DRE-binding Factor Research Articles

Comparative Genomics and Functional Studies of Wheat BED-NLR Loci.

Nucleotide-binding leucine-rich-repeat (LRR) receptors (NLRs) with non-canonical integrated domains (NLR-IDs) are widespread in plant genomes. Zinc-finger BED (named after the Drosophila proteins Boundary Element-Associated Factor and DNA Replication-related Element binding Factor, named BED hereafter) are among the most frequently found IDs. Five BED-NLRs conferring resistance against bacterial and fungal pathogens have been characterized. However, it is unknown whether BED-NLRs function in a manner similar to other NLR-IDs. Here, we used chromosome-level assemblies of wheat to explore the Yr7 and Yr5a genomic regions and show that, unlike known NLR-ID loci, there is no evidence for a NLR-partner in their vicinity. Using neighbor-network analyses, we observed that BED domains from BED-NLRs share more similarities with BED domains from single-BED proteins and from BED-containing proteins harboring domains that are conserved in transposases. We identified a nuclear localization signal (NLS) in Yr7, Yr5, and the other characterized BED-NLRs. We thus propose that this is a feature of BED-NLRs that confer resistance to plant pathogens. We show that the NLS was functional in truncated versions of the Yr7 protein when expressed in N. benthamiana. We did not observe cell-death upon the overexpression of Yr7 full-length, truncated, and ‘MHD’ variants in N. benthamiana. This suggests that either this system is not suitable to study BED-NLR signaling or that BED-NLRs require additional components to trigger cell death. These results define novel future directions to further understand the role of BED domains in BED-NLR mediated resistance.

DREB2 Family Transcription Factors Promote the Expression of Phytocystatin1 in Arabidopsis thaliana During Seed Germination

Phytocystatins (PhyCYSs) are plant-specific proteinaceous inhibitors of cysteine proteases (CPs) that regulate protein turnover and defense responses. Here, we characterized a member of the PhyCYS gene family in Arabidopsis thaliana, AtCYS1, during seed germination. AtCYS1 transcript level and AtCYS1 promoter-driven β-glucuronidase (GUS) activity gradually increased during germination, as measured by quantitative reverse transcription PCR (qRT-PCR) and GUS staining assays, respectively. The level and activity of the AtCYS1 protein were up-regulated during germination, as shown by western blot and CP inhibitory activity analyses, respectively. In addition, a canonical dehydration-responsive element (DRE) was identified in the AtCYS1 promoter. Electrophoretic mobility shift assays (EMSAs) revealed that the DRE-binding factor 2C (DREB2C) binds directly to the AtCYS1 promoter. Transient transactivation assays using Arabidopsis leaf protoplasts showed that DREB2A and DREB2C act as transcriptional activators of AtCYS1. In DREB2C overexpression lines, AtCYS1 protein levels were elevated, while the endogenous CP activity was reduced. However, AtCYS1 overexpression lines (35S:AtCYS1) and the cys1-1 null mutant, characterized by a quantitative imbalance in the AtCYS1 protein level, showed delayed germination under normal growth conditions. These results suggest that DREB2 proteins act as transcriptional activators of the AtCYS1 gene, and an imbalance in the level of AtCYS1 inhibits seed germination.

ZBED1/DREF: A transcription factor that regulates cell proliferation.

Maintenance of genomic diversity is critically dependent on gene regulation at the transcriptional level. This occurs via the interaction of regulatory DNA sequence motifs with DNA-binding transcription factors. The zinc finger, BED-type (ZBED) gene family contains major DNA-binding motifs present in human transcriptional factors. It encodes proteins that present markedly diverse regulatory functions. ZBED1 has similar structural and functional properties to its Drosophila homolog DNA replication-related element-binding factor (DREF) and plays a critical role in the regulation of transcription. ZBED1 regulates the expression of several genes associated with cell proliferation, including cell cycle regulation, chromatin remodeling and protein metabolism, and some genes associated with apoptosis and differentiation. In the present review, the origin, structure and functional role of ZBED1 were comprehensively assessed. In addition, the similarities and differences between ZBED1 and its Drosophila homolog DREF were highlighted, and future research directions, particularly in the area of clinical cancer, were discussed.

Human DREF/ZBED1 is a nuclear protein widely expressed in multiple cell types derived from all three primary germ layers

Drosophila DNA replication-related element binding factor (DREF) is a transcription regulatory factor that binds the promoters of many genes involved in replication and cell proliferation and is required for normal cell cycle progression. Human DREF/zinc finger BED domain-containing protein 1 (ZBED1), an orthologue of Drosophila DREF, also has DNA binding activity, but its cellular functions remain largely uncharacterized. Herein, we show that ZBED1 is a chromatin-associated nuclear protein with a wide expression profile in human tissues from all three primary germ layers. For instance, ZBED1 was expressed in mesodermal-derived epithelial cells of the reproductive system and urinary tract, in endodermal-derived epithelial cells throughout the gastrointestinal tract, and in epidermal epithelium from the ectoderm. ZBED1 was also expressed in connective tissue and smooth muscle cells of multiple organs. To investigate whether ZBED1 is implicated in cell proliferation, similar to Drosophila DREF, we compared the tissue distribution of ZBED1 to that of the proliferation marker Ki-67. ZBED1 and Ki-67 were co-expressed in many epithelial tissues, but ZBED1 expression extended widely beyond that of Ki-67-positive cells. In other tissues, ZBED1 expression was more restricted than Ki-67 expression. These results suggest that ZBED1 is not a cell proliferation-associated factor such as Drosophila DREF, and our study adds to the cumulative understanding of the functions of ZBED1 in human cells and tissues.

The Drosophila histone methyltransferase NSD is positively regulated by the DRE/DREF system.

The Drosophila nuclear receptor-binding SET domain protein (NSD) gene encodes the Drosophila ortholog of mammalian NSD family members that are important in many aspects of development and disease in humans. In this study, we observed that overexpression of Drosophila NSD in imaginal discs induces organ atrophy. Thus, to gain an understanding of the transcriptional regulation of the gene, we analyzed the NSD promoter region. First, we identified the presence of three putative DNA replication-related element (DRE) sequences in its promoter region, where DRE-binding factor (DREF) could bind for transcriptional activation. In the experiments with the fly GAL4-UAS system, we demonstrated that overexpressed DREF increased the endogenous NSD transcription. To confirm the role of DREF as a transcriptional activator on the NSD expression, we generated a series of luciferase reporter gene constructs containing deleted portions of the 5'-flanking regions as well as point mutations in the putative DRE sites. When transiently transfected into S2 cells, the deletion construct containing no DRE sites showed dramatic decrease in the NSD promoter activity, but only two sites near the transcriptional start site were important. Furthermore, we verified the direct interaction of DREF with the two positively cis-acting sequences on the NSD promoter by chromatin immunoprecipitation assay. Taken together, these results demonstrated that NSD is one of the downstream targets of the DRE/DREF pathway that is associated with various cellular processes in Drosophila, indicating that our findings may contribute to the understanding of molecular mechanisms in complex disorders associated with NSD family members in humans.

Transcription Factor hDREF Is a Novel SUMO E3 Ligase of Mi2α

The human transcription factor DNA replication-related element-binding factor (hDREF) is essential for the transcription of a number of housekeeping genes. The mechanisms underlying constitutively active transcription by hDREF were unclear. Here, we provide evidence that hDREF possesses small ubiquitin-like modifier (SUMO) ligase activity and can specifically SUMOylate Mi2α, an ATP-dependent DNA helicase in the nucleosome remodeling and deacetylation complex. Moreover, immunofluorescent staining and biochemical analyses showed that coexpression of hDREF and SUMO-1 resulted in dissociation of Mi2α from chromatin, whereas a SUMOylation-defective Mi2α mutant remained tightly bound to chromatin. Chromatin immunoprecipitation and quantitative RT-PCR analysis demonstrated that Mi2α expression diminished transcription of the ribosomal protein genes, which are positively regulated by hDREF. In contrast, coexpression of hDREF and SUMO-1 suppressed the transcriptional repression by Mi2α. These data indicate that hDREF might incite transcriptional activation by SUMOylating Mi2α, resulting in the dissociation of Mi2α from the gene loci. We propose a novel mechanism for maintaining constitutively active states of a number of hDREF target genes through SUMOylation.

Widespread colocalization of the Drosophila histone acetyltransferase homolog MYST5 with DREF and insulator proteins at active genes.

MYST family histone acetyltransferases play important roles in gene regulation. Here, we have characterized the Drosophila MYST histone acetyltransferase (HAT) encoded by cg1894, whose closest homolog is Drosophila MOF, and which we have termed MYST5. We found it localized to a large number of interbands as well as to the telomeres of polytene chromosomes, and it showed strong colocalization with the interband protein Z4/Putzig and RNA polymerase II. Accordingly, genome-wide location analysis by ChIP-seq showed co-occurrence of MYST5 with the Z4-interacting partner Chriz/Chromator. Interestingly, MYST5 bound to the promoter of actively transcribed genes, and about half of MYST5 sites colocalized with the transcription factor DNA replication-related element-binding factor (DREF), indicating a role for MYST5 in gene expression. Moreover, we observed substantial overlap of MYST5 binding with that of the insulator proteins CP190, dCTCF, and BEAF-32, which mediate the organization of the genome into functionally distinct topological domains. Altogether, our data suggest a broad role for MYST5 both in gene-specific transcriptional regulation and in the organization of the genome into chromatin domains, with the two roles possibly being functionally interconnected.

DUTPase expression correlates with cell division potential in Drosophila melanogaster

dUTP pyrophosphatase (dUTPase) is a dNTP-sanitizing enzyme that prevents the appearance of potentially harmful uracil bases in DNA by hydrolyzing cellular dUTP. This function of dUTPase is found to be essential in many organisms including Drosophila melanogaster. Previously, we showed that the expression pattern of dUTPase determines the extent of uracil accumulation in the genome of different tissues. We wished to find the regulatory mechanism that eventually leaves a set of tissues with a uracil-free and intact genome. We found that the expression pattern established by the promoter of Drosophila dUTPase overlaps with mRNA and protein expression, excluding the involvement of other post-transcriptional contributions. This promoter was found to be active in primordial tissues, such as in the imaginal discs of larvae, in the larval brain and in reproductive organs. In the case of brain and imaginal tissues, we observed that the promoter activity depends on a DNA replication-related element motif, the docking site of DNA replication-related element binding factor, which is known as a transcriptional activator of genes involved in replication and proliferation. These results suggest that dUTPase expression is fine-tuned to meet the requirements of DNA synthesis in tissues where the maintenance of genome integrity is of high importance.

Drosophila myeloid leukemia factor acts with DREF to activate the JNK signaling pathway.

Drosophila myelodysplasia/myeloid leukemia factor (dMLF), a homolog of human MLF1, oncogene was first identified by yeast two-hybrid screen using the DNA replication-related element-binding factor (DREF) as bait. DREF is a transcription factor that regulates proliferation-related genes in Drosophila. It is known that overexpression of dMLF in the wing imaginal discs through the engrailed-GAL4 driver causes an atrophied wing phenotype associated with the induction of apoptosis. However, the precise mechanisms involved have yet to be clarified. Here, we found the atrophied phenotype to be suppressed by loss-of-function mutation of Drosophila Jun N-terminal kinase (JNK), basket (bsk). Overexpression of dMLF induced ectopic JNK activation in the wing disc monitored with the puckered-lacZ reporter line, resulting in induction of apoptosis. The DREF-binding consensus DRE sequence could be shown to exist in the bsk promoter. Chromatin immunoprecipitation assays in S2 cells with anti-dMLF IgG and quantitative real-time PCR revealed that dMLF binds specifically to the bsk promoter region containing the DRE sequence. Furthermore, using a transient luciferase expression assay, we provide evidence that knockdown of dMLF reduced bsk gene promoter activity in S2 cells. Finally, we show that dMLF interacts with DREF in vivo. Altogether, these data indicate that dMLF acts with DREF to stimulate the bsk promoter and consequently activates the JNK pathway to promote apoptosis.

Control of e2f1 and PCNA by Drosophila transcription factor DREF

DREF (DNA replication-related element-binding factor), a zinc finger type transcription factor required for proper cell cycle progression in both mitotic and endocycling cells, is a positive regulator of E2F1, an important transcription factor which regulates genes related to the S-phase of the cell cycle. DREF and E2F1 regulate similar sets of replication-related genes, including proliferating cell nuclear antigen (PCNA), and play roles in the G1 to S phase transition. However, the relationships between dref and e2f1 or PCNA during development are poorly understood. Here, we provided evidence for novel control of e2f1 and PCNA involving DREF in endocycling cells. Somatic clone analysis demonstrated that dref knockdown stabilized E2F1 expression at posttranscriptional levels in endocycling salivary gland cells. Similarly, PCNA expression was up-regulated in the endocycling salivary gland cells. Genetic interaction analysis indicated that the endoreplication defects are partly caused via possible enhancement of E2F1 activity. From these results and previous reports, we conclude that regulation of e2f1 and PCNA by DREF in vivo is complex and the regulation mechanism may differ with the tissue and/or positions in the tissue.

DREB2C acts as a transcriptional activator of the thermo tolerance-related phytocystatin 4 (AtCYS4) gene

Phytocystatins are proteinaceous inhibitors of cysteine proteases. They have been implicated in the regulation of plant protein turnover and in defense against pathogens and insects. Here, we have characterized an Arabidopsis phytocystatin family gene, Arabidopsis thaliana phytocystatin 4 (AtCYS4). AtCYS4 was induced by heat stress. The heat shock tolerance of AtCYS4-overexpressing transgenic plants was greater than that of wild-type and cys4 knock-down plants, as measured by fresh weight and root length. Although no heat shock elements were identified in the 5'-flanking region of the AtCYS4 gene, canonical ABA-responsive elements (ABREs) and dehydration-responsive elements (DREs) were found. Transient promoter activity measurements showed that AtCYS4 expression was up-regulated in unstressed protoplasts by co-expression of DRE-binding factor 2s (DREB2s), especially by DREB2C, but not by bZIP transcription factors that bind to ABREs (ABFs, ABI5 and AREBs). DREB2C bound to and activated transcription from the two DREs on the AtCYS4 promoter although some preference was observed for the GCCGAC DRE element over the ACCGAC element. AtCYS4 transcript and protein levels were elevated in transgenic DREB2C overexpression lines with corresponding decline of endogenous cysteine peptidase activity. We propose that AtCYS4 functions in thermotolerance under the control of the DREB2C cascade.

Dynamic changes in the genomic localization of DNA replication-related element binding factor during the cell cycle

DREF was first characterized for its role in the regulation of transcription of genes encoding proteins involved in DNA replication and found to interact with sequences similar to the DNA recognition motif of the BEAF-32 insulator protein. Insulators are DNA-protein complexes that mediate intra- and inter-chromosome interactions. Several DNA-binding insulator proteins have been described in Drosophila, including BEAF-32, dCTCF and Su(Hw). Here we find that DREF and BEAF-32 co-localize at the same genomic sites, but their enrichment shows an inverse correlation. Furthermore, DREF co-localizes in the genome with other insulator proteins, suggesting that the function of this protein may require components of Drosophila insulators. This is supported by the finding that mutations in insulator proteins modulate DREF-induced cell proliferation. DREF persists bound to chromatin during mitosis at a subset of sites where it also co-localizes with dCTCF, BEAF-32 and CP190. These sites are highly enriched for sites where Orc2 and Mcm2 are present during interphase and at the borders of topological domains of chromosomes defined by Hi-C. The results suggest that DREF and insulator proteins may help maintain chromosome organization during the cell cycle and mark a subset of genomic sites for the assembly of pre-replication complexes and gene bookmarking during the M/G1 transition.

Comparative Analysis of DNA Methylation Polymorphism in Drought Sensitive (HPKC2) and Tolerant (HPK4) Genotypes of Horse Gram (Macrotyloma uniflorum)

DNA methylation is known as an epigenetic modification that affects gene expression in plants. Variation in CpG methylation behavior was studied in two natural horse gram (Macrotyloma uniflorum [Lam.] Verdc.) genotypes, HPKC2 (drought-sensitive) and HPK4 (drought-tolerant). The methylation pattern in both genotypes was studied through methylation-sensitive amplified polymorphism. The results revealed that methylation was higher in HPKC2 (10.1%) than in HPK4 (8.6%). Sequencing demonstrated sequence homology with the DRE binding factor (cbf1), the POZ/BTB protein, and the Ty1-copia retrotransposon among some of the polymorphic fragments showing alteration in methylation behavior. Differences in DNA methylation patterns could explain the differential drought tolerance and the epigenetic signature of these two horse gram genotypes.

Transcriptional regulation of the purine de novo synthesis gene Prat in Drosophila melanogaster

The first step of the purine de novo synthesis pathway is catalyzed by amidophosphoribosyltransferase (E.C.2.4.2.14) which is encoded by two Prat genes in D. melanogaster, Prat and Prat2. Prat is a retrogene duplication of Prat2, where each gene has a distinct expression pattern. Prat transcription is restricted to proliferating tissues such as imaginal discs and the female germ line. Three conserved putative DNA replication-related element binding factor (DREF) sites lie upstream of the Prat coding region. These elements are upstream of many genes important in cell proliferation. We have found that DREF binds directly upstream of Prat and that the DRE sites associated with its activity are necessary for Prat expression; furthermore, we have determined that a second cis-acting element is present upstream of the Prat gene. Finally, the genes Distal-less, Mi-2 and dMyc, which influence Dref activity, do not appear to affect Prat transcription.

Temporal and Spatial Pattern of <i>Dref</i> Expression during <i>Drosophila</i> Bristle Development

The DNA replication-related element-binding factor (DREF) is a BED finger-type transcription factor that has important roles in cell cycle progression. In an earlier study, we showed that DREF is required for endoreplication during posterior scutellar macrochaete development. However, dynamic change in the dref expression in the cell lineage is unclear. In this study, we focused on the spatio-temporal pattern of expression of the dref gene during bristle development. Gene expression analysis using GAL4 enhancer trap lines of dref and the upstream activation sequence-green fluorescent protein with nuclear localization signals (UAS-GFPnls) in combination with immunostaining revealed the half-life of GFPnls in vivo (<6 hours) is short enough to monitor the dref gene expression. The analysis revealed that the dref expression occurs in clusters that include cells consisting of a bristle as well as surrounding epidermal cells. The intensity of GFP signals was almost the same in those cells, suggesting expression of the dref gene in bristle cell lineages occurs simultaneously in clusters. Further analysis showed that GFP signals increased twice during sensory organ precursor development as well as in bristle development at 9 hours and 15 hours after pupal formation, respectively. However, its expression was barely detectable in the cell lineages in and around asymmetric cell division or at other stages of development. For the first time, we clarified a spatio-temporal pattern of expression of the dref gene in vivo and revealed that expression of the dref gene occurs in clusters and is temporally regulated at specific times during bristle development.

D-MTERF5 is a novel factor modulating transcription in Drosophila mitochondria

The MTERF protein family comprises members from Metazoans and plants. All the Metazoan MTERF proteins characterized to date, including the mitochondrial transcription termination factors, play a key role in mitochondrial gene expression. In this study we report the characterization of Drosophila MTERF5 (D-MTERF5), a mitochondrial protein existing only in insects, probably originated from a duplication event of the transcription termination factor DmTTF. D-MTERF5 knock-down in D.Mel-2 cells alters transcript levels with an opposite pattern to that produced by DmTTF knock-down. D-MTERF5 is able to interact with mtDNA at the same sites contacted by DmTTF, but only in the presence of the termination factor. We propose that the two proteins participate in the transcription termination process, with D-MTERF5 engaged in relieving the block exerted by DmTTF. This hypothesis is supported also by D-MTERF5 homology modeling, which suggests that this protein contains protein–protein interaction domains. Co-regulation by DREF (DNA Replication-related Element binding Factor) of D-MTERF5 and DmTTF implies that expression of the two factors needs to be co-ordinated to ensure fine modulation of Drosophila mitochondrial transcription.

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.

Caudal-related homeodomain proteins CDX1/2 bind to DNA replication-related element binding factor

In the intestinal epithelium, the CDX1 and CDX2 homeodomain genes play proliferative and tumor suppressor roles, respectively. The transcription factor DNA replication-related element binding factor (DREF), is an 80kDa polypeptide homodimer that plays an important role in regulating cell proliferation-related genes. Homeodomain genes encode DNA-binding proteins that play crucial roles during development by defining the body plan and determining cell fate. However, until now, the regulation of DREF function by caudal-related homeodomain proteins is poorly understood. In this study, recombinant CDX1/2 homeodomains (CDX1, amino acids [aa] 152–216 and CDX2, aa 184–248) and the DNA-binding domain of Drosophila DREF (dDREF; aa 1–125) were isolated in order to investigate the regulatory mechanism of their interaction. The expression and purification of the truncated CDX1/2 and DREF proteins were successfully performed in Escherichia coli. Models of the CDX1/2 homeodomain and dDREF were constructed using SWISS-MODEL software, a program for relative protein structure modeling. The binding of CDX1/2 and DREF proteins was detected by fluorescence measurement, size-exclusion column (SEC) chromatography, His-tagged pull-down assay, and surface plasmon resonance spectroscopy (BIAcore). In addition, we identified that four different mutants of CDX1 (S185A, N190A, T194A, and V212A) were bound to dDREF with different degrees of interaction. Our results indicate that CDX1/2 homeodomains interact with the DNA-binding domain of dDREF, thereby regulating its transcription activity.

DREF is Critical for Drosophila Bristle Development by Regulating Endoreplication in Shaft Cells

DREF (DNA replication-related element-binding factor) plays important roles in replication and proliferation in vivo by regulating transcription of various genes. However, due to a lack of appropriate cell biological studies in vivo, roles of DREF during a single cell development are poorly understood. To address this question, we focused our attention on macrochaetes bristle development system. Utilizing cell lineage analysis focusing on a single posterior scutellar (PSC) macrochaete sensory organ precursor (SOP) lineages in combination with GAL4/UAS targeted expression system for DREF double strand RNA, we revealed that DREF plays no apparent role in differentiation process during SOP formation. Rather, DREF regulates the timing of asymmetric cell division but perhaps plays no direct role in differentiation during asymmetric cell division. Most importantly, DREF affected replication and growth in shaft cells and/or socket cells. Further analysis revealed that DREF is necessary but not sufficient for nuclear growth and protein synthesis in shaft cells. Finally, it could be demonstrated that DREF plays a critical role in regulating pcna transcription in endocycling shaft cells. All these results provide evidence that DREF plays critical roles, especially in endoreplication process of bristle development, at least in part by regulating the pcna gene expression.

Regulation of the Drosophila p38b gene by transcription factor DREF in the adult midgut

The Drosophila midgut is an excellent model for evaluation of gene networks that regulate adult stem cell proliferation and differentiation. The Drosophila p38b ( D-p38b) gene has been shown to be involved in intestinal stem cell (ISC) proliferation and differentiation in the adult midgut. Here, we report that D-p38b gene expression is regulated by DREF (DNA replication-related element binding factor) in the adult midgut. We have identified a DRE in the 5′-flanking region of the D-p38b gene and showed that DREF could bind to this DRE via a gel mobility shift assay and a ChIP assay. Base-substitution mutations of the D-p38b promoter DRE and analyses of transformants carrying D-p38b-lacZ or D-p38b-DREmut -lacZ indicated that this DRE is required for the activity of the D-p38b gene promoter. Furthermore, by using the GAL4-UAS system, we showed that DREF regulates the activity of the D-p38b gene promoter in adult ISCs and progenitors. In addition, the D-p38b knockdown phenotypes in the midgut were rescued by DREF overexpression, suggesting a functional link between these two factors. Our results suggest that the D-p38b gene is regulated by the DREF pathway and that DREF is involved in the regulation of proliferation and differentiation of Drosophila ISCs and progenitors.