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Introduction: Phosphorus (P) is one of the most important nutrient elements for plant growth and development. Under P starvation, arbuscular mycorrhizal (AM) fungi can promote phosphate (Pi) uptake and homeostasis within host plants. However, the underlying mechanisms by which AM fungal symbiont regulates the AM symbiotic Pi acquisition from soil under P starvation are largely unknown. Here, we identify a HLH domain containing transcription factor RiPho4 from Rhizophagus irregularis ., Methods: To investigate the biological functions of the RiPho4, we combined the subcellular localization and Yeast One-Hybrid (Y1H) experiments in yeasts with gene expression and virus-induced gene silencing approach during AM symbiosis., Results: The approach during AM symbiosis. The results indicated that RiPho4 encodes a conserved transcription factor among different fungi and is induced during the in planta phase. The transcription of RiPho4 is significantly up-regulated by P starvation. The subcellular localization analysis revealed that RiPho4 is located in the nuclei of yeast cells during P starvation. Moreover, knock-down of RiPho4 inhibits the arbuscule development and mycorrhizal Pi uptake under low Pi conditions. Importantly, RiPho4 can positively regulate the downstream components of the phosphate (PHO) pathway in R. irregularis ., Discussion: In summary, these new findings reveal that RiPho4 acts as a transcriptional activator in AM fungus to maintain arbuscule development and regulate Pi uptake and homeostasis in the AM symbiosis during Pi starvation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Nie, Fan, Wei, Chen, Xie and Tang.)

The specificity in gene regulation is controlled by interactions between transcription factors (TFs) and genomic DNA regions such as promoters and enhancers. Enhanced yeast one-hybrid (eY1H) assays are among the methods used for high-throughput detection of transcription factor-DNA interactions. Here, we describe the procedure for screening interactions between DNA regions of interest ("DNA-baits") and an array of transcription factors ("TF-preys"), after DNA-bait and TF-prey yeast strains have been generated. Using a high-density array robotic platform, this method can be used to screen interactions between multiple DNA regions and >1000 TFs within a single experiment., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Ethylene responsive factors (ERFs) are plant-specific transcription factors that are involved in a variety of biological processes. We previously demonstrated that an ERF gene from Tamarix hispida, ThERF1, encodes a protein binding to GCC-box and DRE motifs and negatively modulates abiotic stress tolerance. In the present study, microarray analysis was performed to study the genes regulated by ThERF1 on a genomic scale. There were 154 and 307 genes (respectively representing 134 and 260 unique genes) significantly up- and downregulated by ThERF1 under salt stress conditions, respectively. A novel motif, named TTG, was identified to be recognized by ThERF1, which commonly presents in the promoters of ThERF1-targeted genes. The TTG motif is also bound by other ERFs of a different subfamily from T. hispida and Arabidopsis, indicating that it is commonly recognized by ERF proteins. The binding affinities of ERFs to the TTG motif are significantly induced by salt stress. The TTG motif is more enriched than the GCC-box and DRE motifs in the promoters of ThERF1-targeted genes. Taken together, these studies suggested that the TTG motif plays an important role in the gene expression regulated by ERFs in response to salt stress. [ABSTRACT FROM AUTHOR]

Ethylene response factor (ERF) is a major subfamily of the AP2/ERF family and plays significant roles in the regulation of abiotic- and biotic-stress responses. ERF proteins can interact with the GCC-box cis-element and then initiate a transcriptional cascade activating downstream ethylene response and enhancing plant stress tolerance. In this research, we cloned five StERF genes from potato ( Solanum tuberosum L.). The expressional analysis of StERF genes revealed that they showed tissue- or organ-specific expression patterns and the expression levels in leaf, stem, root, flower, and tuber were different. The assays of quantitative real-time polymerase chain reaction (qRT-PCR) and the reverse transcription-PCR (RT-PCR) showed that the expression of five StERF genes was regulated by ethephon, methyl jasmonate (MeJA), salt and drought stress. The result from the yeast one-hybrid experiment showed that five StERFs had trans-activation activity and could specifically bind to the GCC-box cis-elements. The StERFs responded to abiotic factors and hormones suggested that they possibly had diverse roles in stress and hormone regulation of potato. [ABSTRACT FROM AUTHOR]

The dehydration-responsive element-binding (DREB) proteins play an important role in regulating expression of stress-inducible genes under abiotic stresses. In this study, two genes encoding putative DREB proteins, named SsDREBa and SsDREBb, were cloned from halophyte Suaeda salsa L. using RACE method. The deduced SsDREBa and SsDREBb proteins contain a typical AP2/ERF domain. Multiple sequence alignments and phylogenetic analysis revealed that the two SsDREB genes of S. salsa were highly similar in AP2/ERF domains at the nucleotide and amino acid levels and belong to the A-6 subgroup of the DREB transcription factor subfamily. A subcellular localization assay showed that both SsDREBs localized to the nucleus. Yeast one-hybrid experiments testified that both proteins were able to specifically bind to the DRE sequence and activate the expression of the down-stream HIS reporter gene in yeast. Quantitative real-time PCR analysis demonstrated that under normal conditions, the expression level of SsDREBa was the most high in the roots and no SsDREBa mRNAs were detected in the stems; SsDREBb expressed at relatively higher levels in the leaves than in the roots and stems. The expression of SsDREa and SsDREBb genes in S. salsa roots and leaves was remarkably induced by high-salt and dehydration treatments, but not by cold and ABA, and exhibited stronger induction in roots and leaves, respectively. These results indicate that the SsDREBa and SsDREBb are novel stress-responsive transcription factors, which are involved in the drought and high-salt stress responses through ABA-independent pathways and could be used for production of stress-tolerant transgenic crops. [ABSTRACT FROM AUTHOR]

GmFT2a is a key gene in photoperiodic pathway of soybean. However, the underlying molecular mechanism of GmFT2a expression is not clear. In this study, a photoperiod-sensitive soybean cultivar Zigongdongdou and insensitive cultivar Heihe 27 were used as materials to compare the GmFT2a promoter sequences. It revealed that there were 2 insertion/deletion (InDel) fragments, T1 and T2, which contained light reaction elements in the GmFT2a promoter of Zigongdongdou. However, the same fragments were not in that of Heihe 27. The bait constructs were prepared according to the sequences of light reaction elements of I-box and SP1 to screen cDNA library of Heihe 27 leaves, and a MYB transcription factor bound with T1 was isolated with the yeast one-hybrid system. The obtaining of the MYB transcription factor provided a favorable clue for further study of the molecular mechanism GmFT2a expression regulation. [ABSTRACT FROM AUTHOR]

The interaction between a protein and DNA is involved in almost all cellular functions, and is vitally important in cellular processes. Two complementary approaches are used to detect the interactions between a transcription factor (TF) and DNA, i.e. the TF-centered or protein-DNA approach, and the gene-centered or DNA-protein approach. The yeast one-hybrid (Y1H) is a powerful and widely used system to identify DNA-protein interactions. However, a powerful method to study protein-DNA interactions like Y1H is lacking. Here, we developed a protein-DNA method based on the Y1H system to identify the motifs recognized by a defined TF, termed TF-centered Y1H. In this system, a random short DNA sequence insertion library was generated as the prey DNA sequences to interact with a defined TF as the bait. Using this system, novel interactions were detected between DNA motifs and the AtbZIP53 protein from Arabidopsis. We identified six motifs that were specifically bound by AtbZIP53, including five known motifs (DOF, G-box, I-box, BS1 and MY3) and a novel motif BRS1 [basic leucine zipper (bZIP) Recognized Site 1]. The different subfamily bZIP members also recognize these six motifs, further confirming the reliability of the TF-centered Y1H results. Taken together, these results demonstrated that TF-centered Y1H could identify quickly the motifs bound by a defined TF, representing a reliable and efficient approach with the advantages of Y1H. Therefore, this TF-centered Y1H may have a wide application in protein-DNA interaction studies. [ABSTRACT FROM AUTHOR]

The comprehensive mapping of gene promoters and enhancers has significantly improved our understanding of how the mammalian regulatory genome is organized. An important challenge is to elucidate how these regulatory elements contribute to gene expression by identifying their trans‐regulatory inputs. Here, we present the generation of a mouse‐specific transcription factor (TF) open‐reading frame clone library and its implementation in yeast one‐hybrid assays to enable large‐scale protein–DNA interaction detection with mouse regulatory elements. Once specific interactions are identified, we then use a microfluidics‐based method to validate and precisely map them within the respective DNA sequences. Using well‐described regulatory elements as well as orphan enhancers, we show that this cross‐platform pipeline characterizes known and uncovers many novel TF–DNA interactions. In addition, we provide evidence that several of these novel interactions are relevant in vivo and aid in elucidating the regulatory architecture of enhancers.

Establishing the architecture of the gene regulatory networks (GRNs) responsible for controlling the transcription of all genes in an organism is a natural development that follows elucidation of the genome sequence. Reconstruction of the GRN requires the availability of a series of molecular tools and resources that so far have been limited to a few model organisms. One such resource consists of collections of transcription factor (TF) open reading frames (ORFs) cloned into vectors that facilitate easy expression in plants or microorganisms. In this study, we describe the development of a publicly available maize TF ORF collection (TFome) of 2034 clones corresponding to 2017 unique gene models in recombination-ready vectors that make possible the facile mobilization of the TF sequences into a number of different expression vectors. The collection also includes several hundred co-regulators (CoREGs), which we classified into well-defined families, and for which we propose here a standard nomenclature, as we have previously done for TFs. We describe the strategies employed to overcome the limitations associated with cloning ORFs from a genome that remains incompletely annotated, with a partial full-length cDNA set available, and with many TF/CoREG genes lacking experimental support. In many instances this required the combination of genome-wide expression data with gene synthesis approaches. The strategies developed will be valuable for developing similar resources for other agriculturally important plants. Information on all the clones generated is available through the GRASSIUS knowledgebase (). [ABSTRACT FROM AUTHOR]

Background TCP proteins are plant-specific transcription factors, which are known to have a wide range of functions in different plant species such as in leaf development, flower symmetry, shoot branching, and senescence. Only a small number of TCP genes has been characterised from tomato (Solanum lycopersicum). Here we report several functional features of the members of the entire family present in the tomato genome. Results We have identified 30 Solanum lycopersicum SlTCP genes, most of which have not been described before. Phylogenetic analysis clearly distinguishes two homology classes of the SlTCP transcription factor family - class I and class II. Class II differentiates in two subclasses, the CIN-TCP subclass and the CYC/TB1 subclass, involved in leaf development and axillary shoots formation, respectively. The expression patterns of all members were determined by quantitative PCR. Several SlTCP genes, like SlTCP12, SlTCP15 and SlTCP18 are preferentially expressed in the tomato fruit, suggesting a role during fruit development or ripening. These genes are regulated by RIN (RIPENING INHIBITOR), CNR (COLORLESS NON-RIPENING) and SlAP2a (APETALA2a) proteins, which are transcription factors with key roles in ripening. With a yeast one-hybrid assay we demonstrated that RIN binds the promoter fragments of SlTCP12, SlTCP15 and SlTCP18, and that CNR binds the SlTCP18 promoter. This data strongly suggests that these class I SlTCP proteins are involved in ripening. Furthermore, we demonstrate that SlTCPs bind the promoter fragments of members of their own family, indicating that they regulate each other. Additional yeast-one hybrid studies performed with Arabidopsis transcription factors revealed binding of the promoter fragments by proteins involved in the ethylene signal transduction pathway, contributing to the idea that these SlTCP genes are involved in the ripening process. Yeast two-hybrid data shows that SlTCP proteins can form homo and heterodimers, suggesting that they act together in order to form functional protein complexes and together regulate developmental processes in tomato. Conclusions The comprehensive analysis we performed, like phylogenetic analysis, expression studies, identification of the upstream regulators and the dimerization specificity of the tomato TCP transcription factor family provides the basis for functional studies to reveal the role of this family in tomato development. [ABSTRACT FROM AUTHOR]

Background Using motif detection programs it is fairly straightforward to identify conserved cissequences in promoters of co-regulated genes. In contrast, the identification of the transcription factors (TFs) interacting with these cis-sequences is much more elaborate. To facilitate this, we explore the possibility of using several bioinformatic and experimental approaches for TF identification. This starts with the selection of co-regulated gene sets and leads first to the prediction and then to the experimental validation of TFs interacting with cis-sequences conserved in the promoters of these co-regulated genes. Results Using the PathoPlant database, 32 up-regulated gene groups were identified with microarray data for drought-responsive gene expression from Arabidopsis thaliana. Application of the binding site estimation suite of tools (BEST) discovered 179 conserved sequence motifs within the corresponding promoters. Using the STAMP web-server, 49 sequence motifs were classified into 7 motif families for which similarities with known cis-regulatory sequences were identified. All motifs were subjected to a footprintDB analysis to predict interacting DNA binding domains from plant TF families. Predictions were confirmed by using a yeastone- hybrid approach to select interacting TFs belonging to the predicted TF families. TFDNA interactions were further experimentally validated in yeast and with a Physcomitrella patens transient expression system, leading to the discovery of several novel TF-DNA interactions. Conclusions The present work demonstrates the successful integration of several bioinformatic resources with experimental approaches to predict and validate TFs interacting with conserved sequence motifs in co-regulated genes. [ABSTRACT FROM AUTHOR]

In this study, a full-length cDNA sequence of DREB transcription factor gene, HhDREB2 (GenBank No.: EU872018), was isolated from Halimodendron halodendron. Voss by homology-based cloning and RACE (rapid amplification of cDNA end) technology. Sequence analysis showed that the cDNA sequence was 873 bp without introns and had a maximum open reading frame from 108 to 626 bp, encoding 172 amino acid residues. Molecular weight of the gene encoding protein was 19.702 kDa, isoelectric point was 8.92. Phylogenetic tree and homologous sequence alignment analysis indicated that this protein contained a typical AP2/ERF conservative domain, and was most closely related to soybean GmDREB2, belonging to the A5 subgroups. Yeast one-hybrid experiment demonstrated that HhDREB2 gene encoded a transcription activator. In addition, subcellular localization analysis of a construct with the transient expression vector of the HhDREB2 gene showed that the HhDREB2 protein was localized in the nucleus. The expression level of HhDREB2 gene was greatly induced by high salt, drought and low temperature through semi-quantitative RT-PCR, and had no significant change with GA3, NAA, ABA and 6BA treatments. [ABSTRACT FROM AUTHOR]

Using a combination of bioinformatics and synthetic promoters, novel elicitor-responsive cis-sequences were discovered in promoters of pathogen-upregulated genes from Arabidopsis thaliana. One group of functional sequences contains the conserved core sequence GACTTTT. This core sequence and adjacent nucleotides are essential for elicitor-responsive gene expression in a parsley protoplast system. By yeast one-hybrid screening, WRKY70 was selected with a cis-sequence harbouring the core sequence GACTTTT but no known WRKY binding site (W-box). Transactivation experiments, mutation analyses, and electrophoretic mobility shift assays demonstrate that the sequence CGACTTTT is the binding site for WRKY70 in the investigated cis-sequence and is required for WRKY70-activated gene expression. Using several cis-sequences in transactivation experiments and binding studies, the CGACTTTT sequence can be extended to propose YGACTTTT as WRKY70 binding site. This binding site, designated WT-box, is enriched in promoters of genes upregulated in a WRKY70 overexpressing line. Interestingly, functional WRKY70 binding sites are present in the promoter of WRKY30, supporting recent evidence that both factors play a role in the same regulatory network. [ABSTRACT FROM AUTHOR]

Abstract: Insulin self-tolerance is, to a large extent, assured by the expression of small quantities of insulin by medullary thymic epithelial cells (mTECs). Regulation of thymic insulin expression differs from that in pancreas and its therapeutic manipulation could play an important role in the prevention of type 1 diabetes (T1D). Knowledge of the transcriptional regulators involved in the mTEC nuclear environment is essential for the development of such therapeutics. The yeast one-hybrid (Y1H) approach was used in order to identify such mTEC-specific nuclear proteins. We used a target composed of the human insulin gene promoter joined to the upstream class III VNTR allele, which is associated with both protection from T1D and higher thymic insulin expression, and a cDNA library from our insulin-producing mouse mTEC line. The Y1H screening allowed the identification of eleven proteins. An in vitro assay was used to confirm and quantify protein–DNA binding to the human insulin gene promoter alone or joined to a class I or class III VNTR allele, and identified the transcription factors ZBTB7A, JUN and EWSR1 as strong interacting partners. All three proteins could induce insulin expression in transfected HEK-293 cells, but ZBTB7A provided the most robust results especially in the presence of AIRE, with an additional 11-fold increase of the insulin mRNA levels from a co-transfected reporter driven by the class III VNTR allele. Thus, ZBTB7A is identified as a strong candidate for regulation of thymic insulin expression. [Copyright &y& Elsevier]

Background: Tamarix spp. are woody halophyte, which are very tolerant to abiotic stresses such as salinity and drought, but little is known about their specific stress response systems. Basic leucine zipper proteins (bZIPs) play important roles in the ability of plants to withstand adverse environmental conditions. However, their exact roles in abiotic stress tolerance are still not fully known. In the current study, we functionally characterized a bZIP gene (ThbZIP1) from Tamarix hispida in response to abiotic stresses. Results: We addressed the regulatory network of ThbZIP1 in three levels, i.e. its upstream regulators, the cis-acting elements recognized by ThbZIP1, and its downstream target genes. Two MYCs were found to bind to E-box, in the promoter of ThbZIP1 to activate its expression. Expression of ThbZIP1 is induced by ABA, salt, drought, methyl viologen and cold. ThbZIP1 can specifically bind to ACGT elements, with the highest binding affinity to the C-box, followed by the G-box and lastly the A-box. Compared with wild-type (Col-0) Arabidopsis, transgenic plants expressing ThbZIP1 had an increased tolerance to drought and salt, but had an increased sensitivity to ABA during seed germination and root growth; meanwhile, ROS level, cell death and water loss rate in transgenic plants were significantly reduced. Microarray analyses showed that many ROS scavenging genes were up-regulated by ThbZIP1 under salt stress conditions. Conclusions: Based on these data, we suggest that ThbZIP1 confers abiotic stress tolerance through activating stress tolerance genes to modulate ROS scavenging ability and other physiological changes involved in stress tolerance, and plays an important role in the ABA-mediated stress response of T. hispida. [ABSTRACT FROM AUTHOR]

High salinity causes remarkable losses in rice productivity worldwide mainly because it inhibits growth and reduces grain yield. To cope with environmental changes, plants evolved several adaptive mechanisms, which involve the regulation of many stress-responsive genes. Among these, we have chosen OsRMC to study its transcriptional regulation in rice seedlings subjected to high salinity. Its transcription was highly induced by salt treatment and showed a stress-dose-dependent pattern. OsRMC encodes a receptor-like kinase described as a negative regulator of salt stress responses in rice. To investigate how OsRMC is regulated in response to high salinity, a salt-induced rice cDNA expression library was constructed and subsequently screened using the yeast one-hybrid system and the OsRMC promoter as bait. Thereby, two transcription factors (TFs), OsEREBP1 and OsEREBP2, belonging to the AP2/ERF family were identified. Both TFs were shown to bind to the same GCC-like DNA motif in OsRMC promoter and to negatively regulate its gene expression. The identified TFs were characterized regarding their gene expression under different abiotic stress conditions. This study revealed that OsEREBP1 transcript level is not significantly affected by salt, ABA or severe cold (5 °C) and is only slightly regulated by drought and moderate cold. On the other hand, the OsEREBP2 transcript level increased after cold, ABA, drought and high salinity treatments, indicating that OsEREBP2 may play a central role mediating the response to different abiotic stresses. Gene expression analysis in rice varieties with contrasting salt tolerance further suggests that OsEREBP2 is involved in salt stress response in rice. [ABSTRACT FROM AUTHOR]

Abstract: Botrytis cinerea, the grey mould fungus, secretes non-host-specific phytotoxins that kill the cells of many plant species. Phytotoxic assays performed about ten years ago, have highlighted the role in the infection mechanism of one of these secondary metabolites, the sesquiterpene botrydial. We recently showed that BcBOT1 to BcBOT5 genes, which are required for botrydial biosynthesis, are organised into a physical cluster. However, this cluster includes no gene encoding a transcription factor (TF) that might specifically coregulate the expression of BcBOT genes. To identify which TF(s) are implicated in the regulation of this cluster and thereby to decipher DNA-protein interactions in the phytopathogenic fungus B. cinerea, we developed a strategy based on the yeast one-hybrid (Y1H) method. In this study, a Y1H library was generated with the TFs predicted from complete genome sequencing. The screening of this library revealed an interaction between a promoter of the botrydial biosynthesis gene cluster and a new Cys2His2 zinc finger TF, that we called BcYOH1. Inactivation of the BcYOH1 gene and expression analyses demonstrated the involvement of this TF in regulating expression of the botrydial biosynthesis gene cluster. Furthermore, whole-transcriptome analysis suggested that BcYOH1 might act as a global transcriptional regulator of phytotoxin and other secondary metabolism gene clusters, and of genes involved in carbohydrate metabolism, transport, virulence and detoxification mechanisms. [Copyright &y& Elsevier]

The comprehensive mapping of gene promoters and enhancers has significantly improved our understanding of how the mammalian regulatory genome is organized. An important challenge is to elucidate how these regulatory elements contribute to gene expression by identifying their trans-regulatory inputs. Here, we present the generation of a mouse-specific transcription factor (TF) open-reading frame clone library and its implementation in yeast one-hybrid assays to enable large-scale protein–DNA interaction detection with mouse regulatory elements. Once specific interactions are identified, we then use a microfluidics-based method to validate and precisely map them within the respective DNA sequences. Using well-described regulatory elements as well as orphan enhancers, we show that this cross-platform pipeline characterizes known and uncovers many novel TF–DNA interactions. In addition, we provide evidence that several of these novel interactions are relevant in vivo and aid in elucidating the regulatory architecture of enhancers. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) is an important phytohormone involved in the abiotic stress resistance in plants. The ABA-responsive element (ABRE) binding factors play significant roles in the plant development and response to abiotic stresses, but none so far have been isolated and characterized from the oil palm. Two ABA-responsive cDNA clones, named EABF and EABF1, were isolated from the oil palm fruits using yeast one-hybrid system. The EABF had a conserved AP2/EREBP DNA-binding domain (DNA-BD) and a potential nuclear localization sequence (NLS). No previously known DNA-BD was identified from the EABF1 sequence. The EABF and EABF1 proteins were classified as DREB/CBF and bZIP family members based on the multiple sequence alignment and phylogenetic analysis. Both proteins showed ABRE-binding and transcriptional activation properties in yeast. Furthermore, both proteins were able to trans-activate the down-stream expression of the LacZ reporter gene in yeast. An electrophoretic mobility shift assay revealed that in addition to the ABRE sequence, both proteins could bind to the DRE sequence as well. Transcriptional analysis revealed that the expression of EABF was induced in response to the ABA in the oil palm fruits and leaves, but not in roots, while the EABF1 was constitutively induced in all tissues. The expressions of both genes were strongly induced in fruits in response to the ABA, ethylene, methyl jasmonate, drought, cold and high-salinity treatments, indicating that the EABF and EABF1 might act as connectors among different stress signal transduction pathways. Our results indicate that the EABF and EABF1 are novel stress-responsive transcription factors, which are involved in the abiotic stress response and ABA signaling in the oil palm and could be used for production of stress-tolerant transgenic crops. [ABSTRACT FROM AUTHOR]

Sulphur deficiency severely affects plant growth and their agricultural productivity leading to diverse changes in development and metabolisms. Molecular mechanisms regulating gene expression under low sulphur conditions remain largely unknown. AtSLIM1, a member of the EIN3-like (EIL) family was reported to be a central transcriptional regulator of the plant sulphur response, however, no direct interaction of this protein with any sulphur-responsive promoters was demonstrated. The focus of this study was on the analysis of a promoter region of UP9C, a tobacco gene strongly induced by sulphur limitation. Cloning and subsequent examination of this promoter resulted in the identification of a 20-nt sequence (UPE-box), also present in the promoters of several Arabidopsis genes, including three out of four homologues of UP9C. The UPE-box, consisting of two parallel tebs sequences (TEIL binding site), proved to be necessary to bind the transcription factors belonging to the EIL family and of a 5-nt conserved sequence at the 3′-end. The yeast one-hybrid analysis resulted in the identification of one transcription factor (NtEIL2) capable of binding to the UPE-box. The interactions of NtEIL2, and its homologue from Arabidopsis, AtSLIM1, with DNA were affected by mutations within the UPE-box. Transient expression assays in Nicotiana benthamiana have further shown that both factors, NtEIL2 and AtSLIM1, activate the UP9C promoter. Interestingly, activation by NtEIL2, but not by AtSLIM1, was dependent on the sulphur-deficiency of the plants. [ABSTRACT FROM PUBLISHER]

A new full-length cDNA encoding an AP2/EREBP domain-containing transcription factor named AoDREB was isolated from Asparagus officinalis L. using the RACE-PCR method. It is a homolog to the dehydration-responsive element binding protein (DREB) and classified to the A6 subgroup of the DREB subfamily. Using the yeast one-hybrid system, we conducted a DRE binding assay and demonstrated that AoDREB can bind the DRE element specifically. A transcriptional activity assay showed that AoDREB is a transcription factor capable of activating expression of the reporter gene in yeast. RT-PCR analysis revealed that expression of the AoDREB gene is induced under 20% PEG and high salinity stress, whereas no obvious response to low temperature was observed. Overexpression of AoDREB in transgenic Arabidopsis caused no growth retardation and induced stronger expression of the target genes, including RD29A and COR15A, than in wild type, resulting in increased tolerance of transgenic Arabidopsis to drought and high salinity. These results suggest that introduction of an exogenous AoDREB into plants is an effective way to improve plant drought and salinity tolerance. [ABSTRACT FROM AUTHOR]

A novel transcription factor, TcAP2, was isolated from Taxus cuspidata by yeast one-hybrid strategy. This factor interacts with jasmonate- and elicitor-responsive element. Analysis of the deduced TcAP2 amino acid sequence revealed that TcAP2 contained a conserved AP2/ethylene-responsive element binding protein domain that consisted of 268 amino acids in a potential nuclear localization sequence. The factor of TcAP2 had a high homology, in its AP2 domain, to other AP2 family members. Based on phylogenetic analysis, it was different from other five DRE-binding proteins in their evolutionary relationship. The transcription of TcAP2 gene in yew accumulated primarily in young organs, such as young stems. Quantitative real-time RT-PCR analysis indicated that TcAP2 gene was inducible to express by treatments with methyl jasmonate plus salicylic acid, high salinity, and cold. This gene showed no response to either abscisic acid or drought treatment. [ABSTRACT FROM AUTHOR]

Gene regulatory networks (GRNs) play a vital role in metazoan development and function, and deregulation of these networks is often implicated in disease. GRNs depict the dynamic interactions between genomic and regulatory state components. The genomic components comprise genes and their associated cis-regulatory elements. The regulatory state components consist primarily of transcriptional complexes that bind the latter elements. With the availability of complete genome sequences, several approaches have recently been developed which promise to significantly enhance our ability to identify either the genomic or regulatory state' components, or the interactions between these two. In this review, I provide an in-depth overview of these approaches and detail how each contributes to a more comprehensive understanding of GRN composition and function. [ABSTRACT FROM AUTHOR]

Abstract: Given the frequent occurrence of false negatives in yeast genetic assays, it is both interesting and practical to address the possible mechanisms of false negatives and, more important, to turn false negatives into true positives. We recently developed a modified yeast one-hybrid system (MY1H) useful for investigation of simultaneous protein–protein and protein:DNA interactions in vivo. We coexpressed the basic helix–loop–helix/Per-Arnt-Sim (bHLH/PAS) domains of aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (Arnt)—namely NAhR and NArnt, respectively—which are known to form heterodimers and bind the cognate xenobiotic response element (XRE) sequence both in vitro and in vivo, as a positive control in the study of XRE-binding proteins in the MY1H system. However, we observed negative results, that is, no positive signal detected from binding of the NAhR/NArnt heterodimer and XRE site. We demonstrate that by increasing the copy number of XRE sites integrated into the yeast genome and using double GAL4 activation domains, the NAhR/NArnt heterodimer forms and specifically binds the cognate XRE sequence, an interaction that is now clearly detectable in the MY1H system. This methodology may be helpful in troubleshooting and correcting false negatives that arise from unproductive transcription in yeast genetic assays. [Copyright &y& Elsevier]

Using a yeast one-hybrid method, a transcription factor, OsAP25, which interacts specifically with a GCC box was isolated from rice. The OsAP25 protein contained a conserved ethylene-responsive element binding factor (ERF) domain which shared identity with other reported ERF domains. Phylogenetic analysis showed that OsAP25 could be categorized into class III ERF of the previously characterized ERF proteins on an evolutionary relationship. The semi-quantitative RT-PCR analysis revealed that OsAP25 gene was constitutively expressed in leaves, roots, growing points, flower, bolting stage and grain filling stage. In addition, OsAP25 gene was induced by NaCl, cold, drought, abscisic acid and exogenous ethylene. [ABSTRACT FROM AUTHOR]

A transcription factor RCBF2 which interacts with C-repeat/DRE was isolated from Oryza sativa L. by a yeast one-hybrid method. Analysis of the deduced RCBF2 amino acid sequence revealed that RCBF2 contained a conserved ethylene-responsive element binding protein (EREBP)/AP2 domain of 59 amino acids and a potential nuclear localization sequence. RCBF2 showed a high level of homology with other CBF family members only in AP2 domain. Phylogenetic analysis showed that RCBF2 might be different from other eight DRE-binding proteins on evolutionary relationship. The semi-quantitative RT-PCR (s-Q RT-PCR) analysis indicated the expression of RCBF2 gene was induced by cold, dehydration and high-salinity, but not by abscisic acid, and the transcription of RCBF2 gene accumulated primarily in rice immature seeds, growing point and shoots. [ABSTRACT FROM AUTHOR]

Petal and stamen identity of the Antirrhinum majus flower is under the genetic control of the floral homeotic gene DEFICIENS ( DEF). To isolate factors involved in the regulation of DEF gene activity, a promoter segment of this B-function gene, containing cis-acting regulatory elements, was used to identify the novel trans-acting factor ROSINA (RSI). RSI does not show an extended similarity with any gene product present in the database. Rather RSI constitutes a protein that contains domains similar to known proteins from organisms of different phyla. The capacity of RSI to bind a sequence element of the DEF promoter, its spatial and temporal expression pattern together with the phenotype of RSI-RNAi interference plants as well as RSI over-expression in Arabidopsis thaliana suggest that RSI is a putative regulator of DEF gene activity in A. majus. [ABSTRACT FROM AUTHOR]

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase in human, has been identified as the rate-limiting factor in telomerase activity, but its detailed mechanism of transcription regulation remains unclear. In this investigation, a novel human gene telomerase transcriptional elements-interacting factor (TEIF) was isolated from HeLa cell based on hTERT promoter yeast one-hybrid assay. TEIF has a 2358-bp open reading frame encoding a predicted protein of 786 amino acids, which is functionally conserved in general eukaryotic species. The transcription of TEIF was detected in cells and expressed an 86-kDa native protein, distributing mainly in cellular nuclei. Its interaction with hTERT promoter was identified and the DNA binding potential was credited to C-terminus of TEIF. Transfection of TEIF into HeLa cells presented marked transactivation of hTERT promoter and stimulated both endogenous hTERT transcription and telomerase activities. These results suggest that TEIF protein might be a transcription regulator and take part in the activation of hTERT. [Copyright &y& Elsevier]

A major goal of molecular biology is to understand protein interactions and how interaction networks form the functional circuitry of cells. This goal is also relevant to drug development, as molecular target discovery and validation require an understanding of the function and disease relevance of proteins. One of the best approaches available to achieve this goal is the yeast two-hybrid system and its variants. This powerful tool can be used not only to identify and characterize individual drug targets, but also to dissect whole pathways and map protein interactions on a proteomic scale. [Copyright &y& Elsevier]

The dopamine transporter (DAT) terminates dopaminergic neurotransmission via reuptake of released dopamine into presynaptic neurons. We have cloned 2.5 kb of the regulatory region upstream of human DAT (hDAT) and constructed a series of deletion mutants to test promoter activity. A comparison of promoter activity between non-neural and neuronal cell lines reveals an interesting difference in pattern. In the PC12 cell line, activity of the proximal promoter is strongly silenced by one or more unidentified elements spanning positions −395 to −2465 of the hDAT gene. Our studies focus on identifying and characterizing the activating factor for hDAT transcription in the sequence between −2511 and −2492 (5′-CTA CCT GCA CAG TTC ACG GA-3′, termed HY1). In this investigation, we cloned the zinc finger protein 161 (ZFP161) gene as a HY1-binding factor, using the yeast one-hybrid screen. Recombinant ZFP161 was produced to evaluate the DNA-binding properties of the protein. The ability of ZFP161 to directly bind HY1 was examined in an electrophoretic mobility shift assay. RT-PCR analyses revealed that transfection of ZFP161 induced hDAT mRNA expression in HEK293 cells. We additionally confirmed the expression and localization of the DAT protein, using a specific antibody. Both the HY1 sequence and the downstream region were necessary for activation of the hDAT promoter by ZFP161. This finding suggests that the site of cofactor interaction with ZFP161 may exist downstream of HY1. [Copyright &y& Elsevier]

To isolate proteins involved in hTERT transcriptional regulation, the HeLa cDNA library was screened using the hTERT promoter-based yeast one-hybrid assay. A positive clone was rescued and proved to contain an open reading frame and the upstream coding sequences were obtained by 5′-RACE. The assembled full cDNA consisted of a 2.5 kb reading frame encoding 834 amino acids, in which a conserved N-acetyltransferase domain (GNAT family) was searched out in bioinformatics, and thus named as hALP (human N-acetyltransferase-like protein, GenBank Accession No. AF 489535). The expression of native hALP was identified in HeLa cells and proved to distribute in the cellular nucleus. The binding potential of hALP to hTERT promoter was confirmed by EMSA and the interacting sequence involved to −201- to −56-nt upstream region of the promoter. On transfection assay, hALP could obviously transactivate hTERT promoter and stimulate endogenous telomerase activity of cells. The analysis on histone acetyltransferase showed that hALP could specifically acetylate free histones in vitro. The investigation suggested that hALP influences the activity of histone acetylation and could up-regulate telomerase activity through transactivation of hTERT promoter. [Copyright &y& Elsevier]

Abstract Although yeast two-hybrid experiments are commonly used to identify protein interactions, the frequent occurrence of false negatives and false positives hampers data interpretation. Using both yeast one-hybrid and two-hybrid experiments, we have identified potential sources of these problems: the media preparation protocol and the source of the yeast nitrogen base may not only impact signal range but also effect whether a result appears positive or negative. While altering media preparation may optimize signal differences for individual experiments, media preparation must be reported in detail to replicate studies and accurately compare results from different experiments.

Solanum lycopersicum phenylalanine ammonia-lyase 5 (SlPAL5) gene regulates the metabolism of phenolic compounds. The study of transcription factors that regulate the expression of SlPAL5 gene is of great significance to elucidate the regulatory mechanism underlying the biosynthesis of phenolic compounds in tomato fruit induced by UV-C irradiation. Here, yeast one-hybrid library of tomato fruit was constructed, and the yeast one-hybrid technology was used to screen the transcription factors that regulate the expression of SlPAL5, the key gene related to the synthesis of phenolic compounds in tomato fruit. As a result, a transcription factor, SlERF7, was obtained and sequenced, followed by the blast homology analysis. Further experiments confirmed that SlERF7 interacted with the promoter of SlPAL5 gene. In addition, UV-C irradiation significantly increased the expression level of SlERF7. These results indicate that SlERF7, which is regulated by UV-C irradiation, might be involved in regulating the transcription of SlPAL5, which provided foundations for further studying the regulation mechanism of the biosynthesis of phenolic compounds in tomato fruit induced by UV-C irradiation.