDD site 1 and examination of lacZ expression from PCNA promoter-lacZ fusion genes carrying mutations in site 1, introduced into flies by germ line transformation, revealed that CFDD site 1 plays an important role for the promoter activity both in cultured cells and in living flies. In addition to the PCNA gene, multiple CFDD sites were found in promoters of the DNA polymerase alpha and DREF genes, and CFDD binding to the DREF promoter was confirmed. Therefore, CFDD may play important roles in regulation of Drosophila DNA replication-related genes (Hayashi, 1997).
Upstream regions containing a novel common 8-base pair (bp) palindromic sequence, 5'-TATCGATA (Drosophila DNA replication-related element (DRE)), are required for the high expression of Drosophila genes for DNA polymerase alpha and PCNA. Three DREs and one DRE are present in the DNA polymerase alpha gene (nucleotides-217, -83, and -30 with respect to the transcription initiation site) and in the PCNA gene (nucleotide-100), respectively. Deletions or 2-bp insertional mutations of DRE sequences led to an extensive reduction of promoter activities of both genes. Chemically synthesized oligonucleotides containing DRE sequences greatly stimulated the activity of the heterologous promoter of the Drosophila metallothionein gene, in addition to the promoter of the PCNA gene, when they were placed upstream from these promoters in a normal or a reverse orientation. The stimulatory effect increased synergistically and depended on the number of DREs. DRE activated the promoter when placed within 1.4 kilobases upstream from the promoter, but was much less active when placed 2.5 kilobases or more apart from the promoter. Using a gel mobility shift assay method, evidence was obtained for a protein factor (DREF) in the nuclear extract of cultured Drosophila cells (Kc cells), and this factor specifically binds to DREs of both genes. DNase I footprinting analysis indicated that DREF binds to the 24-bp DRE region of the DNA polymerase alpha gene in which 8-bp palindromic sequences are centered. A UV cross-linking experiment revealed that a polypeptide of approximately 90 kDa in the nuclear extract interacts directly with the DRE sequence. Using DRE-conjugated latex particles, DREF was affinity-purified from the Kc cell nuclear extract. By comparing results obtained by SDS-polyacrylamide gel electrophoresis and gel mobility shift experiments, it is concluded that DREF is associated with the 86-kDa polypeptide. On gel filtration chromatography, a single peak of DREF activity was recovered in fractions corresponding to a molecular mass of 170 kDa, and the 86-kDa polypeptide was detected only in the corresponding fractions; thus, active DREF is probably a homodimeric form of the 86-kDa polypeptide. DREF may play important roles in coordinating expressions of Drosophila DNA replication-related genes (Hirose, 1993).
The gene promoter of Drosophila PCNA contains several transcriptional regulatory elements, such as upstream regulatory element (URE), DNA replication-related element (DRE, 5'-TATCGATA), and E2F recognition sites. In the present study, a yeast one-hybrid screen using three tandem repeats of DRE in PCNA promoter was used as the bait allowed isolation of a cDNA encoding Cut, a Drosophila homolog of mammalian CCAAT-displacement protein (CDP)/Cux. Electrophoretic mobility shift assays showed that Cut binds to both DRE and the sequence 5'-AATCAAAC in URE, with much higher affinity to the former. Measurement of PCNA promoter activity by transient luciferase expression assays in Drosophila S2 cells after an RNA interference for Cut or DREF showed DREF activates the PCNA promoter while Cut functions as a repressor. Chromatin immunoprecipitation assays in the presence or absence of 20-hydroxyecdysone further showed both DREF and Cut proteins to be localized in the genomic region containing the PCNA promoter in S2 cells, especially in the Cut case upon induction of differentiation. These results indicate that Cut functions as a transcriptional repressor of PCNA gene by binding to the promoter region in the differentiated state, while DREF binds to DRE to promote expression of PCNA during cell proliferation (Seto, 2006).
The DRE/DREF system plays an important role in transcription of DNA replication genes, such as those encoding the 180 and 73 kDa subunits of DNA polymerase alpha as well as the gene that encodes PCNA. Two sequences were found homologous to DNA replication-related element (DRE; 5'-TATCGATA) in the 5'-flanking region (-370 to -357 with respect to the transcription initiation site) of the D-raf gene. Transcriptional activity was confirmed through gel mobility shift assays, transient CAT assays, and spatial patterns of lacZ expression in transgenic larval tissues carrying D-raf and lacZ fusion genes. The D-raf gene was found to be another target of the Zerknullt (Zen) protein with the observation of D-raf repression by Zen protein in cultured cells and its ectopic expression in the dorsal region of the homozygous zen mutant embryo. The evidence of DRE/DREF involvement in regulation of the D-raf gene strongly supports the idea that the DRE/DREF system is responsible for the coordinated regulation of cell proliferation-related genes in Drosophila (Ryu, 1997).
Two mRNA species were observed for the Drosophila E2F (dE2F) gene, differing with regard to the first exons (exon 1-a and exon 1-b), which were expressed differently during development. A single transcription initiation site for mRNA containing exon 1-b was mapped by primer extension analysis and numbered +1. Three tandemly aligned sequences were found, similar to the DNA replication-related element (DRE; 5'-TATCGATA), which is commonly required for transcription of genes related to DNA replication and cell proliferation, in the region upstream of this site. Band mobility shift analyses using oligonucleotides containing the DRE-related sequences with or without various base substitutions revealed that two out of three DRE-related sequences are especially important for binding to the DRE-binding factor (DREF). On footprinting analysis with Kc cell nuclear extracts and a glutathione S-transferase fusion protein with the N-terminal fragment (1-125 amino acid residues) of DREF, all three DRE-related sequences were found to be protected. Transient luciferase expression assays in Kc cells demonstrated that the region containing the three DRE-related sequences is required for high promoter activity. Transgenic lines of Drosophila were established in which ectopic expression of DREF was targeted to the eye imaginal disc cells. Overexpression of DREF in eye imaginal disc cells enhanced the promoter activity of dE2F. The obtained results indicate that the DRE/DREF system activates transcription of the dE2F gene (Sawado, 1998).
Boundary elements interfere with communication between enhancers and promoters, but only when interposed. Understanding this activity will require identifying the proteins involved. The boundary element-associated factor BEAF is one protein that is implicated in boundary element function. Three genomic fragments (scs', BE76 and BE28) containing BEAF binding sites function as boundary elements in transgenic Drosophila, suggesting that this is an intrinsic property of the numerous genomic regions to which BEAF binds. To characterize additional proteins that interact with boundary elements, a protein was isolated that binds to two of these boundary elements (BE76 and BE28); and it was identified as the transcription factor DREF. Evidence is presented that BEAF and DREF compete for binding to overlapping binding sites, and that this competition occurs in vivo. DREF is believed to regulate genes whose products are involved in DNA replication and cell proliferation, suggesting that the activation of transcription predicted to result from the displacement of BEAF by DREF might be limited to certain rapidly proliferating tissues. This is the first suggestion that the activity of a subset of boundary elements might be regulated (Hart, 1999).
The developmental pattern of expression of the genes encoding the catalytic (alpha) and accessory (beta) subunits of mitochondrial DNA polymerase (pol gamma) has been examined in Drosophila. The steady-state level of pol gamma-beta mRNA increases during the first hours of development, reaching its maximum value at the start of mtDNA replication in Drosophila embryos. In contrast, the steady-state level of pol gamma-alpha mRNA decreases as development proceeds and is low in stages of active mtDNA replication. This difference in mRNA abundance results at least in part from differences in the rates of mRNA synthesis. The pol gamma genes are located in a compact cluster of five genes that contains three promoter regions (P1-P3). The P1 region directs divergent transcription of the pol gamma-beta gene and the adjacent rpII33 gene. P1 contains a DNA replication-related element (DRE) that is essential for pol gamma-beta promoter activity, but not for rpII33 promoter activity in Schneider's cells. A second divergent promoter region (P2) controls the expression of the orc5 and sop2 genes. The P2 region contains two DREs that are essential for orc5 promoter activity, but not for sop2 promoter activity. The expression of the pol gamma-alpha gene is directed by P3, a weak promoter that does not contain DREs. Electrophoretic mobility shift experiments demonstrate that the DRE-binding factor (DREF) regulatory protein binds to the DREs in P1 and P2. DREF regulates the expression of several genes encoding key factors involved in nuclear DNA replication. Its role in controlling the expression of the pol gamma-beta and orc5 genes establishes a common regulatory mechanism linking nuclear and mitochondrial DNA replication. Overall, these results suggest that the accessory subunit of mtDNA polymerase plays an important role in the control of mtDNA replication in Drosophila (Lefai, 2000).
The TATA box binding protein (TBP) is a general transcription factor required for initiation by all three eukaryotic RNA polymerases. The promoter region of the Drosophila melanogaster TBP gene contains three sequences similar to the DNA replication-related element (DRE) (5'-TATCGATA). The DRE-like sequences are also present in the promoter of the Drosophila virilis TBP gene, suggesting a role for these sequences in TBP expression. Band mobility shift assays revealed that oligonucleotides containing sequences similar to the DRE of D. melanogaster TBP gene promoter form specific complexes with a factor in a Kc cell nuclear extract and with recombinant DRE-binding factor (DREF). Furthermore, these complexes were either supershifted or diminished by monoclonal antibodies to DREF. Transient luciferase assays demonstrated that induction of mutations in two DRE-related sequences at positions -223 and -63 resulted in an extensive reduction of promoter activity. Thus, the DRE-DREF system appears to be involved in the expression of the D. melanogaster TBP gene (Choi, 2000).
The structural organization of the Drosophila gene encoding mitochondrial single-stranded DNA-binding protein (mtSSB) has been determined and its pattern of expression evaluated during Drosophila development. The Drosophila mtSSB gene contains four exons and three small introns. The transcriptional initiation site is located 22 nucleotides upstream from the initiator translation codon in adults, whereas several initiation sites are found in embryos. No consensus TATA or CAAT sequences are located at canonical positions, although an AT-rich sequence was identified flanking the major transcriptional initiation site. Northern analyses indicated that the mtSSB transcript is present at variable levels throughout development. In situ hybridization analysis shows that maternally deposited mtSSB mRNA is distributed homogeneously in the early embryo, whereas de novo transcript is produced specifically at an elevated level in the developing midgut. Transfection assays in cultured Schneider cells with promoter region deletion constructs revealed that the proximal 230 nucleotides contain cis-acting elements required for efficient gene expression. Putative transcription factor binding sites clustered within this region include two Drosophila DNA replication-related elements (DRE) and a single putative E2F binding site. Deletion and base substitution mutagenesis of the DRE sites demonstrated that they are required for efficient promoter activity, and gel electrophoretic mobility shift analyses showed that DRE binding factor (DREF) binds to these sites. These data suggest strongly that the Drosophila mtSSB gene is regulated by the DRE/DREF system. This finding represents a first link between nuclear and mitochondrial DNA replication (Ruiz De Mena, 2000).
A cDNA for Drosophila mitochondrial transcription factor A (D-mtTFA) was cloned and the recombinant protein was characterized. In Drosophila Kc cells, D-mtTFA was localized in the mitochondria, but not in the nucleus. By repetitive precipitation with His-tag and PCR amplification, the consensus nucleotide sequence for D-mtTFA-binding was determined to be 5'-TTATC/G. The binding sequence was found to be clustered in the A + T region of mitochondrial DNA which is suggested to be a replication origin and promoter region for light strand and heavy strand. A DNA replication-related element (DRE)-like sequence was found located upstream of the transcription initiation site of the D-mtTFA gene and results were obtained indicating that DRE-binding factor (DREF) can bind to the DRE-like sequence of the D-mtTFA gene. The data suggest that transcription of the D-mtTFA gene is under control of the DRE/DREF regulatory system. Based on these results, the functions of D-mtTFA were discussed in relation to mitochondrial biogenesis of Drosophila (Takata, 2001)
Drosophila melanogaster possesses a single gene, Dm myb, that is closely related to the vertebrate proto-oncogene c-Myb, and its other family members (A-Myb and B-Myb), all of which encode transcription factors. Dm myb is expressed in all proliferating cells throughout development, and previous studies demonstrate that Dm myb promotes both S-phase and M-phase in proliferating cells, while preserving diploidy by suppressing endoreduplication. A characterization of the mechanisms that regulate Dm myb expression has been initiated, and the transcriptional activator DREF was found to activate Dm myb transcription via two binding sites located in the 5' flanking region. The Dm myb promoter lacks a prototypical TATA box sequence and instead appears to use an initiator/downstream promoter element (Inr/DPE) type promoter. Dm myb expression is regulated at the translational as well as transcriptional level (Sharkov, 2002).
DDB1, for a Drosophila homolog of the p127 subunit of the human damage-specific DNA-binding protein, is thought to recognize (6-4) photoproducts and related structures. In Drosophila, the gene product also appeared to play a role as a repair factor. DDB1 knockout Kc cells generated with a RNAi method were sensitive to UV. In addition, UV or methyl methanesulfonate treatment increased DDB1 transcripts. However, it was found that the gene is controlled by the DRE/DREF system, which is generally responsible for activating the promoters of proliferation-related genes. Moreover, during Drosophila development, the transcription of DDB1 changed greatly, with the highest levels in unfertilized eggs, indicating that external injury to DNA is not essential to DDB1 function. Interestingly, as with UV irradiation-induced transfer of DDB1 to the nucleus from the cytoplasm, during spermatogenesis the protein transiently shifted from one cell compartment to the other. The results indicate that D-DDB1 not only contributes to the DNA repair system, but also has a role in cell proliferation and development (Takata, 2002).
Organogenesis involves cell proliferation followed by complex determination and differentiation events that are intricately controlled in time and space. The instructions for these different steps are, to a large degree, implicit in the gene expression profiles of the cells that partake in organogenesis. Combining fluorescence-activated cell sorting and SAGE, genomic expression patterns were analyzed in the developing eye of Drosophila. Genomic activity changes as cells pass from an uncommitted proliferating progenitor state through determination and differentiation steps toward a specialized cell fate. Analysis of the upstream sequences of genes specifically expressed during the proliferation phase of eye development implicates the transcription factor DREF and its inhibitor dMLF in the control of cell growth in this organ (Jasper, 2002).
To monitor the genome-wide gene transcription profiles associated with the different phases of eye development, defined subsets of cells isolated from eye imaginal discs were analyzed. These groups of cells were distinguished by the specific expression of green fluorescent protein (GFP) under the control of the Gal4-UAS system. Three distinct cell populations were purified from dissected third instar eye imaginal discs by fluorescence-activated cell sorting (FACS) of trypsin-dissociated cells. The first pool (referred to as GMR−; see below) contained cells from the region before the MF and represents the pluripotent, proliferative stage of eye development. The second pool of cells (GMR+) includes cells in the morphogenetic furrow, the second mitotic wave, as well as cells engaged in differentiation and patterning programs. Expression of GFP under the control of the GMR-Gal4 driver is restricted to the second pool of cells and can be used to distinguish the two cell populations. The third cell pool that was isolated represents a late stage of organogenesis, a group of already determined cells that are undergoing differentiation into specialized photoreceptor and cone cells. These cells were sorted based on GFP expression under the control of the sevenless enhancer/promoter (using sevGal4), which is transiently active in R3/R4 photoreceptor precursors and whose expression during ommatidial development becomes confined to R1, R6, R7, and the cone cells (Jasper, 2002).
The transcriptome of the three cell pools was quantitatively analyzed by serial analysis of gene expression (SAGE). SAGE was chosen as a method, since it allows accurate genome-wide quantification of mRNA levels in minute amounts of cellular material, without the need for amplification of the RNA pool by strategies that are prone to distortion of relative RNA representation. SAGE libraries were constructed from the sorted GMR−, GMR+, and Sev+ cell pools. Close to 20,000 tags were sequenced from each library, generating expression data for 4,279 different genes (tags present twice or more times in the 57,441 tags of the combined libraries) (Jasper, 2002).
SAGE tags were annotated using recently described databases and by BLAST searches against the Drosophila genome. Similar to results in the analysis of embryonic expression patterns, about 20% of the identified tags had no match to the Drosophila genome. Six percent had multiple matches and 4% matched the genome in regions without predicted genes. A large fraction (34% of all tags) matched the genome 3′ to a predicted gene, indicating alternative 3′ end processing and incomplete annotation of the genome sequence (Jasper, 2002).
The majority of tags appeared at comparable frequency in the three libraries, indicating constant expression levels of the corresponding genes. A tag derived from the transgene RNAs encoding GFP and Gal4 was abundant in the GMR+ and Sev+ libraries, while found only once in the GMR− library, illustrating the validity of the data and the purity of the sorted cell preparations. The SAGE data was confirmed by performing RNA in situ hybridization on eye imaginal discs for selected genes that were differentially represented in the different libraries. These experiments corroborated the differential expression of virtually all genes for which an informative signal could be obtained (28 out of 29). For many other genes, the data matched earlier reports of specific expression in the analyzed cell populations (e.g., toy, capt, sdk, lz; mdelta, B-H1 and ru (Jasper, 2002).
Classification of the differentially expressed genes into functional categories based on published data or on sequence similarities provides an overview of the general changes in cellular functions as cells transit from proliferation to the patterning and differentiation stages of organ development. Not surprisingly, many of the genes that are downregulated upon cessation of cell proliferation and at the onset of differentiation encode proteins involved in DNA replication and cell proliferation. These include genes specifically induced at the transition from G1 to S phase of the cell cycle, such as pcna (mus209) and ribonucleoside-diphosphate reductase (rnrL), as well as the replication licensing factors mcm2 and mcm5 (Jasper, 2002).
Other genes that are expressed at elevated levels in the proliferating cells of the GMR− pool encode products with functions in metabolism and the regulation of protein synthesis. This is consistent with the reported deleterious effect of mutations in some of these genes on cell proliferation and growth, such as for und, eif4A, Asp-tRNA synthetase, bellwether, and bonsai. The similar expression patterns of a group of proteasome subunits can be rationalized by the high degree of regulated protein turnover in proliferating tissues. Altogether, 93 genes were identified that are upregulated significantly in the GMR− pool and that have tentatively assigned functions in cell growth and proliferation (Jasper, 2002).
When eye imaginal disc cells enter the MF, they transit from the growth phase to the patterning phase of organogenesis and initiate specific differentiation programs. Consistent with this change of function, the cells posterior to the furrow upregulate specific cell adhesion and signal transduction molecules. These include proteins involved in the regulation of cellular adhesiveness and the cortical cytoskeleton such as Paxillin, Spectrin, Ankyrin, and α-Actinin, which show elevated expression levels in the GMR+ and Sev+ libraries. It is conceivable that such proteins mediate dynamically changing cell contacts as ommatidial clusters undergo rotation movements within the plane of the epithelium. Furthermore, differentiation markers such as genes involved in synaptic organization and axonal pathfinding begin to be upregulated in the GMR+ library and are yet more highly represented in the Sev+ library. Many of the mRNAs that are most prevalent in the latter library are involved in neuronal differentiation and signaling. Genes that are selectively transcribed in differentiating photoreceptors, as identified by their exclusive expression in the Sev+ cell population, include the cell type-specific transcription factors rough, lozenge, BarH1, and E(spl)mdelta. rough encodes a homeodomain transcription factor expressed in photoreceptors R2, R3, R4, and R5, whereas lozenge encodes a Runt domain transcription factor known to be expressed in cone cells and in all photoreceptors that arise from the second mitotic wave (R1, R6, and R7). The homeodomain transcription factor BarH1 is specifically expressed in R1 and R6 cells. E(spl)mdelta is a bHLH transcription factor expressed in R4 and R7. These transcription factors act in combination with specific signaling events to direct cell fate decisions within ommatidial clusters. The expression of the AT-rich interaction domain (ARID) transcription factor Retained, in a subset of photoreceptors as identified in this study, might contribute to this combinatorial genetic control of cell specification (Jasper, 2002).
In summary, the group of genes that was identified by SAGE to be specifically expressed in the differentiating cells of the eye imaginal disc overlaps to a significant degree with the regulators of photoreceptor differentiation previously identified by genetic means. This underscores the reliability of the method and supports the notion that genes that were designated as differentiation specific by SAGE, but have not yet been characterized genetically, may make important contributions to eye development. A further analysis of these genes thus holds the promise of providing significant new insights into the molecular biology of retinal development (Jasper, 2002).
It was reasoned that the coordinated regulation of groups of genes at specific stages of organogenesis might correlate with the presence of similar regulatory sequence motifs in their promoter regions. To identify such putative cis-acting elements, an unbiased computational approach was employed that would identify nonrandom sequence patterns in sequences proximal to the transcription start site of coregulated genes. Such algorithms have been employed successfully to identify genetic regulatory networks in the yeast genome. The AlignACE server was used to screen for nonrandom patterns within 1,000 bp upstream of the transcription start site of a set of 23 coregulated growth-related genes as well as a set of 23 differentiation-specific genes. In this way, one DNA element (TATCGATA) was identified that occurs in the upstream regions of genes implicated in cell growth and proliferation ahead of the MF. This motif is identical to the DNA replication-related element (DRE). DREs, in combination with E2F-responsive elements, control expression of genes involved in DNA replication including pcna. DREF, the transcription factor that binds to DREs, acts as a regulator of DNA synthesis in the Drosophila eye imaginal disc and is expressed predominantly in proliferating cells of the eye disc. The AlignACE results were confirmed by searching for DREs in the upstream region of a larger group of GMR−-specific genes as well as in the 23 differentiation-specific genes used for the second AlignACE search. Strikingly, 14 of 41 tested GMR−-specific genes contain a perfect match and 10 more contain a sequence closely resembling the 8 bp consensus DRE sequence within 1,000 bp of their transcription start site. In many cases, DREs or DRE-related sequences are found clustered with other DREs or with consensus binding sequences for E2F, another cell cycle-promoting transcription factor. In contrast, only 1 out of 23 tested differentiation-specific genes contained a DRE in the examined promoter regions. However, in the upstream sequences of this group of genes, a different motif resembling the binding site for the transcription factor Glass was found frequently. Glass is required for photoreceptor differentiation and is expressed in all cells posterior to the MF (Jasper, 2002).
The prevalence of DREs in genes that are associated with the proliferative state of the GMR− cell population suggests that the transcription factor DREF, possibly in concert with E2F, regulates a genetic program of cellular proliferation and growth during the early stages of eye development. In such a scenario, the downregulation of genes containing DRE sequences in their promoter region in the cells in and behind the MF (represented by the GMR+ and Sev+ pools) is likely to be a consequence of a suppression of DREF activity. One mechanism to explain the downregulation of DREF activity in the MF involves a known inhibitor of DREF, myelodysplasia/myeloid leukemia factor (dmlf). As indicated by the increased presence of dMLF-derived SAGE tags in the GMR+ and Sev+ libraries, and confirmed by in situ hybridization, dMLF expression is specifically upregulated in the MF and to a lesser degree posterior to the MF, thus coincident with the proposed suppression of DREF activity. Induction of dmlf in the MF might thus limit DREF function when cells prepare for differentiation. To test this model, DREF was ectopically expressed in the cells behind the MF. Earlier reports suggested that DREF overexpression leads to increased DNA synthesis behind the MF. Additionally, a significant increase of mitotic cells in this area was found, as visualized by immunostaining for phosphorylated histone 3, a specific marker for mitotic cells. These data thus suggest a function of the DREF/dMLF system in the control of a cell growth and proliferation program during organogenesis (Jasper, 2002).
The proteasome regulator REG (PA28gamma) is a conserved complex present in metazoan nuclei and is able to stimulate the trypsin-like activity of the proteasome in a non-ATP dependent manner. However, the in vivo function for REGgamma in metazoan cells is currently unknown. To understand the role of Drosophila REGgamma attempts were made to identify the type of promoter elements regulating its transcription. Mapping the site of the transcription initiation revealed a TATA-less promoter, and a sequence search identified elements found typically in Drosophila genes involved in cell cycle progression and DNA replication. In order to test the relevance of the motifs, REGgamma transcriptional assays were carried out with mutations in the proposed promoter. The results indicate that a single Drosophila replication-related element sequence, DRE, is essential for REGgamma transcription. To confirm that REGgamma has a role in cell cycle progression, the effect of removing REGgamma from S2 cells was tested using RNA interference. Drosophila cells depleted of REGgamma showed partial arrests in G1/S cell cycle transition. Immuno-staining of Drosophila embryos revealed that REGgamma is typically localized to the nucleus during embryogenesis with increased levels present in invaginating cells during gastrulation. The REGgamma was found dispersed throughout the cell volume within mitotic domains undergoing cell division. Finally, database searches suggest that the DRE system may regulate key members of the proteasome system in Drosophila (Masson, 2003).
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. A DNA replication-related element (DRE) has been identified 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 (Y.-J. Choi, 2004).
Selenophosphate synthetase catalyzes the synthesis of selenophosphate which is a selenium donor for Sec biosynthesis. In Drosophila, there are two types of selenophosphate synthetases designated dSPS1 and dSPS2, where dSPS2 is a selenoprotein. The mechanism of gene expression of dSPS2 as well as other selenoproteins in Drosophila has not been elucidated. This study reports an essential regulator system that regulates the transcription of the dSPS2 gene (dsps2). Through deletion/substitution mutagenesis, the downstream DNA replication-related element (DRE) located at +71 has been identified as an essential element for dsps2 promoter activity. Furthermore, double-stranded RNA interference (dsRNAi) experiments were performed to ablate transcription factors such as TBP, TRF1, TRF2 and DREF in Schneider cells. The dsRNAi experiments showed that dsps2 promoter activities in DREF- and TRF2-depleted cells were significantly decreased by 90% and 50%, respectively. However, the depletion of TBP or TRF1 did not affect the expression level of dsps2 even though there is a putative TATA box at -20. These results strongly suggest that the DRE/DREF system controls the basal level of transcription of dsps2 by interacting with TRF2 (Jin, 2004).
Reactive oxygen species (ROS) cause oxidative stress and aging. The catalase gene is a key component of the cellular antioxidant defense network. However, the molecular mechanisms that regulate catalase gene expression are poorly understood. In this study, a DNA replication-related element (DRE; 5'-TATCGATA) was identified in the 5'-flanking region of the Drosophila catalase gene. Gel mobility shift assays revealed that DREF binds to the DRE sequence in the Drosophila catalase gene. Site-directed mutagenesis and in vitro transient transfection assays were used to establish that expression of the catalase gene is regulated by DREF through the DRE site. To explore the role of DRE/DREF in vivo, transgenic flies were established carrying a catalase-lacZ fusion gene with or without mutation in the DRE. The beta-galactosidase expression patterns of these reporter transgenic lines demonstrated that the catalase gene is upregulated by DREF through the DRE sequence. In addition, suppression of the ectopic DREF-induced rough eye phenotype was induced by a catalase amorphic Cat(n1) allele, indicating that DREF activity is modulated by the intracellular redox state. These results indicate that the DRE/DREF system is a key regulator of catalase gene expression and provide evidence of cross-talk between the DRE/DREF system and the antioxidant defense system (Park, 2004).
PIMT, a transcriptional coactivator which interacts with and enhances nuclear receptor coactivator PRIP function, was identified recently in mammalian cells and suggested to function as a link between two major multiprotein complexes anchored by CBP/p300 and PBP. This study finds that the Drosophila homologue of PIMT, designated as Dtl, is closely associated and has an overlapping promoter with a gene encoding another transcriptional coactivator, ADA2a, which in turn participates in GCN5 HAT-containing complexes. Ada2a also produces an RNA polII subunit, RPB4, via alternative splicing; consequently, an overlapping regulatory region serves for the production of three proteins, each involved in transcription. By studying expression of reporter gene fusions in tissue culture cells and transgenic animals it has been demonstrated that the regulatory regions of Ada2a/Rpb4 and Dtl overlap and the Dtl promoter is partly within the Ada2a/Rpb4 coding region. The shared regulatory region contains a DRE element, binding site of DREF, the protein factor involved in the regulation of a number of genes which play a role in DNA replication and cell proliferation. Despite the perfectly symmetrical DRE, DREF seems to have a more decisive role in Ada2a/Rpb4 transcription than in the transcription of Dtl (Papai, 2005).
DNA replication-related element (DRE) and the DRE-binding factor (DREF) play an important role in regulating DNA replication-related genes such as PCNA and DNA polymerase alpha in Drosophila. Overexpression of DREF in developing eye imaginal discs induces ectopic DNA synthesis and apoptosis, which results in rough eyes. To identify genetic interactants with the DREF gene, a screen was carried out for modifiers of the rough eye phenotype. One of the suppressor genes identified was the Drosophila orc2 gene. A search for known transcription factor recognition sites revealed that the orc2 gene contains three DREs, named DRE1 (+14 to +21), DRE2 (-205 to -198), and DRE3 (-709 to -702). Band mobility shift analysis using Kc cell nuclear extracts detected the specific complex formed between DREF and the DRE1 or DRE2. Specific binding of DREF to genomic region containing the DRE1 or DRE2 was further demonstrated by chromatin immunoprecipitation assays, suggesting that these are the genuine complexes formed in vivo. The luciferase assay in Kc cells indicated that the DRE sites in the orc2 promoter are involved in a transcriptional regulation of the orc2 gene. The results, taken together, demonstrate that the orc2 gene is under the control of DREF pathway (Okudaira, 2005).
SKPa is component of a Drosophila SCF complex that functions in combination with the ubiquitin-conjugating enzyme UbcD1. skpA null mutation results in centrosome overduplication, unusual chromatin condensation, defective endoreduplication and cell-cycle progression. While the molecular mechanisms that regulate expression of the skpA gene are poorly understood, the DNA replication-related element (DRE) and the DRE-binding factor (DREF) play important roles in regulating proliferation-related genes in Drosophila and DRE (5'-TATCGATA) and DRE-like (5'-CATCGATT) sequences were here found to be involved in skpA promoter activity. Thus both luciferase transient expression assays in cultured Drosophila S2 cells using skpA promoter-luciferase fusion plasmids and anti-lacZ immunostaining of various tissues from transgenic third instar larvae carrying the skpA promoter-lacZ fusion genes provided supportive evidence. Furthermore, anti-SKPa immunostaining of eye imaginal discs from flies overexpressing DREF showed ectopic expression of protein in the region posterior to the morphogenetic furrow where DREF is overexpressed. Knockdown of DREF in some tissues where SKPa distribution is well known almost completely abrogated the skpA gene expression. These findings, taken together, indicate that the Drosophila skpA gene is a novel target of the transcription factor DREF (Phuong Thao, 2006).
DREF is an 80 kDa polypeptide homodimer which plays an important role in regulating cell proliferation-related genes. Both DNA binding and dimer formation activities are associated with residues 16-115 of the N-terminal region. However, the mechanisms by which DREF dimerization and DNA binding are regulated remain unknown. This study reports that the DNA binding activity of DREF is regulated by a redox mechanism, and that the cysteine residues are involved in this regulation. Electrophoretic mobility shift analysis using Drosophila Kc cell extracts or recombinant DREF proteins indicated that the DNA binding domain is sufficient for redox regulation. Site-directed mutagenesis and transient transfection assays showed that Cys59 and/or Cys62 are critical both for DNA binding and for redox regulation, whereas Cys91 is dispensable. In addition, experiments using Kc cells indicated that the DNA binding activity and function of DREF are affected by the intracellular redox state. These findings give insight into the exact nature of DREF function in the regulation of target genes by the intracellular redox state (T. Y. Choi, 2004).
SKPa is component of a Drosophila SCF complex that functions in combination with the ubiquitin-conjugating enzyme UbcD1. skpA null mutation results in centrosome overduplication, unusual chromatin condensation, defective endoreduplication and cell-cycle progression. While the molecular mechanisms that regulate expression of the skpA gene are poorly understood, the DNA replication-related element (DRE) and the DRE-binding factor (DREF) play important roles in regulating proliferation-related genes in Drosophila and DRE (5'-TATCGATA) and DRE-like (5'-CATCGATT) sequences were here found to be involved in skpA promoter activity. Thus both luciferase transient expression assays in cultured Drosophila S2 cells using skpA promoter-luciferase fusion plasmids and anti-lacZ immunostaining of various tissues from transgenic third instar larvae carrying the skpA promoter-lacZ fusion genes provided supportive evidence. Furthermore, anti-SKPa immunostaining of eye imaginal discs from flies overexpressing DREF showed ectopic expression of protein in the region posterior to the morphogenetic furrow where DREF is overexpressed. Knockdown of DREF in some tissues where SKPa distribution is well known almost completely abrogated the skpA gene expression. These findings, taken together, indicate that the Drosophila skpA gene is a novel target of the transcription factor DREF (Phuong Thao, 2006).
The transcription factor DREF regulates proliferation-related genes in Drosophila. With two-hybrid screening using DREF as a bait, a clone was obtained encoding a protein homologous to human myelodysplasia/myeloid leukemia factor 1 (hMLF1). The protein was termed Drosophila MLF (dMLF); it consists of a polypeptide of 309 amino acid residues, whose sequence shares 23.1% identity with hMLF1. High conservation of 54.2% identity over 107 amino acids was found in the central region. The dMLF gene was mapped to 52D on the second chromosome by in situ hybridization. Interaction between dMLF and DREF in vitro was confirmed by glutathione S-transferase pull-down assay, with the conserved central region appearing to play an important role in this. Northern blot hybridization analysis revealed dMLF mRNA levels to be high in unfertilized eggs, early embryos, pupae and adult males, and relatively low in adult females and larvae. This fluctuation of mRNA during Drosophila development is similar to that observed for DREF mRNA, except in the pupa and adult male. Using a specific antibody against the dMLF, immunofluorescent staining of Drosophila Kc cells was performed and showed a primarily cytoplasmic staining, whereas DREF localizes in the nucleus. However, dMLF protein contains a putative 14-3-3 binding motif involved in the subcellular localization of various regulatory molecules, and interaction with DREF could be regulated through this motif. The transgenic fly data suggesting the genetic interaction between DREF and dMLF support this possibility. Characterization of dMLF in the present study provides the molecular basis for analysis of its significance in Drosophila (Ohno, 2000).
In human, the myeloid leukemia factor 1 (hMLF1) has been shown to be involved in acute leukemia, and mlf related genes are present in many animals. Despite their extensive representation and their good conservation, very little is understood about their function. In Drosophila, dMLF physically interacts with both the transcription regulatory factor DREF and an antagonist of the Hedgehog pathway, Suppressor of Fused, whose over-expression in the fly suppresses the toxicity induced by polyglutamine. No connection between these data has, however, been established. This study showed that dmlf is widely and dynamically expressed during fly development. The first dmlf mutants were isolated and analyzed: embryos lacking maternal dmlf product have a low viability with no specific defect, and dmlf-/- adults display weak phenotypes. dMLF subcellular localization was monitored in the fly and cultured cells. Although generally nuclear, dMLF can also be cytoplasmic, depending on the developmental context. Furthermore, two differently spliced variants of dMLF display differential subcellular localization, allowing the identification of regions of dMLF potentially important for its localization. Finally, dMLF can act developmentally and postdevelopmentally to suppress neurodegeneration and premature aging in a cerebellar ataxia model (Martin-Lanneree, 2006).
Drosophila TATA-box-binding protein (TBP)-related factor 2 (TRF2) is a member of a family of TBP-related factors present in metazoan organisms. Recent evidence suggests that TRF2s are required for proper embryonic development and differentiation. However, true target promoters and the mechanisms by which TRF2 operates to control transcription remain elusive. A Drosophila TRF2-containing complex has been purified by antibody affinity; this complex contains components of the nucleosome remodelling factor (NURF) chromatin remodelling complex as well as the DNA replication-related element (DRE)-binding factor DREF. This latter finding leads to potential target genes containing TRF2-responsive promoters. A combination of in vitro and in vivo assays has been used to show that the DREF-containing TRF2 complex directs core promoter recognition of the proliferating cell nuclear antigen (PCNA) gene. Additional TRF2-responsive target genes involved in DNA replication and cell proliferation have also been identified. These data suggest that TRF2 functions as a core promoter-selectivity factor responsible for coordinating transcription of a subset of genes in Drosophila (Hochheimer, 2002).
Metazoan organisms have evolved diverse mechanisms to control the spatial and temporal patterns of gene expression during growth, differentiation and development. It has become increasingly evident that cell-type-specific components of the general transcriptional apparatus, for example the mammalian TFIID component TAFII105 or the Drosophila TAFII80 homolog Cannonball contribute significantly to tissue-specific and gene-selective transcriptional regulation in metazoan organisms. Recent studies have also established that TBP-related factors like Drosophila TRF1 can direct transcription from an alternative core promoter and a TRF1:BRF complex is required for RNA polymerase III transcription of transfer RNA genes. TRF2 is a third member of the TBP family in Drosophila and, like TBP and TRF1, TRF2 interacts with the basal transcription factors TFIIA and TFIIB10. However, the primary amino acid sequence of the putative TRF2 DNA-binding domain has diverged from TBP and TRF1 and, not surprisingly, TRF2 fails to bind to DNA containing canonical TATA boxes. But TRF2 is associated with loci on Drosophila chromosomes that are distinct from TBP and TRF1. This suggested that TRF2 may direct promoter specificity and perhaps coordinate a subset of target genes. Size exclusion chromatography indicated that Drosophila TRF2 is likely to be part of a macromolecular complex. Unlike TRF1, Drosophila TRF2 has amino-terminal and carboxy-terminal extensions flanking the putative DNA-binding core domain. This suggested that TRF2 may be associated with a set of proteins that are distinct from TBP- and TRF1-associated factors. It was reasoned that the purification and identification of TRF2-associated factors might enable the identification of TRF2-specific promoters and reveal how TRF2 operates to execute transcriptional specificity (Hochheimer, 2002).
In the absence of a functional assay allowing conventional purification of TRF2-associated factors a panel of monoclonal antibodies was generated directed against several domains of TRF2 to affinity purify TRF2 and its putative associated subunits. Approximately 3,500 hybridomas were screened and a clone was isolated that efficiently immunoprecipitated TRF2 and its associated factors from Drosophila embryo nuclear extract. A Sarkosyl-eluted complex containing TRF2 was analysed by SDS-polyacrylamide gradient gel electrophoresis (PAGE) that revealed a set of 18 polypeptides with relative molecular mass (Mr) ranging from 300K to 29K that co-immunoprecipitated consistently with TRF2 even under very stringent conditions (Hochheimer, 2002).
An 80K protein associated with TRF2 is identical to DREF1. DREF and its corresponding response element DRE have been well documented to be important for the regulation of cell-cycle and cell-proliferation genes in Drosophila (that is, genes for PCNA and the 180K and 73K subunits of DNA polymerase). The identification of the promoter-selective DNA-binding protein DREF was intriguing because Drosophila TRF2 thus far had failed to bind to canonical TATA-box elements, which suggests that TRF2 may cooperate with DREF to execute promoter specificity and perhaps operate like a metazoan sigma factor (Hochheimer, 2002).
The 140K protein associated with TRF2 is identical to Drosophila Iswi, which is the catalytic ATPase subunit of NURF, ACF and CHRAC chromatin remodelling complexes. Moreover, the 55K and 38K proteins associated with TRF2 turned out to be NURF-55/CAF-1 and NURF-38/inorganic pyrophosphatase, respectively. Notably, the peptide sequences obtained from the three largest (300K, 250K and 230K) proteins associated with TRF2 do not match NURF-301, suggesting that the presence of some NURF subunits is not merely a result of contaminating NURF in the TRF2 complex. However, analysis of the cDNAs encoding the 190K and 160K proteins associated with TRF2 revealed that both proteins contain conserved sequence motifs for 11 and 5 zinc finger motifs (C2H2), respectively; these smaller proteins thus resemble factors like the CCCTC-binding factor CTCF that has been implicated in mediating chromatin-dependent processes such as the regulation of insulator function. It is therefore possible that the TRF2 complex encompasses both promoter-selectivity functions and NURF-like components as well as other activities with distinct subunits and specificity (Hochheimer, 2002).
Sequence analysis of the cDNA encoding the 65K protein revealed a significant similarity to the RNA-binding protein Rap55 isolated from Pleurodeles waltl and Xenopus laevis, whereas sequence analysis of the 70K, 116K and 118K proteins showed no significant similarity to known proteins in the databases. The functional relevance of ß-tubulin in the TRF2 complex is at present unclear and the 47K and the 29K polypeptides associated with TRF2 are yet to be characterized (Hochheimer, 2002).
Having identified DREF as a tightly associated component of the TRF2 complex, it was next asked whether TRF2 can function as a true core promoter recognition factor and selectively initiate transcription at a promoter that is documented to be stimulated by the DRE/DREF system. The DREF-responsive PCNA promoter, which contains at least three promoter-proximal regulatory elements including an upstream regulatory element URE, DRE and two E2F recognition sites located within 200 bp upstream of the start site, was chosen (Hochheimer, 2002).
To test the responsiveness of the PCNA promoter in vitro and to map the transcription start site(s), a -580 PCNA (-580 to +56) promoter fragment, which contains all known regulatory elements, and a -64 PCNA (-64 to +56) promoter fragment, which lacks all regulatory elements except for the E2F-binding sites were used as DNA templates for in vitro transcription. Increasing amounts of a partially purified Drosophila embryo nuclear extract (H.4) that contains all the necessary basal factors were added, as well as both the TRF2 complex and limiting amounts of TFIID to the transcription reaction. Using the -580 PCNA template two distinct transcription start sites separated by 63 nucleotides were detected. Promoter 1 (with start site at position +1) was stimulated with increasing amounts of H.4 supplemented with TFIID whereas promoter 2 (with start site at position -63) was detected only with the lowest amounts of H.4 added. Using the truncated -64 PCNA, template, transcription from promoter 2 was essentially abolished, whereas a weak activity could be detected from promoter 1 by adding the maximum amount of H.4 + TFIID. In vitro and in vivo results suggest that promoter 2 might be TRF2- and DRE-dependent, whereas promoter 1 appears to be mediated by TFIID (Hochheimer, 2002).
It was next asked whether TRF2 can contribute to the enhancer-dependent activated transcription of the PCNA promoters by E2F and DP, which cooperate in DNA-binding and transcriptional activation. The co-expression of just the trancriptional activators E2F and DP in the absence of exogenous TRF2/DREF results in a substantial transcriptional activation of the PCNA reporter. It is likely that this activation by E2F/DP is mediated by endogenous TRF2. This activation is abolished with the -64 PCNA reporter, which lacks the DRE-binding sites but still contains the E2F-binding sites. As expected, inducing the co-expression of all three promoter recognition factors -- TRF2, DREF and E2F -- results in a strong synergistic activation of the PCNA promoters (80-fold) in a DRE-dependent fashion. These results suggest that in SL2 cells TRF2 and DREF can work together to stimulate the PCNA reporter in a DRE-dependent fashion. This is consistent with the finding in vitro that the TRF2 complex can selectively initiate transcription from promoter 2 of the PCNA gene in a DRE-dependent manner (Hochheimer, 2002).
To investigate whether TRF2 is involved in the coordinate regulation of other DNA replication and cell cycle genes in the Drosophila genome, oligonucleotide-based microarrays representing 13,500 Drosophila genes were hybridized with RNA probes isolated at different time points after induction of TRF2 expression in SL2 cells. The microarray analysis revealed that only 1.9% of all genes analysed were upregulated more than 2-fold and only 1.6% downregulated by more than 2-fold. These biochemical studies and cell based assays suggested that TRF2 functions as a core promoter-selectivity factor that collaborates with DREF. It was therefore asked whether there are other TRF2-responsive genes that also contain a promoter-proximal DRE. An analysis of the distribution of DREs in the Drosophila genome revealed that about 100 genes bear a consensus DRE within 1 kb of the predicted promoter region. Microarray analysis revealed that 38 of these DRE-containing genes were also responsive to TRF2 overexpression. For example, genes encoding PCNA, the 180K subunit of DNA polymerase, the a-subunit of mitochondrial DNA polymerase, and E2F were all found to be upregulated 2-5-fold by TRF2 in the microarray analysis and confirmed by RNase protection assays. Three additional DRE-regulated genes encoding TBP, the 73K subunit of DNA polymerase, and the 50K subunit of DNA polymerase were found to be downregulated (Hochheimer, 2002).
These data suggest that in addition to the PCNA gene a number of other Drosophila DRE-containing genes may also be regulated by TRF2 and further support the model that TRF2 can function as a core promoter-selectivity factor that governs a restricted subset of genes that are coordinately regulated. A recent bioinformatics study of core promoter sequences in the Drosophila genome identified a consensus DRE as the second most frequent control core element (other than TATA and INR) providing independent evidence for DRE as a core promoter element (Hochheimer, 2002).
Because these studies have relied largely on 'gain of function' assays double stranded RNA interference (dsRNAi) was imployed in flies and SL2 cells25 to determine the consequences of ablating TRF2/DREF on transcription of putative target genes such as PCNA and DNApol 180 in cultured cells. SL2 cells were treated with in vitro synthesized dsRNA and the depletion of TRF2 and DREF proteins was monitored by immunoblot analysis. After 2 days of incubation the specifically targeted TRF2 and DREF proteins were severely depleted. After 48 h of dsRNA treatment, a significant number of cells sloughed off the plate and died. This is in accordance with previous finding that TRF2 RNAi in Drosophila embryos is lethal for embryos (Hochheimer, 2002).
However, between 24 and 48 h of dsRNAi treatment it was possible to reproducably measure the activity of transiently transfected luciferase reporters fused to either the PCNA or DNApol 180 promoters and compare them to an internal control reporter gene. The activities from both the PCNA (-580 to +56) and DNApol 180 (-620 to +20) promoters were significantly reduced in TRF2-depleted cells (4.2-fold and 3-fold, respectively) relative to a Renilla luciferase control reporter driven by the HSV TK promoter. Likewise, these two target gene promoters were downregulated in DREF-depleted cells (3.5-fold and 5-fold, respectively). These depletion experiments using dsRNAi thus support the findings that TRF2 and DREF participate in directing transcription of a select subset of genes that include PCNA and DNApol 180 (Hochheimer, 2002).
Although the dsRNAi studies provide an independent line of evidence to support the notion that TRF2/DREF play a role in promoter selectivity in vivo, they fail to provide a direct mechanistic link between TRF2/DREF and the PCNA and DNApol 180 promoters. Chromatin immunoprecipitation (ChIP) experiments were carried to determine the occupancy of TRF2/DREF at the PCNA and DNApol 180 promoters in formaldehyde-treated SL2 cells using antibodies raised against TRF2, DREF and TBP. The precipitated DNA fragments with an average length of 500-1,000 base pairs (bp) were analysed directly by polymerase chain reaction (PCR). The PCNA promoter region was specifically precipitated by anti-TRF2 and anti-DREF and to a lesser extent by anti-TBP; this is consistent with previous findings and confirms that TRF2, DREF and the TFIID subunit TBP can co-localize and directly interact with the PCNA promoter region in living cells. As further evidence for the targeting of specific promoters by TRF2 and DREF, an analysis was carried out of the DRE-containing DNApol 180 promoter region, which is selectively precipitated by the TRF2-, DREF- and TBP-specific antibodies. These ChIP experiments strongly support the in vitro transcription results as well as the cell-based dsRNAi transcription assays in establishing a core promoter selectivity for the TRF2/DREF complex (Hochheimer, 2002).
DREF is a transcriptional regulatory factor required for the expression of genes carrying the 5'-TATCGATA DRE. DREF has been reported to bind to a sequence in the chromatin boundary element, and thus may play a part in regulating insulator activity. To generate further insights into DREF function, a Saccharomyces cerevisiae two-hybrid screening was screened with DREF polypeptide as bait, and Mi-2 was identified as a DREF-interacting protein. Biochemical analyses revealed that the C-terminal region of Drosophila Mi-2 (dMi-2) specifically binds to the DNA-binding domain of DREF. Electrophoretic mobility shift assays showed that dMi-2 thereby inhibits the DNA-binding activity of DREF. Ectopic expression of DREF and dMi-2 in eye imaginal discs resulted in severe and mild rough-eye phenotypes, respectively, whereas flies simultaneously expressing both proteins exhibited almost-normal eye phenotypes. Half-dose reduction of the dMi-2 gene enhanced the DREF-induced rough-eye phenotype. Immunostaining of polytene chromosomes of salivary glands showed that DREF and dMi-2 bind in mutually exclusive ways. These lines of evidence define a novel function of dMi-2 in the negative regulation of DREF by its DNA-binding activity. Finally, it is postulated that DREF and dMi-2 may demonstrate reciprocal regulation of their functions (Hirose, 2002).
This report proposes a novel mechanism whereby dMi-2 is involved in repressing transcription of DRE-containing genes by inhibiting the DNA binding of DREF. The observations point to a first example of a member of the SWI/SNF2 family of DNA-stimulated ATPases directly interacting with a transcription factor to attenuate its activity. Although the present biochemical and genetic analyses clearly indicated direct interaction between DREF and dMi-2, it is uncertain whether the dMi-2 polypeptide alone or in association with another subunit of the chromatin-remodeling complex, such as HDAC, binds to DREF in vivo. It is worth noting that treatment of Drosophila cultured cells with trichostatin A, a microbial metabolite generally used as an inhibitor of HAT, did not affect the PCNA promoter activity, whereas cotransfection of a dMi-2-expressing plasmid with reporter plasmid significantly decreased PCNA promoter activity depending on the presence of the DRE sequence. This indicates that accompanying histone acetyltransferase activity might not be involved in repression by dMi-2 (or the dMi-2 complex). However, a requirement for other subunits cannot be ruled out. Although previous studies on mammalian Mi-2 (Mi-2 ß, CHD4) complexes characterized the Mi-2 polypeptide as a major component, biological functions of separate components have not been examined. Importantly, several different strategies resulted in the purification of slightly different Mi-2 complexes. In the case of the original NRD complex, the purification was performed by pursuing HDAC activity by conventional chromatography, followed by affinity chromatography for Mi-2 ß (CHD4). With this purification method, the bulk of the tightly associated NRD core complex does not contain sequence-specific DNA-binding protein. Recently, MeCP1 complexes have been purified to homogeneity and the presence of the core polypeptide of the known NRD complex was demonstrated, indicating that several kinds of complexes, including the Mi-2 polypeptide, might exist in cells. Considering that only 5% of the total dMi-2 polypeptide was estimated to be associated with DREF by immunoprecipitation experiments, it is hypothesized that binding with DREF in vivo may also be limited. Furthermore, it is interesting to note that the amino-terminal region of dMi-2 exhibits inhibitory effects on its binding to DREF, suggesting a possible regulation by change in the structure of the molecule. To assess this possibility, a challenge for the future will be the determination of the three-dimensional structure of Mi-2 (or the Mi-2 complex) that binds and modulates DREF activity. Transgenic fly lines have been established expressing HA epitope-tagged dMi-2 and Flag epitope-tagged DREF by using the GAL4-UAS system. These flies should be powerful tools for the purification of DREF/dMi-2 complexes (Hirose, 2002).
dMi-2 protein is localized at several hundred loci of the polytene chromosomes of salivary glands. A model of dMi-2 protein function has been proposed featuring repression of transcription by binding to a Polycomb group protein in the form of a Hunchback-dMi-2 complex, with consequent recruitment to DNA. However, this study observed dMi-2 in interbands and regions associated with high transcriptional activity (puffs), suggesting an ability to enhance as well as to repress gene expression. To address this question, it is important that genes that are positively regulated by dMi-2 be identified (Hirose, 2002).
Another important finding of the immunostaining is that DREF and dMi-2 bind to polytene chromosomes in a mutually exclusive manner. This seems contrary to the results of immunoprecipitation and in vitro binding experiments but can be explained as follows. Since the DNA-binding domain and the Mi-2-binding domain of DREF overlap, dMi-2 cannot interact with DREF bound to DNA. In contrast, dMi-2 presumably has access to free DREF. If dMi-2 cannot disrupt DREF/DNA complexes, genes adjacent to DREF binding sites will be kept in a transcriptionally active state. Furthermore, overexpression of DREF or dMi-2 in eye imaginal discs induces a rough-eye phenotype although the eyes of transgenic flies simultaneously expressing DREF and dMi-2 appear normal. These results suggest that DREF and dMi-2 negatively regulate each other's functions. To date, although there is no evidence that molecules recruit dMi-2 to specific loci of polytene chromosomes, it can be speculated that DREF could be involved in the regulation of such dMi-2 recruitment. If this is the case, an important mechanism for the maintenance of epigenetic activation (or silencing) of genes can be envisaged. This idea is not contradictory to the model in which DREF contributes to the cancellation of chromatin boundary function by displacing BEAF from its binding sites (Hirose, 2002).
In summary, evidence has been provided for a novel function of dMi-2 in repressing transcription of DRE-containing genes by attenuating the DRE-binding activity of DREF. In addition, it is hypothesized that DREF and dMi-2 may demonstrate reciprocal regulation of their functions. To probe this possibility, efforts to isolate DREF mutant flies and determine the dMi-2 (complex) structure in association with DREF are necessary in the future (Hirose, 2002).
DREF is required for expression of many proliferation-related genes carrying the DRE sequence, 5'-TATCGATA. Over-expression of DREF in the eye imaginal disc induces ectopic DNA synthesis, apoptosis and inhibition of photoreceptor cell specification, and results in rough eye phenotype in adults. In the present study, half dose reduction of the Distal-less (Dll) gene enhanced the DREF-induced rough eye phenotype, suggesting that Dll negatively regulates DREF activity in eye imaginal disc cells. Biochemical analyses revealed the N-terminal (30aa to 124aa) and C-terminal (190aa to 327aa) regions of Dll interact with the DNA binding domain (16aa to 125aa) of DREF, although it is not clear yet whether the interaction is direct or indirect. Electrophoretic mobility shift assays showed that Dll thereby inhibits DNA binding. The repression of this DREF-function by a homeodomain protein like Dll may contribute to the differentiation-coupled repression of cell proliferation during development (Hayashi, 2006).
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