InteractiveFly: GeneBrief

DNA replication-related element factor: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory element DRE (5'-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DNA replication-related element factor or DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs' chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, transgenic flies were established in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induces ectopic DNA synthesis in the cells behind the morphogenetic furrow that are normally postmitotic, and abolishes photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype is suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, it was found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppresses and that reduction of Distal-less enhances the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection of Drosophila deficiency stocks allowed identification of several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators (Hirose, 2001).

The promoters of Drosophila genes related to DNA replication, such as those for the 180-kDa catalytic subunit and 73-kDa subunit polypeptide of DNA polymerase α and proliferating-cell nuclear antigen (PCNA), contain a common 8-bp palindromic sequence (5'-TATCGATA), named the DNA replication-related element (DRE) (2003) in addition to E2F recognition sites. The DRE requirement for promoter activation has been confirmed in both cultured cells and transgenic flies (Yamaguchi, 1995a; Yamaguchi, 1996). Studies using the latter have shown that DRE is required for the function of the PCNA promoter throughout development except in adult females. A specific DRE-binding factor (DREF) was identified consisting of an 80-kDa polypeptide homodimer, and molecular cloning of its cDNA has led to confirmation that DREF is a trans activator for DRE-containing genes (Hirose, 1996; Hirose, 2001 and references therein).

The N-terminal fragment of the DREF polypeptide containing a region responsible for DRE binding and dimer formation acts as dominant negative effector against DREF (1999). Expression of a dominant-negative form of DREF in cells of salivary glands and eye imaginal discs using the GAL4-upstream activation site (UAS) system inhibits endoreplication of larval salivary gland cells and significantly reduces DNA replication in the second mitotic wave, respectively (1999). The results indicate that DREF is required for DNA replication in both the mitotic cell cycle and the endo cell cycle. However, progression of DNA replication requires a number of genes involved in growth signal transduction, cell cycle regulation, DNA replication itself, or transcription regulation. So far, no clues habe been available to determine which of these genes is a critical target of the dominant negative form of DREF (Hirose, 2001).

Screening for the DRE sequence in the Drosophila genome permitted an estimate that more than 10³ Drosophila genes are regulated by DREF (Matsukage, 1995). These genes were classified into five groups by their functional category: (1) DNA replication related, (2) translation related, (3) signal transduction/cell cycle regulation related, (4) transcription factor, and (5) others. The fourth category contains specific transcriptional regulators required for normal development or determination of cell differentiation, such as zeste, caudal, Antennapedia, serendipity-beta, and some RNA-biding proteins. These observations suggest a possibility that DREF may play an important role in cell differentiation other than activation of genes involved in cell proliferation (Hirose, 2001).

Hart haa proposed a novel function of DREF as an antagonist of boundary element-associated factor (BEAF), which is involved in boundary activity of the scs' region of the Drosophila hsp 70 gene (Hart, 1997; Hart, 1999). Staining of polytene chromosomes with anti-DREF and anti-BEAF antibodies revealed that about half of the signals from the two proteins overlapped and that the other half were from only BEAF or only DREF. Furthermore, using a chromatin precipitation method, Hart demonstrated that DREF can bind to the same sequences as BEAF, establishing the chromatin boundary in the scs' special chromatin domain present in Drosophila melanogaster 87A7 hsp 70. From the results, Hart assumed that competition of binding between DREF and BEAF is important for regulation of activity at the chromatin boundary (Hirose, 2001).

To clarify in vivo functions of DREF, transgenic flies expressing DREF in eye imaginal disc cells by using the GAL4-UAS system. If overexpression of DREF in eye imaginal discs confers a specific phenotype in the eyes of adult flies, the flies can be utilized to screen for modifiers of DREF activity or its target genes. Such an approach has been successfully undertaken with dE2F-overexpressing flies. This study analyzed the consequences of ectopic expression of DREF polypeptide in cells of eye imaginal discs. Ectopic expression of DREF in the cells posterior to the MF induced ectopic DNA synthesis and apoptosis in normally postmitotic cells, inhibited differentiation of the photoreceptor cells, and resulted in a severe rough eye phenotype in the adult flies. Furthermore, mutations were identified that enhance or suppress the DREF-induced phenotype. Candidates for DREF interaction include cell cycle regulators and regulators of chromatin-structure, thus giving clues to resolve the molecular mechanisms of how DREF activates many genes related to cell proliferation (Hirose, 2001).

Involvement of the DRE-DREF system in the regulation of a considerable variety of genes has been suggested by the results of DNA database searches. In about 3.5% of the Drosophila genome, 73 copies of 5'-TATCGATA sequences were found to be localized within 0.6 kb upstream regions of 61 genes, including those involved in transcription, translation, growth signal transduction, cell cycle regulation, and transcriptional regulation, in addition to genes related to DNA replication (Matsukage, 1995). Genes related to cell proliferation, such as those for PCNA, DNA polymerase alpha 180- and 73-kDa subunits, CycA, D-raf, and E2F, have been shown to be under DRE-DREF system regulation (Ohno, 1996; Ryu, 1997; Sawado, 1998). In addition, ectopic expression of the truncated form of DREF, possessing dominant negative activity, in salivary glands or eye imaginal discs significantly reduces DNA replication. Evidence suggests that DREF is required for DNA replication in both the endo and mitotic cell cycles. However, it has hitherto not been clear whether the accumulation of DREF is sufficient to induce DNA replication as well as ectopic expression of dE2F to drive normally quiescent cells to enter S phase (Hirose, 2001).

This study demonstrates that ectopic expression of DREF in eye imaginal discs, using the Glass-responsive GMR promoter, induces ectopic DNA synthesis and apoptosis in the cells behind the morphogenetic furrow. The results appear to be very similar to phenomena observed in eye imaginal discs expressing dE2F/dDP. One possible molecular mechanism is direct up-regulation of genes involved in DNA replication such as those encoding PCNA and DNA polymerase alpha. Another possible mechanism is indirect up-regulation of genes related to DNA replication or genes required for the G₁/S transition by means of activation of dE2F expression. Overexpression of DREF in eye imaginal disc cells enhances the promoter activity of dE2F (Sawado, 1998). Therefore, overexpression of DREF may result in E2F accumulation behind the morphogenetic furrow and consequently induce an ectopic S phase. This view is consistent with the observation that reduction of the dE2F gene dose at least partially suppresses the rough eye phenotype induced by DREF. However, it should be noted that there are some differences between the DREF- and dE2F/dDP-induced phenotypes: (1) In eye imaginal discs expressing dE2F/dDP under control of the GMR promoter, most S phases are seen in uncommitted cells, whereas in imaginal discs expressing DREF, nuclei of cells under differentiation process located apically with respect to the imaginal disc appear to incorporate BrdU; (2) it has been reported that coexpression of baculovirus p35 protein, an inhibitor of cell death, strongly enhances the dE2F/dDP phenotype, indicating that the majority of cells ectopically entering S phase as a result of E2F/dDP expression are eliminated by apoptosis, while the DREF phenotype, in contrast, was significantly suppressed by coexpression of p35; and (3) overexpression of dE2F/dDP does not grossly interfere with the initiation of neuronal cell differentiation or the stepwise recruitment of cells into preclusters, whereas expression of DREF perturbs photoreceptor specifications of R1, R6, and R7, whose differentiation normally begins after those of R8, R2, R5, R3, and R4. The molecular mechanisms underlying these differences are unclear, but it is speculated that elevated amounts of DREF not only affect DNA replication-related genes but also disturb the transcription of genes required for normal processes of differentiation, consequently inducing apoptosis (Hirose, 2001).

The present study focused on the use of flies for screening interaction with Polycomb and trithorax group genes. It is known that DREF and BEAF share binding sites with functions as boundary elements in Drosophila (scs' region of hsp70, BE76, and BE28) and also the promoter region of DNA polymerase alpha 180-kDa subunit gene. Polycomb and trithorax group proteins appear to regulate chromatin boundary activity by establishing higher-order domains of chromatin organization required for the assembly of functional insulators at the nuclear matrix, and the present genetic screening showed that a half-dose reduction of the trithorax group genes brm, osa, mor, and E(Pc) suppressed while a half-dose reduction of Dll enhanced the DREF-induced rough eye phenotype (Hirose, 2001).

brm is the Drosophila homologue of the yeast SWI2/SNF2 gene, isolated as a dominant suppressor of Pc mutations. osa shows strong genetic interactions with brm, suggesting that its gene product may cooperate closely with the BRM complex. The product of mor is homologous to yeast Swi3p and has also recently been identified as a BRM complex-associated protein, BAP. BRM, OSA, and MOR are part of a large multiprotein complex containing several other proteins with extensive homology to yeast SWI/SNF or the related chromatin-remodeling complex, and therefore the BRM complex has been predicted to be one of the Drosophila chromatin-remodeling complexes. It has been demonstrated that the BRM complex containing OSA and MOR is an essential coactivator for the trithorax group protein Zeste, a sequence-specific transcription regulator. Several homeotic and other genes carrying the Zeste-binding sequence in their promoter regions thus appear to be candidate target genes (or chromatin regions) for the BRM complex (Hirose, 2001).

Suppression of the DREF-induced rough eye phenotype by a half-dose reduction of some members of BRM complex genes suggests that DREF activity may be positively regulated by the BRM complex. The molecular basis for any interaction is not yet clear, but several possibilities exist. The first is that genetic interaction may result from a direct physical interaction between the two. To test this possibility, biochemical analyses are being undertaken. An alternative possibility is that the BRM complex is a regulator of the expression of some genes critical for induction of the rough eye phenotype by DREF overexpression. This is difficult to test because it is estimated that several hundred genes are under DREF control. Finally, it is possible that the BRM complex generally activates transcription by remodeling chromatin. However, a half-dose reduction of brm, mor, or osa did not change the severity of the rough eye phenotype induced by BEAF32, and mutations in brm, mor or osa enhance the rough eye phenotype induced by overexpression of dE2F/dDP/p35. Therefore, suppression of the DREF-induced rough eye phenotype by reduction of the dosage of brm, mor, or osa may reflect specific interactions between DREF and the BRM complex. Further studies are necessary to clarify the molecular basis and biological meaning of the interaction between DREF and the BRM complex (Hirose, 2001).

REGULATION

Promoter Structure

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).

Functionally distinct promoter classes initiate transcription via different mechanisms reflected in focused versus dispersed initiation patterns

Recruitment of RNA polymerase II (Pol II) to promoters is essential for transcription. Despite conflicting evidence, the Pol II preinitiation complex (PIC) is often thought to have a uniform composition and to assemble at all promoters via an identical mechanism. Using Drosophila melanogaster S2 cells as a model, this study demonstratea that different promoter classes function via distinct PICs. Promoter DNA of developmentally regulated genes readily associates with the canonical Pol II PIC, whereas housekeeping promoters do not, and instead recruit other factors such as DREF. Consistently, TBP and DREF are differentially required by distinct promoter types. TBP and its paralog TRF2 also function at different promoter types in a partially redundant manner. In contrast, TFIIA is required at all promoters, and this study identified factors that can recruit and/or stabilize TFIIA at housekeeping promoters and activate transcription. Promoter activation by tethering these factors is sufficient to induce the dispersed transcription initiation patterns characteristic of housekeeping promoters. Thus, different promoter classes utilize distinct mechanisms of transcription initiation, which translate into different focused versus dispersed initiation patterns (Serebreni, 2023).

Transcription of protein-coding genes by RNA polymerase II (Pol II) is a highly regulated process orchestrated by noncoding regulatory elements, namely enhancers and promoters. Pol II recruitment at promoters leads to transcription initiation from the core promoter region, a roughly 100 base-pair region around the transcription start site (TSS) at the 5' end of protein-coding genes. Although core promoter DNA fragments on their own are typically not sufficient for activity in vivo and support only low levels of transcription in vitro, the TATA-box core promoter is sufficient to bind the TATA-binding protein (TBP) and assemble the Pol II preinitiation complex ). This finding suggests that the core promoter DNA sequence has a crucially important function for PIC assembly and transcription and made the TATA-box core promoter subtype a prominent model for studies of PIC assembly and transcription initiation. Based on multiple lines of evidence, promoters in Drosophila melanogaster can be categorized into two broad classes (i) developmental promoters of developmentally regulated or cell-type-restricted genes that contain TATA-boxes, downstream promoter elements (DPEs), and/or Initiator (INR) motifs and (ii) housekeeping promoters of broadly or ubiquitously expressed genes that contain TCT, DRE, and Ohler1/6 motifs. These two classes of promoters exhibit distinctive regulatory properties, respond differently toward activating cues, and are activated by distinct sets of coactivators. In addition, developmental promoters typically display focused initiation at a single, dominant TSS, whereas housekeeping promoters typically display dispersed initiation at multiple TSSs) (Serebreni, 2023).

The general transcription factors (GTFs: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) assemble the PIC hierarchically at TATA-box core promoters: the TATA-binding protein (TBP) within TFIID binds to the TATA-box motif in promoter DNA and recruits TFIIA, followed by the remaining GTFs and Pol II. TFIIA cooperates with TFIID to commit PIC assembly into an active state on promoters in vitro. However, the nature of the PIC and PIC assembly at different core promoter subtypes and whether they relate to these promoters' distinct functions, remain unknown; moreover, the distinct properties of core promoter subtypes seem incompatible with a single mechanism of PIC assembly and transcription initiation (Serebreni, 2023).

Some evidence indeed suggests that different promoters utilize different PIC components. For example, some cells do not seem to require TBP, and some promoters require only a subset of GTFs for transcription in vitro or in cells, which is in line with the existence of different stable intermediates or alternative arrangements of the PIC on promoter DNA. Further, promoter-bound multi-subunit protein complexes that are part of the PIC, such as TFIID, can exhibit different arrangements. For instance, the Taf9 subunit of TFIID regulates cell-type-specific genes in neural stem cells, whereas the Taf3 subunit of TFIID activates cell-type-specific genes in myoblasts (Serebreni, 2023).

In addition, some GTFs might not be required in all cells and/or GTF paralogs may regulate transcription in distinct cell types or at specific promoters. The TBP-related factors TBP2 (also known as TRF3) and TBPL1 (TRF2 in Drosophila) have, for example, been implicated in transcription in early steps of mouse oocyte differentiation and during spermatogenesis, respectively, In Drosophila, Trf2 has been suggested to regulate the transcription of ribosomal protein genes, histone H1, and DPE motif-containing promoters. This cumulative evidence suggests that different promoter-bound GTF assemblies may exist on different promoter types and/or in different cell types, which potentially relates to these promoters' distinct properties (Serebreni, 2023).

This study used DNA affinity purification to identify proteins that closely interact with core promoters, combined with protein depletion and PRO-seq to identify proteins that are required for the transcriptional function of core promoters. Differential use of TBP and Trf2 was found at different promoter subtypes, and distinct recruitment mechanisms of TFIIA were found: TFIIA was enriched at developmental promoters in vitro and required for their activity in vivo, suggesting a direct recruitment mechanism and compact PIC architecture at this promoter class. In contrast, TFIIA was not enriched at housekeeping promoters in vitro but still required for their activity in vivo, suggesting an indirect recruitment mechanism and/or dispersed PIC architecture at these promoters. This work suggests that direct recruitment of TFIIA at developmental promoters leads to their focused initiation pattern, whereas indirect recruitment of TFIIA at housekeeping promoters leads to their dispersed initiation pattern (Serebreni, 2023).

In contrast to a prevalent model that Pol II PIC assembly and transcription activation occur similarly at all promoters, this study found that different core promoter types recruit and activate Pol II via distinct strategies that depend on different factors. Developmental promoter DNA is sufficient to recruit and assemble a Pol II PIC from nuclear extract in vitro, by having high affinity to GTFs such as TBP. Found as part of a soluble Pol II holoenzyme in yeast, TBP in complex with TFIIA is tightly associated with chromatin in metazoans and important in directing Pol II PIC assembly on DNA and cofactor mediated transcription in vitro (Serebreni, 2023).

The data indicate that most TATA-less promoters are independent of TBP and utilize TRF2, or TBP and TRF2 in a redundant fashion. Transcription in the absence of TBP has been observed for particular promoters, potentially involving TBP paralogs such as TRF2 in flies. Even though TRF2 has been reported to be unable to bind DNA directly, it may be recruited indirectly to promoters, potentially through interactions with TFIIA and/or TFIID. This is analogous to transcription initiation during oocyte growth when the mammalian TBP paralog TBPL2 cooperates with TFIIA to initiate transcription independently of TFIID. The promoters of snRNA genes also function independently of TBP yet depend on SNAPc. At these promoters, SNAPc seems to directly bind TFIIA and/or TFIIB via an interface shared with TBP (Serebreni, 2023).

The partial redundancy of TBP and TRF2, especially when one of the two is depleted reconciles the current results with recent structural studies of PIC assembly at non-TATA-box promoters: as TBPL1 or other TBP paralogs had not been considered during complex assembly in vitro, TBP was included in the PIC, irrespective of the promoter type. This might have been possible given the flexibility of the PIC, including TFIID that has been reported as sufficiently flexible to accommodate either TBP or TRF2 at different classes of promoters (Serebreni, 2023).

Interestingly, several proteins were found that had been described as insulator or architectural proteins bound to housekeeping promoters, both in vitro and in vivo. This is consistent with the observations that topological chromatin boundaries in Drosophila coincide with housekeeping genes. This could either be a coincidence or—more likely—reflect that these genomic regions and proteins mediate both functions. At least Chromator has transcription-activating activity toward housekeeping core promoters. It is interesting to speculate whether the housekeeping transcriptional program, which is inherently incompatible with cell-type-specific or developmental transcriptional regulation, can per se mediate insulation or if the respective factors have evolved both functions independently (Serebreni, 2023).

Housekeeping promoters also bind sequence-specific TFs such as DREF and M1BP, which in turn interact with cofactors such as GFZF, Chromator and Putzig that—directly or indirectly—recruit GTFs (e.g., TFIIA) and Pol II. These differences in the assembly and stability of the DNA–protein interface and protein complexes might explain the distinct transcription initiation patterns at developmental and housekeeping promoters, which generally exhibit focused and dispersed initiation patterns, respectively. Indeed, forced recruitment of housekeeping activators such as GFZF to arbitrary DNA sequences is sufficient to induce broad transcription initiation patterns, consistent with the initiation patterns observed at housekeeping promoters in vivo and with alternative PIC recruitment. This directly links the transcription-activating cofactors of developmental and housekeeping programs to the distinct initiation patterns observed for the respective promoters. It is note that even for dispersed housekeeping promoters, TSS choice is not entirely random or arbitrary but that certain positions seem to be favored, likely relating to local DNA structure, the energy barrier landscape for both DNA helix melting and phospho-diester-bond formation (Serebreni, 2023).

Given that key features of the promoter types, such as their initiation patterns, sequence motifs and their enhancer responsiveness is observed in Drosophila cell types as different as embryonic S2 cells and adult ovarian OSCs, and because GTFs are typically broadly expressed across cell types, the relative utilization of cofactors is expected to be similar in most cellular contexts. Moreover, while some of the specific TFs do not have one-to-one orthologs outside insects, focused and dispersed initiation patterns are widely observed across a wide range of species, including mammals. It will be exciting to see how homologous and analogous factors function at these distinct promoter types in different species (Serebreni, 2023).

The alternative mechanisms converge on TFIIA that is essential for transcription initiation at all promoter types. A central role of TFIIA recruitment for transcription initiation is consistent with the direct interaction of the TBP paralog TBPL2 with TFIIA in oocyte transcription, the direct interaction of SNAPc with TFIIA and/or TFIIB and noncanonical Pol II transcription of transposon-rich and H3K9me3-marked piRNA source loci in Drosophila germ cells through the TFIIA paralog moonshiner and TRF2. Essentiality for some or all promoter types might extend to other GTFs that could not be tested in this study, including TFIIB that is required at most promoters in human HAP1 cells (Serebreni, 2023).

Some features of Drosophila housekeeping promoters, including the dispersed patterns of transcription initiation, are similarly observed for the majority of vertebrate CpG island promoters comprising roughly 70% of all promoters; FANTOM Consortium and the RIKEN PMI and CLST. The functional regulatory dichotomy of these promoters combined with the evidence of distinct PIC composition and initiation mechanisms here and in other recent studies suggest that it is necessary to challenge the notion of a universal model of rigid and uniform PIC assembly. It will be exciting to see future functional, biochemical, and structural studies revealing more diverse transcription initiation mechanisms at the different promoter types in our genomes (Serebreni, 2023).

This study used two complementary strategies to explore the flexibility of enhancers with regard to nucleotide and motif identity at specific enhancer positions as well as the position dependence of motif activity. Even though median enhancer activity drops significantly when randomizing an 8-nt stretch at important positions, many sequence variants, including variants of the wild-type motif but also other TF motifs, can achieve strong enhancer activity. The diverse solutions at each position show that enhancers exhibit some degree of flexibility. However, as only a few hundred out of the >65,000 tested sequences work, the flexibility at any given position is constrained. Similarly, systematically pasting different motifs into hundreds of enhancer positions revealed that motif activity is strongly modulated by the enhancer sequence context. Therefore, constrained sequence flexibility and the modulation of motif function by the sequence context seem to be key features of enhancers (Serebreni, 2023).

The observation that both Drosophila and human TF motifs require specific enhancer sequence contexts suggests that this is a general principle of enhancers. Even though motifs possess some intrinsic strengths, their potential to activate transcription strongly depends on the sequence context and follows certain syntax rules, including motif flanks, combinations, and distances. Although this study cannot assess the mechanistic causes for these rules, they might be related to local DNA shape or to more general enhancer DNA properties such as DNA bending. The observation that homotypic interactions of certain motifs at close distances (e.g., GATA or ETS) are negatively associated with enhancer activity is consistent with repressive homotypic interactions between pluripotency TFs found by thermodynamic modeling; the mechanisms, however, are still unclear. Intermotif distances can impact the synergy between TFs at the level of DNA binding or after binding, such as cofactor recruitment and activation, which could explain both positive and negative TF-TF interactions. Although these syntax rules seem to be stricter for some TF motifs (e.g., GATA) and more relaxed for others (e.g., P53), the results show that motifs are not simply independent modules. Instead, they interact with all enhancer features in a highly cooperative manner, which can modulate motif activity by more than 100-fold. This is an important result that supports a model where enhancer activity is encoded through a complex interdependence between motifs and context, rather than motifs acting independently and additively. Whereas tissue- or cell type–specificity can already be predicted by motif presence-absence patterns alone, the encoding of different enhancer strengths seems to depend on more complex cis-regulatory syntax rules. The functional implications of mutations in TF motifs or elsewhere within enhancer sequences can therefore only be assessed in the context of these syntax features (Serebreni, 2023).

The motif syntax rules described in this study agree well with the ones learned by DeepSTARR trained on genome-wide enhancer activity data and the BPNet model trained on endogenous TF binding and cooperativity, suggesting that these rules are important in wild-type enhancer sequences. As an ectopic reporter assay STARR-seq measures the potential of sequences to act as enhancers, even if the sequences might be repressed endogenously at the chromatin level, making it a powerful tool to uncover the sequence determinants for enhancer activity. It will be interesting to explore the sequence rules and mechanisms by which chromatin modulates endogenous enhancer activities and gene expression using complementary methods. In addition, DeepSTARR also predicted with good accuracy the activity of all randomized sequence variants and of motifs pasted in different enhancer contexts. This supports the validity of computational models such as DeepSTARR and their use in in-silico-like experiments (e.g., motif pasting experiments with a larger set of TF motifs across many more genomic positions) to improve understanding of the regulatory information encoded in enhancer sequences and the impact of mutations (Serebreni, 2023).

This study shows that enhancer sequences are flexible enough for enhancer strength to be achieved by a small yet diverse set of sequence variants, and that mutations in information-poor positions have little impact on the enhancer activity in a single cell type. This flexibility allows many different sequences to achieve similar enhancer activities in a single cell type, which might be an important prerequisite for the evolution of developmental enhancers that operate under many additional constraints, for example, regarding the precise spatiotemporal control of enhancer activities. As the activity in a given cell can be achieved by many solutions, the specific solutions that fulfill additional requirements can be explored during evolution. Indeed, previous studies that have analyzed expression changes of enhancer mutations across different cell types in vivo have observed that the cell type–specific expression patterns of enhancers can change upon (minimal) sequence perturbations. The fact that enhancer strength in any given cell type and enhancer specificity across cell types and developmental time are subject to different yet overlapping sequence constraints highlights the complexity of the regulatory code. It is expected that the combination of quantitative enhancer-sequence-to-function models in individual cell types and qualitative predictions of enhancer activities across cell types will provide unprecedented progress in understanding of enhancer biology and the ability to read and write enhancer sequences (Serebreni, 2023).

Transcriptional Regulation

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).

Targets of Activity

Regulation of zerknüllt by DREF

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).

Transcriptional regulation of the Drosophila CycA gene by the DNA replication-related element (DRE) and DRE binding factor (DREF)

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).

DNA replication-related elements cooperate to enhance promoter activity of the drosophila DNA polymerase alpha 73-kDa subunit gene

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).

Regulation of PCNA by DREF

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).

Transcriptional regulation of the Drosophila-raf proto-oncogene by the DNA replication-related element (DRE)/DRE-binding factor (DREF) system

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).

The DNA replication-related element (DRE)/DRE-binding factor system is a transcriptional regulator of the Drosophila E2F gene

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).

An antagonistic relationship between the boundary element-associated factor BEAF and DREF

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).

Differential regulation of the catalytic and accessory subunit genes of Drosophila mitochondrial DNA polymerase

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).

DREF targets TBP

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).

Regulation of mitochondrial single-stranded DNA-binding protein gene expression links nuclear and mitochondrial DNA replication in Drosophila

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).

Drosophila mitochondrial transcription factor A: characterization of its cDNA and expression pattern during development

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)

The DNA replication-related element-binding factor (DREF) is a transcriptional regulator of the Drosophila myb gene

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).

Drosophila damage-specific DNA-binding protein 1 (DDB1) is controlled by the DRE/DREF system

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).

A genomic switch at the transition from cell proliferation to terminal differentiation in the Drosophila eye

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 Myelodysplasia/myeloid leukemia factor (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).

Drosophila proteasome regulator REGgamma: transcriptional activation by DNA replication-related factor DREF and evidence for a role in cell cycle progression

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).

Transcriptional regulation of the caudal gene by DRE/DREF

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).

A DNA replication-related element downstream from the initiation site of Drosophila selenophosphate synthetase 2 gene is essential for its transcription

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).

Transcriptional regulation of the Drosophila catalase gene by the DRE/DREF system

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).

Intimate relationship between the genes of two transcriptional coactivators, ADA2a and PIMT, of Drosophila: regulation of ADA2a by DREF

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).

Transcriptional regulation of the Drosophila orc2 gene by the DREF pathway

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).

Identification of the Drosophila skpA gene as a novel target of the transcription factor DREF

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).

Redox regulation of DNA binding activity of DREF

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).

Identification of the Drosophila skpA gene as a novel target of the transcription factor DREF

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 DRE motif is a key component in the expression regulation of the importin-β encoding Ketel gene in Drosophila

Importin-β, encoded by the Ketel gene in Drosophila, is a key component of nuclear protein import, the formation of the spindle microtubules and the assembly of the nuclear envelope. The Drosophila embryos rely on the maternal importin-β dowry at the beginning of their life. Expression of the zygotic Ketel gene commences during gastrulation in every cell and while the expression is maintained in the mitotically active diploid cells it ceases in the non-dividing larval cells in which nuclear protein import is assured by the long persisting importin-β molecules. How is the expression of the Ketel gene regulated? In silico analysis revealed several conserved transcription factor binding sequences in the Ketel gene promoter. Reporter genes in which different segments of the promoter ensured transient expression of the luciferase gene in S2 cells identified the sequences required for normal Ketel gene expression level. Gel retardation and band shift assays revealed that the DREF and the CFDD transcription factors play key roles in the regulation of Ketel gene expression. Transgenic LacZ reporter genes revealed the sequences that ensure tissue-specific gene expression. Apparently, the regulation of Ketel gene expression depends largely on a DRE motif and action of the DREF, CFDD, CF2-II and BEAF transcription factors (Villanyi, 2008).

The Ketel gene has been known to be expressed (1) in the egg primordia to provide importin-β for oogenesis and early embryogenesis, (2) in every cell of the gastrulating embryo and (3) in the diploid cells during larval life, but not in the polytenic larval cells. The polytenic cells need relatively few importin-β molecules to accomplish nuclear protein import, and this duty is accomplished by the unusually long-lived importin-β molecules, some of which are maternally provided others are produced during early gastrulation. Intensive expression of the Ketel gene in the diploid cells is comprehensible since these cells need importin-β not only for nuclear protein import but also for the formation of the spindle microtubules and the reassembly of the nuclear envelope at the end of mitosis. The aim of the present study was to understand the mechanisms that ensure the characteristic expression pattern of the Ketel gene. To achieve this goal, cis-acting control elements were that are engaged in (1) the proper loading of the egg cytoplasm with the Ketel gene products, (2) the regulation of the all-over type of importin-β production during gastrulation and (3) controlling tissue-specific expression of the Ketel gene during the later stages of development (Villanyi, 2008).

Computer analysis revealed several evolutionarily conserved transcription factor binding sites in the Ketel promoter of which only the CF2-II, the CFDD, the DREF and perhaps the BEAF binding sites are of relevance. The CFDD, the DREF and the BEAF transcription factors have been known to be involved in the expression regulation of a number of genes engaged in cell cycle regulation. In fact, the CFDD binding sites are commonly present in the promoters of a number of DNA replication-related genes like PCNA and DREF. Since importin-β is required for spindle formation and nuclear envelope assembly, which are essential events in cell proliferation, it may not be surprising that the expression of the Ketel gene is regulated by the same transcription factors that control the expression of several genes engaged in cell cycle regulation (Villanyi, 2008).

Transient expression of a luciferase reporter gene in S2 cells clearly showed that all the sequences which regulate Ketel gene expression reside within a 750 bp sequence towards the 5' region of the Ketel gene. The 'active' transcription factor binding sequences within the region were identified in gel-shift experiments, and the sequences that ensure tissue-specific expression of the Ketel gene were determined through the analysis of the expression patterns of LacZ reporter transgenes (Villanyi, 2008).

It appears that the presence of an approximately 140 bp long sequence around the transcription start site is sufficient for a basic expression of the Ketel gene in the gonial cells. The simultaneous presence of two sequences is required for the expression of the Ketel gene in the nurse cells and for the loading of the egg cell cytoplasm with the Ketel gene products: a CFDD binding site in the first intron (around +247) and the DRE motif around −74. (Note that the importin-β-related maternal effect depends on the expression of the Ketel gene in the germ line components of the egg primordia; Tirian, 2000) Removal of either of these sequences leads to an absence of Ketel gene expression in the nurse cells. Similarly, the concurrent presence of the DRE motif at −74 and the CFDD site(s) around −250 is necessary for the expression of the Ketel gene in every cell of the gastrulating embryo. Removal of any of these sequences abolishes Ketel gene expression during early gastrulation. It appears that cooperative binding of transcription factors to the DRE motif and to either of the CFDD recognition sites establishes favourable conditions for tissue-specific expression of the Ketel gene. A CF2-II binding site around −483 is sufficient and necessary for the expression of the Ketel gene in the diploid cells of the imaginal discs, the neuroblasts and the follicle epithelium. CF2-II has been reported to be expressed in the follicle cells and seems to be the only candidate to control Ketel gene expression in the imaginal disc cells and in the neuroblasts (Villanyi, 2008).

Interestingly, none of the six different types of LacZ reporter transgenes are expressed in any polytenic larval cell types. One possible explanation could be the different modes of action of DREF in the larval and in the diploid cells: DREF does not displace BEAF from the DRE motif in the larval cells and, thus, an insulator can form which blocks transcription of the Ketel gene. Three BEAF binding sites are necessary for the formation of an insulator, and the promoter of the Ketel gene contains three BEAF binding sites, one of which is part of the DRE motif. In the diploid cells, where DREF binds to the DRE motif and competes with BEAF, the insulator cannot form and, hence, there is no block to prevent expression of the Ketel gene. However, the above model is rather unlikely since when the DRE motif, and along with it one of the BEAF binding sites, is abolished the BEAF insulator cannot form. Yet, the Ketel gene is not expressed in the larval cells. The lack of Ketel gene expression in the larval cells can also be explained by the absence of CF2-II transcription factor in that cell type. Further studies are needed to ascertain whether this assumption is correct (Villanyi, 2008).

In summary, the DRE motif is a key component in the regulation of Ketel gene expression: transcription factors that bind to the DRE motif interact with different CFDDs, which are bound to different CFDD binding sites, ensuring tissue-specific expression of the gene. The DRE motif and the CFDD sites are commonly present in the promoter of several genes engaged in DNA replication and cell cycle control, and interaction of DREF and CFDD could be a key component in the regulation of those genes as well (Villanyi, 2008).

Transcriptional regulation of the Drosophila moira and osa genes by the DREF pathway

The DNA replication-related element binding factor (DREF) plays an important role in regulation of cell proliferation in Drosophila, binding to DRE and activating transcription of genes carrying this element in their promoter regions. Overexpression of DREF in eye imaginal discs induces a rough eye phenotype in adults, which can be suppressed by half dose reduction of the osa or moira (mor) genes encoding subunits of the BRM complex. This ATP-dependent chromatin remodeling complex is known to control gene expression and the cell cycle. In the 5' flanking regions of the osa and mor genes, DRE and DRE-like sequences exist which contribute to their promoter activities. Expression levels and promoter activities of osa and mor are decreased in DREF knockdown cells and the results in vitro and in cultured cells indicate that transcription of osa and mor is regulated by the DRE/DREF regulatory pathway. In addition, mRNA levels of other BRM complex subunits and a target gene, string/cdc25, were found to be decreased by knockdown of DREF. These results indicate that DREF is involved in regulation of the BRM complex and thereby the cell cycle (Nakamura, 2008).

This study demonstrated that both osa and mor are DREF target genes. Thus osa and mor promoters exhibited decreased activities when carrying mutations in their DREs and after knockdown of DREF in cultured cells. In addition, levels of osa and mor mRNAs were reduced in DREF knockdown cells. Third, DREF can bind to DREs of osa and mor in vitro, and binding of DREF to the genomic regions containing DREs of both genes was observed in cultured cells. These results showed that DRE and DREF are important for osa and mor promoter activation. Promoters having mutations in all DREs of both osa and mor genes, however, still retained some activity. It is therefore possible that another element(s) and/or unknown factor(s) regulated by DREF are involved in osa and mor transcriptional activation. The observed rescue of the DREF-induced rough eye phenotype by a reduction in the osa and moire gene dosage is consistent with the idea that the osa and moire gene transcription is activated by DREF. However,the possibility cannot be excluded that the rescue could also be affected by a mechanism involving protein-protein interactions between DREF and BAP/PBAP at the promoters of cell cycle-regulated genes. Further analyses are necessary to address this point (Nakamura, 2008).

Both osa and mor encode components of the BRM complex, which is a SWI/SNF type ATP-dependent chromatin remodeling complex conserved from yeast to human, with two forms, BAP and PBAP. Osa is a signature subunit of BAP, while PBAP contains Polybromo and BAP170 in its place. Localization patterns of Osa and Polybromo on polytene chromosomes differ, though several sites overlap. Whole-genome expression analysis also demonstrated that BAP and PBAP differentially regulate gene expression. For example, Osa negatively regulates expression of the Wingless-target genes and the achaete/scute gene. Osa, Polybromo and BAP170 are all required for function of BRM complex. It is thought that Osa functions in recruitment of BAP to its target genes. Mor, a subunit common to both BAP and PBAP, is presumed to be essential for complex integrity, since its absence results in degradation of both forms. SRG3, which is a homolog of Mor in mammals, also acts for complex stabilization by protecting against proteasomal degradation. Therefore, Osa and Mor are essential subunits for function and stabilization of BRM complexes and DREF may control integrity of the BRM complex through activating osa and mor gene expression (Nakamura, 2008).

BAP and PBAP share seven subunits, Brm, Mor, Snr1, BAP111, BAP60, BAP55 and Actin. Brm is a catalytic subunit harboring the ATPase domain and it was previously reported that reduction of the brm gene dose suppressed the DREF-induced rough eye phenotype. It was also found the the mRNA level of brm is decreased in DREF knockdown cells. However, DRE-like sequences in the second intron, do not appear to function as regulatory elements, since DREF does not bind to the genomic region containing these sites in vivo. DREF may therefore indirectly control brm gene expression (Nakamura, 2008).

The genes coding for BAP55 and BAP60, common subunits for BAP and PBAP, also contain DRE or DRE-like sequences in their 5' flanking regions and are affected by DREF knockdown. DREF binds to the genomic regions containing their DREs in vivo and it is, therefore, possible that BAP55 and BAP60 are directly regulated by DREF. Furthermore, the PBAP-specific subunit BAP170 carries a DRE in its 5' flanking region. Reduction of mRNA levels of osa, polybromo and BAP170 in DREF knockdown cells also is evidence that DREF contributes to the transcriptional regulation of both BAP and PBAP complexes. Therefore, DREF may regulate expression of genes coding for most subunits for both BAP and PBAP complexes and influence expression of many genes through chromatin remodeling (Nakamura, 2008).

It has been reported that OSA-containing BAP complexes are necessary for G2/M progression through stg promoter activation while PBAP complexes are not. stg encodes a CDC25 phosphatase, which is required for G2/M progression. It is well known that DREF predominantly regulates the transcription of DNA replication-related genes. Reduced stg mRNA has been reported in DREF-eliminated cells and this study also observed reduction of stg mRNA levels in DREF knockdown cells, as with brm, osa and mor. In addition to regulation of S phase entry, DREF thus appears to play an important role in G2/M transition by activating the BAP complex to promote cell cycling. Two DRE-like sequences were found in the stg gene upstream region, -219 to -212 (5'-aATCGATg) and -591 to -584 (5'-TATCGATt). Therefore, DREF could regulate stg gene expression directly via binding to DRE-like and/or indirectly via activation of genes coding for BAP complexes. Further analysis is necessary to distinguish these possibilities (Nakamura, 2008).

BRM complexes are thought to inhibit S phase entry and mutations of brm, osa and mor suppress the rough eye phenotype induced by E2F/DP/p35 overexpression. The rough eye phenotype of a cyclin E hypomorphic mutant was also suppressed by BRM complex mutation through increase in the S phase. Therefore, BRM complexes appear to negatively regulate S phase entry, while DREF activates E2F gene transcription and promotes G1/S progression. Although osa is ubiquitously expressed in eye imaginal discs, it is most intensely expressed anterior to the morphogenic furrow where cells enter the G1 phase. Similarly, DREF is strongly expressed in this region. It is conceivable that DREF simultaneously activates both positive and negative regulators of G1/S progression. This kind of regulation may be necessary for fine tuning of cell cycle progression to inhibit excess S phase induction (Nakamura, 2008).

Drosophila DREF acting via the JNK pathway is required for thorax development

The Drosophila Jun N-terminal kinase (JNK) gene basket (bsk) promoter contains a DNA replication-related element (DRE) like sequence, raising the possibility of regulation by the DNA replication-related element-binding factor (DREF). Chromatin immunoprecipitation assays with anti-DREF IgG showed the bsk gene promoter region to be effectively amplified. Luciferase transient expression assays revealed the DRE like sequence to be important for bsk gene promoter activity, and knockdown of DREF decreased the bsk mRNA level and the bsk gene promoter activity. Furthermore, knockdown of DREF in the notum compartment of wing discs by pannier-GAL4 and UAS-DREFIR resulted in a split thorax phenotype. Monitoring of JNK activity in the wing disc by LacZ expression in a puckered (puc)-LacZ enhancer trap line revealed the reduction in DREF knockdown clones. These findings indicate that DREF is involved in regulation of Drosophila thorax development via actions on the JNK pathway (Yoshioka, 2012).

DREF is involved in the steroidogenesis via regulation of shadow gene

The Drosophila DNA replication-related element-binding factor (dDREF) has been identified as a master regulator of cell proliferation-related genes via its binding to the DRE sequence, 5'-TATCGATA. However, the biological roles of DREF are still to be clarified. This study show that DREF mutant females have steroid hormone ecdysone-deficient phenotypes, such as the loss of vitellogenic egg chambers. Furthermore, DREF knockdown in the prothoracic gland of larva prevented pupation and this was rescued via 20-hydroxyecdysone treatment. A DRE-like sequence (-625 to -632) was found in the 5'-flanking region of the Drosophila shadow gene, which catalyzes the conversion of 2-deoxyecdysone to ecdysone, and it was demonstrated that shadow is a novel target gene of dDREF using quantitative RT-PCR and Chip assays. In addition, the level of dDREF protein was shown to be correlated with age-related changes in the level of shadow mRNA in the ovaries of wild-type flies. Taken together, these data indicate that dDREF plays a key role in steroid synthesis via regulation of the shadow gene (Park, 2012).

Enhancer--core-promoter specificity separates developmental and housekeeping gene regulation

Gene transcription in animals involves the assembly of RNA polymerase II at core promoters and its cell-type-specific activation by enhancers that can be located more distally. However, how ubiquitous expression of housekeeping genes is achieved has been less clear. In particular, it is unknown whether ubiquitously active enhancers exist and how developmental and housekeeping gene regulation is separated. An attractive hypothesis is that different core promoters might exhibit an intrinsic specificity to certain enhancers. This is conceivable, as various core promoter sequence elements are differentially distributed between genes of different functions, including elements that are predominantly found at either developmentally regulated or at housekeeping genes. This study shows that thousands of enhancers in Drosophila melanogaster S2 and ovarian somatic cells (OSCs) exhibit a marked specificity to one of two core promoters-one derived from a ubiquitously expressed ribosomal protein gene and another from a developmentally regulated transcription factor-and confirm the existence of these two classes for five additional core promoters from genes with diverse functions. Housekeeping enhancers are active across the two cell types, while developmental enhancers exhibit strong cell-type specificity. Both enhancer classes differ in their genomic distribution, the functions of neighbouring genes, and the core promoter elements of these neighbouring genes. In addition, two transcription factors -- Dref and Trl -- were identified that bind and activate housekeeping versus developmental enhancers, respectively. These results provide evidence for a sequence-encoded enhancer-core-promoter specificity that separates developmental and housekeeping gene regulatory programs for thousands of enhancers and their target genes across the entire genome (Zabidi, 2014).

The core promoter of Ribosomal protein gene 12 (RpS12) and a synthetic core promoter derived from the even skipped transcription factor were chosen as representative 'housekeeping' and 'developmental' core promoters, respectively (hereafter termed hkCP and dCP), and the ability of all candidate enhancers genome wide to activate transcription from these core promoters was tested using self-transcribing active regulatory region sequencing (STARR-seq) in D. melanogaster S2 cells. This set-up allows the testing of all candidates in a defined sequence environment, which differs only in the core promoter sequences but is otherwise constant (Zabidi, 2015).

Two hkCP STARR-seq replicates were highly similar [genome-wide Pearson correlation coefficient (PCC) 0.98] and yielded 5,956 enhancers, compared with 5,408 enhancers obtained when dCP STARR-seq data was reanalyzed. Interestingly, the hkCP and dCP enhancers were largely non-overlapping and the genome-wide enhancer activity profiles differed (PCC 0.38), as did the individual enhancer strengths: of the 11,364 enhancers, 8,144 (72%) activated one core promoter at least twofold more strongly than the other, a difference rarely seen in the replicate experiments for each of the core promoters. Indeed, 21 out of 24 hkCP-specific enhancers activated luciferase expression (>1.5-fold) from the hkCP versus 1 out of 24 from the dCP. Consistently, 10 out of 12 dCP-specific enhancers were positive with the dCP but only 2 out of 12 with the hkCP, a highly significant difference that confirms the enhancer–core-promoter specificity observed for thousands of enhancers across the entire genome (Zabidi, 2015).

Enhancers that were specific to either the hkCP or the dCP showed markedly different genomic distributions: whereas the majority (58.4%) of hkCP-specific enhancers overlapped with a transcription start site (TSS) or were proximal to a TSS (<200 bp upstream), dCP-specific enhancers located predominantly to introns (56.5%) and intergenic regions (26.9%). Importantly, despite the TSS-proximal location of most hkCP-specific enhancers, they activated transcription from a distal core promoter in STARR-seq. Luciferase assays confirmed that they function from a distal position (>2 kb from the TSS) downstream of the luciferase gene and independently of their orientation towards the luciferase TSS. These results show that TSS-proximal sequences can act as bona fide enhancers and that developmental and housekeeping genes are both regulated through core promoters and enhancers, yet with a substantially different fraction of TSS-proximal enhancers (3.4% versus 58.4%; Zabidi, 2015).

hkCP and dCP enhancers were also located next to functionally distinct classes of genes according to gene ontology (GO) analyses: genes next to hkCP enhancers were enriched in diverse housekeeping functions including metabolism, RNA processing and the cell cycle, whereas genes next to dCP enhancers were enriched for terms associated with developmental regulation and cell-type-specific functions. Consistently, hkCP enhancers were preferentially near ubiquitously expressed genes and dCP enhancers were near genes with tissue-specific expression (Zabidi, 2015).

The core promoters of the putative endogenous target genes of hkCP and dCP enhancers were also differentially enriched in known core promoter elements: TSSs next to hkCP enhancers were enriched in Ohler motifs 1, 5, 6 and 7, consistent with the ubiquitous expression and housekeeping functions of these genes. In contrast, TSSs next to dCP enhancers were enriched in TATA box, initiator (Inr), motif ten element (MTE) and downstream promoter element (DPE) motifs, which are associated with cell-type-specific gene expression (Zabidi, 2015).

Whether the specificity that hkCP and dCP show to the two enhancer classes applies more generally was tested. Three additional core promoters were chosed from housekeeping genes with different functions: from the eukaryotic translation elongation factor 1δ (eEF1δ) , the putative splicing factor x16, and the cohesin loader Nipped-B (NipB). Importantly, all three contained combinations of core promoter elements that differed from that of hkCP, namely TCT and DNA-replication-related element (DRE) motifs (eEF1δ), and Ohler motifs 1 and 6 (x16 and NipB). In addition, a DPE-containing core promoter of the transcription factor pannier (pnr) and the TATA-box core promoter of Heat shock protein 70 (Hsp70) , which can be activated by tissue-specific enhancers, were tested, thus covering the two most prominent core promoter types of regulated genes (Zabidi, 2015).

To assess whether the marked core promoter specificities of the hkCP and dCP enhancers are encoded in their sequences, the cis-regulatory motif content of both classes of enhancers was examined. This revealed a strong enrichment of the DRE motif in hkCP enhancers, whereas dCP enhancers were strongly enriched in the GAGA motif of Trithorax-like (Trl) and other motifs previously described to be important for dCP enhancers. Published genome-wide chromatin immunoprecipitation (ChIP) data confirmed that DRE-binding factor (Dref) bound significantly more strongly to hkCP enhancers than to dCP enhancers, while the opposite was true for Trl. Considering only distal enhancers (>500 bp from the closest TSS) yielded the same results, suggesting that the differential occupancy is a property of both classes of enhancers rather than a consequence of the different extents to which they overlap with TSSs. Disrupting the DRE motifs in four different hkCP enhancers substantially reduced the activities of the enhancers as measured by luciferase assays in S2 cells (between 2.3- and 24.5-fold reduction), while dCP enhancers depend on GAGA motifs. Adding DRE motifs to 11 different dCP enhancers significantly increased luciferase expression from the hkCP for 9 of them (82%), and changing the GAGA motifs of two dCP enhancers to DRE motifs significantly increased the activities of both enhancers towards the hkCP but decreased their activities towards the dCP. Furthermore, an array of six DRE motifs was sufficient to activate luciferase expression from the hkCP but not the dCP. Together, these results show that hkCP and dCP enhancers depend on DRE and GAGA motifs, respectively, and demonstrate that DRE motifs are required and sufficient for hkCP enhancer function (Zabidi, 2015).

These results show that developmental and housekeeping gene regulation is separated genome wide by sequence-encoded specificities of thousands of enhancers to one of two types of core promoter, supporting the longstanding 'enhancer–core-promoter specificity' hypothesis. The findings indicate that these specificities are probably mediated by defined biochemical compatibilities between different trans-acting factors such as Dref versus Trl (at enhancers) and the different paralogues that exist for several components of the general transcription apparatus (at core promoters), presumably including the TATA-box-binding protein-related factor 2 (Trf2) at housekeeping core promoters. As such paralogues can have tissue-specific expression and stage-specific or promoter-selective functions, sequence-encoded enhancer-core-promoter specificities could be used more widely to define and separate different transcriptional programs (Zabidi, 2015).

The Hippo pathway as a target of the Drosophila DRE/DREF transcriptional regulatory pathway

The DRE/DREF transcriptional regulatory system has been demonstrated to activate a wide variety of genes with various functions. In Drosophila, the Hippo pathway is known to suppress cell proliferation by inducing apoptosis and cell cycle arrest through inactivation of Yorkie, a transcription co-activator. This study found that half dose reduction of the hippo (hpo) gene induces ectopic DNA synthesis in eye discs that is suppressed by overexpression of DREF. Half reduction of the hpo gene dose reduced apoptosis in DREF-overexpressing flies. Consistent with these observations, overexpression of DREF increased the levels of hpo and phosphorylated Yorkie in eye discs. Interestingly, the diap1-lacZ reporter was seen to be significantly decreased by overexpression of DREF. Luciferase reporter assays in cultured S2 cells revealed that one of two DREs identified in the hpo gene promoter region was responsible for promoter activity in S2 cells. Furthermore, endogenous hpo mRNA was reduced in DREF knockdown S2 cells, and chromatin immnunoprecipitation assays with anti-DREF antibodies proved that DREF binds specifically to the hpo gene promoter region containing DREs in vivo. Together, these results indicate that the DRE/DREF pathway is required for transcriptional activation of the hpo gene to positively control Hippo pathways (Vo, 2014: PubMed).

A transcriptional network controlling glial development in the Drosophila visual system

In the nervous system, glial cells need to be specified from a set of progenitor cells. In the developing Drosophila eye, perineurial glia proliferate and differentiate as wrapping glia in response to a neuronal signal conveyed by the FGF receptor pathway. To unravel the underlying transcriptional network, this study silenced all genes encoding predicted DNA-binding proteins in glial cells using RNAi. Dref and other factors of the TATA box-binding protein-related factor 2 (TRF2) complex were previously predicted to be involved in cellular metabolism and cell growth. Silencing of these genes impaired early glia proliferation and subsequent differentiation. Dref was found to control proliferation via activation of the Pdm3 transcription factor, whereas glial differentiation was regulated via Dref and the homeodomain protein Cut. Cut expression was controlled independently of Dref by FGF receptor activity. Loss- and gain-of-function studies showed that Cut was required for glial differentiation and was sufficient to instruct the formation of membrane protrusions, a hallmark of wrapping glial morphology. This work discloses a network of transcriptional regulators controlling the progression of a naïve perineurial glia towards the fully differentiated wrapping glia (Bauke, 2015).

Using a genome-wide RNAi-based screen this study has unravelled the transcriptional machinery responsible for such a switch during gliogenesis in the Drosophila eye. During embryonic development the anlage of the eye imaginal disc is formed. It is attached to the forming brain through the so-called Bolwig's nerve. A few glial cells reside along this nerve, presumably generated in the segmental nerves, as are most of the glia. These glial cells proliferate extensively during larval stages to form ~300 glial cells within each eye imaginal disc. During the third larval stage ~50 of these cells differentiate into wrapping glia in an FGFR-dependent manner. This study shows that the proliferation of the glial progenitor pool requires the activity of Pdm3 and the DNA replication-related element-binding factor (Dref), which are both strongly expressed by proliferating perineurial glia. Dref was first identified as an important factor required for efficient transcription of the proliferating cell nuclear antigen (PCNA), a key regulator of replication. Dref protein associates with the TATA box-binding protein related factor 2 (TRF2), which functions as a core promoter selectivity factor that governs a restricted subset of genes co-ordinately regulated. Interestingly, pan-glial knockdown of TRF2 also results in lethality, suggesting that the Dref/TRF2 complex is active in glia. Knockdown of CG30020, encoding a member of the Dref/TRF2 complex, or osa and moira, which had been shown previously to interact with Dref, caused similar glial phenotypes in the visual system (Bauke, 2015).

TRF2 targets several classes of TATA-less promoters present in more than 1000 genes, including a cluster of ribosomal protein genes. Likewise, Dref was found to associate with many genes involved in protein synthesis and cell growth, and loss of Dref results in reduced organismal growth rates. Most likely, dividing glial cells as well as differentiating wrapping glia have an increased protein synthesis demand, which might explain the observed defects in proliferation and differentiation. This study shows that expression of the transcription factor Pdm3 depends on Dref. Previously, Pdm3 has been associated with axonal pathfinding. The current results indicate that Pdm3 also regulates cell number. In contrast to Dref, Pdm3 expression is repressed by FGFR signalling, ensuring that perineurial glia routed to differentiation do not express Pdm3 anymore (Bauke, 2015).

Previous work suggested that in the Drosophila eye imaginal disc perineurial glial cells at the anterior margin of the eye field are competent to react to a neuronal signal inducing their glial differentiation. During this phase glial cells have reduced Dref expression but increased FGFR activity. Whereas in the absence of FGFR signalling no glial differentiation can be observed, high levels of FGF signalling trigger the expression of Cut specifically in wrapping glia. In addition to Cut, Dref is essential for proper glial differentiation. Dref is required for normal Cut expression levels but gain of Dref function is unable to activate Cut ectopically in perineurial glial cells. This requires additional FGFR signalling, indicating that two parallel molecular pathways converge on the activation of the transcription factor Cut to orchestrate wrapping glial differentiation (Bauke, 2015).

In the Drosophila PNS Cut controls the ES/ChO lineage decision. By contrast, during glial cell development this work defined Cut as a master regulator organizing elaborated membrane growth, which is required during the wrapping of axons. Similarly, Cut instructs the morphogenesis of multi-dendritic neurons. In mammals, the Cut homologues Cux1/2 also control dendritic branching, the number of dendritic spines and synapses. The number of filopodial extensions correlates to the level of Cut expression, corresponding to these findings. It was recently shown that Cut-dependent filopodia formation depends on the function of CrebA, which activates components of the secretory pathway. Cut might not only orchestrate membrane organization through the modulation of the secretory pathway, it also directly controls cytoskeletal dynamics. In larval sensory da neurons, the actin bundling protein Fascin is necessary for a Cut-dependent induction of spiked cell protrusions. However, eye disc glial cells still form long cell processes when fascin expression is suppressed by RNAi. Further understanding of wrapping glial cell differentiation will require the identification of the transcriptional targets of Cut (Bauke, 2015).

In conclusion, this study demonstrates that the specification of wrapping glial cells in the developing visual system does not require a single lineage switch gene but rather appears as a gradual process. The specification of wrapping glia is orchestrated by a transcriptional network comprising Pdm3, Dref and Cut that is modulated by the activity of the FGFR (Bauke, 2015).

Protein Interactions

Drosophila myeloid leukemia factor interacts with DREF

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).

Characterization of the Drosophila myeloid leukemia factor

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, Myelodysplasia/myeloid leukemia factor (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 shows 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 mutant adults display weak phenotypes. dMLF subcellular localization in the fly and cultured cells was monitored. 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, it was demonstrated that dMLF can act developmentally and postdevelopmentally to suppress neurodegeneration and premature aging in a cerebellar ataxia model (Martin-Lanneree, 2006).

TRF2 associates with DREF and Iswi and regulates the PCNA promoter

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).

Drosophila Mi-2 negatively regulates dDREF by inhibiting its DNA-binding activity

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).

Drosophila distal-less negatively regulates dDREF by inhibiting its DNA binding activity

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).

XNP/dATRX interacts with DREF in the chromatin to regulate gene expression

The ATRX gene encodes a chromatin remodeling protein that has two important domains, a helicase/ATPase domain and a domain composed of two zinc fingers called the ADD domain. The ADD domain binds to histone tails and has been proposed to mediate their binding to chromatin. The putative ATRX homolog in Drosophila (XNP/dATRX) has a conserved helicase/ATPase domain but lacks the ADD domain. In this study, it is proposed that XNP/dATRX interacts with other proteins with chromatin-binding domains to recognize specific regions of chromatin to regulate gene expression. A novel functional interaction is reported between XNP/dATRX and the cell proliferation factor DREF in the expression of pannier (pnr). DREF binds to DNA-replication elements (DRE) at the pnr promoter to modulate pnr expression. XNP/dATRX interacts with DREF, and the contact between the two factors occurs at the DRE sites, resulting in transcriptional repression of pnr. The occupancy of XNP/dATRX at the DRE, depends on DNA binding of DREF at this site. Interestingly, XNP/dATRX regulates some, but not all of the genes modulated by DREF, suggesting a promoter-specific role of XNP/dATRX in gene regulation. This work establishes that XNP/dATRX directly contacts the transcriptional activator DREF in the chromatin to regulate gene expression (Valadez-Graham, 2012; full text of article).

DEVELOPMENTAL BIOLOGY

Embryonic

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).

Larval

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).

EFFECTS OF MUTATION

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

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-DREF_1-709). 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-DREF_1-709 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-DREF_1-709. To confirm that ectopic expression of DREF-directly affects eye development, transgenic flies were established carrying pGMR-DREF_1-709. Flies carrying pGMR-DREF_1-709 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-DREF_1-709, 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).

Over-expression of DREF in the Drosophila wing imaginal disc induces apoptosis and a notching wing phenotype

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).

DREF is required for efficient growth and cell cycle progression in Drosophila imaginal discs

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 DREF^RNAi). 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 DREF^RNAi 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 DREF^RNAi 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 DREF^RNAi 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 DREF^RNAi 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 DREF^RNAi 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 DREF^RNAi expression. At higher levels of DREF^RNAi 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 DREF^RNAi 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, DREF^RNAi 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 DREF^RNAi 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 DREF^RNAi 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, DREF^RNAi expression was induced ubiquitously in third instar larvae using the TS-Gal80 TARGET system. At various time points after DREF^RNAi 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 DREF^RNAi, 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, DREF^RNAi 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 DREF^RNAi 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 DREF^RNAi 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 DREF^RNAi-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 DREF^RNAi 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).

DREF is required for EGFR signalling during Drosophila wing vein development

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).

REFERENCES

Search PubMed for articles about Drosophila DNA replication-related element factor

Bauke, A.C., Sasse, S., Matzat, T. and Klämbt, C. (2015). A transcriptional network controlling glial development in the Drosophila visual system. Development 142(12):2184-93. PubMed ID: 26015542

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

Nakamura, K., Ida, H. and Yamaguchi, M. (2008). Transcriptional regulation of the Drosophila moira and osa genes by the DREF pathway. Nucleic Acids Res. 36: 3905-3915. PubMed Citation: 18511465

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, J. S., Choi, Y. J., Thao, D. T., Kim, Y. S., Yamaguchi, M. and Yoo, M. A. (2012). DREF is involved in the steroidogenesis via regulation of shadow gene. Am J Cancer Res 2: 714-725. PubMed ID: 23226617

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

Serebreni, L., Pleyer, L. M., Haberle, V., Hendy, O., Vlasova, A., Loubiere, V., Nemcko, F., Bergauer, K., Roitinger, E., Mechtler, K. and Stark, A. (2023). Functionally distinct promoter classes initiate transcription via different mechanisms reflected in focused versus dispersed initiation patterns. Embo J: e113519. PubMed ID: 37013908

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

Valadez-Graham, V., et al. (2012). XNP/dATRX interacts with DREF in the chromatin to regulate gene expression. Nucleic Acids Res. 40(4): 1460-74. PubMed Citation: 22021382

Villányi, Z., Debec, A., Timinszky, G., Tirián, L. and Szabad, J. (2008). Long persistence of importin-β explains extended survival of cells and zygotes that lack the encoding gene. Mech. Dev. 125(3-4): 196-206. PubMed Citation: 18221858

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

Vo, N., Horii, T., Yanai, H., Yoshida, H. and Yamaguchi, M. (2014). The Hippo pathway as a target of the Drosophila DRE/DREF transcriptional regulatory pathway. Sci Rep 4: 7196. PubMed ID: 25424907

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

Yoshioka, Y., et al. (2012). Drosophila DREF acting via the JNK pathway is required for thorax development. Genesis [Epub ahead of print]. PubMed Citation: 22307950

Zabidi, M. A., Arnold, C. D., Schernhuber, K., Pagani, M., Rath, M., Frank, O. and Stark, A. (2015). Enhancer--core-promoter specificity separates developmental and housekeeping gene regulation. Nature 518(7540):556-9. PubMed ID: 25517091

date revised: 25 March 2015

Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.