Transcriptional Activation In Yeast Cells Research Articles

CfSGR1 and CfSGR2 from Cryptomeria fortunei exhibit contrasting responses to hormones and abiotic stress in transgenic Arabidopsis

Stay-green (SGR) genes are pivotal regulatory genes in the context of plant chlorophyll metabolism, but few studies on SGR homologues in Cryptomeria fortunei have been previously reported. We cloned two CfSGR genes and overexpressed them in Arabidopsis to explore their functions. Full-length CfSGR1 and CfSGR2 are 1265 and 1197 bp, encompassing open reading frames (ORFs) encoding 274 and 276 amino acids, respectively. SGRs exhibited high conservation in higher plants, and phylogenetic analysis indicated that SGRs from monocots and gymnosperms cluster in a clade. The proteins localized to chloroplasts and showed no transcriptional activity in yeast cells. The CfSGR gene expressions were induced by abiotic stresses and hormones. Under conditions of darkness, abscisic acid (ABA), salt, drought, or freezing stress, CfSGR2-transgenic Arabidopsis exhibited a delay in leaf yellowing compared to the WT, which was attributed to increased chlorophyll content and enhanced photosynthetic capacity. These transgenic plants exhibited improved resistance to stress via upregulated expression of resistance-related genes, increased antioxidant enzyme activities, and reduced malondialdehyde content and electrolyte leakage rate. In contrast, CfSGR1-transgenic plants may accelerate leaf yellowing and exhibit reduced stress resistance. Our findings highlight potential divergence in the functions of CfSGR genes concerning plant growth and development and responses to abiotic stresses or hormones, providing a scientific foundation for future breeding of stress-resistant C. fortunei cultivars.

Exploring the GRAS gene family in Taraxacum kok-saghyz Rodin：characterization, evolutionary relationships, and expression analyses in response to abiotic stresses

The GRAS gene is an important specific transcription factor in plants, which has multiple functions such as signal transduction, cell morphogenesis and stress response. Although it is widely distributed in plants and has been characterized in several species, however, information about the GRAS family in Taraxacum kok-saghyz Rodin remains unknown. Here, TkGRAS family members were identified and analyzed for molecular characterization, tissue expression patterns and induced expression patterns. A total of 64 GRAS family members were identified at the genome-wide level, which could be categorized into 14 subfamilies by phylogenetic analysis. Most TkGRASs were intronless and had essentially the same gene structure in the same subfamily. Meanwhile, there were multiple response elements found in the promoters of TkGRASs. Tissue expression patterns and induced expression patterns showed that TkGRASs were expressed in different tissues and induced by abiotic stresses. Notably, the expression level of TkGRAS20 was up-regulated under different stresses, suggesting that this gene plays a pivotal role in the stress response. TkGRAS20 showed transcriptional activity in yeast cells and localized in the nucleus and plasma membrane. In conclusion, our study provided valuable insights into the genetic mechanisms underlying stress tolerance in TKS, and several key genes may be used for genetic breeding to improve stress tolerance.

Comprehensive analysis of the Spartina alterniflora WD40 gene family reveals the regulatory role of SaTTG1 in plant development.

The WD40 gene family, prevalent in eukaryotes, assumes diverse roles in cellular processes. Spartina alterniflora, a halophyte with exceptional salt tolerance, flood tolerance, reproduction, and diffusion ability, offers great potential for industrial applications and crop breeding analysis. The exploration of growth and development-related genes in this species offers immense potential for enhancing crop yield and environmental adaptability, particularly in industrialized plantations. However, the understanding of their role in regulating plant growth and development remains limited. In this study, we conducted a comprehensive analysis of WD40 genes in S. alterniflora at the whole-genome level, delving into their characteristics such as physicochemical properties, phylogenetic relationships, gene architecture, and expression patterns. Additionally, we cloned the TTG1 gene, a gene in plant growth and development across diverse species. We identified a total of 582 WD40 proteins in the S. alterniflora genome, exhibiting an uneven distribution across chromosomes. Through phylogenetic analysis, we categorized the 582 SaWD40 proteins into 12 distinct clades. Examining the duplication patterns of SaWD40 genes, we observed a predominant role of segmental duplication in their expansion. A substantial proportion of SaWD40 gene duplication pairs underwent purifying selection through evolution. To explore the functional aspects, we selected SaTTG1, a homolog of Arabidopsis TTG1, for overexpression in Arabidopsis. Subcellular localization analysis revealed that the SaTTG1 protein localized in the nucleus and plasma membrane, exhibiting transcriptional activation in yeast cells. The overexpression of SaTTG1 in Arabidopsis resulted in early flowering and increased seed size. These outcomes significantly contribute to our understanding of WD40 gene functions in halophyte species. The findings not only serve as a valuable foundation for further investigations into WD40 genes in halophyte but also offer insights into the molecular mechanisms governing plant development, offering potential avenues in molecular breeding.

Role of RcTINY2 in the Regulation of Drought and Salt Stress Response in Arabidopsis and Rose

In plants, transcription factors (TFs) belonging to the APETALA2/ethylene responsive factor (AP2/ERF) superfamily regulate a variety of life processes, including germination, maturation, and stress response. In the present study, RcTINY2, a novel dehydration response element binding protein (DREB) belonging to the A-4 group, was identified and characterized in rose (Rosa chinensis). RcTINY2 shares high homology with AtTINY2 of Arabidopsis (Arabidopsis thaliana), with several abiotic stress-responsive cis-regulatory elements. Transcript levels of RcTINY2 were induced by exposure to abscisic acid (ABA) in rose leaves and repressed by exposure to ABA, polyethylene glycol (PEG), and NaCl in rose roots. RcTINY2 is localized in the nucleus and showed transcriptional activation in yeast cells. Further analysis of transgenic Arabidopsis demonstrated that plants overexpressing RcTINY2 displayed increased ABA, PEG, and NaCl sensitivity in both germinating seeds and seedlings with reduced root growth and lateral root number. RcTINY2-silenced rose plants were found to be increasingly intolerant of both drought and salt stress. Furthermore, the transcript levels of several ABA- and abiotic stress-related genes were suppressed in RcTINY2-silenced rose plants. The results suggested that RcTINY2 may serve as a candidate gene for genetic improvement of abiotic stress tolerance in rose and other plant species.

CfAPX, a cytosolic ascorbate peroxidase gene from Cryptomeria fortunei, confers tolerance to abiotic stress in transgenic Arabidopsis

Plants subjected to biotic or abiotic stresses produce a large amount of reactive oxygen species (ROS). If ROS cannot be cleared in time, they cause a series of harmful reactions in plants. Ascorbate peroxidase (APX) is a key enzyme that removes ROS from plant cells and plays a vital role in plant stress resistance. However, to date, no studies on APX homologs in Cryptomeria fortunei have been reported. In this study, we isolated complementary DNA (cDNA) encoding APXfrom C. fortunei needles, which is referred to as CfAPX, by rapid amplification of cDNA ends (RACE). The full-length CfAPX sequence was 1226 bp in length and included a 750-bp open reading frame (ORF) encoding a protein of 249 amino acids. Phylogenetic analysis showed that APXs of different plant species have been highly evolutionarily conserved. CfAPX was shown to belong to the cytoplasmic subgroup and was more closely related to GbAPX of the gymnosperm Ginkgo biloba. CfAPX showed no transcriptional activity in yeast cells but was highly expressed in cones. To better handle abiotic stresses, compared with wild-type (WT) Arabidopsis thaliana, 35S::CfAPX transgenic Arabidopsis strongly expressed CfAPX, presented increased antioxidant enzyme activities, ascorbic acid (AsA) contents, chlorophyll levels and fluorescence parameter and reduced malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents. In addition, CfAPX expression in C. fortunei was mostly upregulated under stress. In summary, CfAPX confers abiotic stress responses to plants, which provides a scientific basis for subsequent breeding for increased stress resistance in C. fortunei.

Characterization and functional analysis of LoUDT1, a bHLH transcription factor related to anther development in the lily oriental hybrid Siberia (Lilium spp.)

Lily (Lilium spp.), with its beautiful flower, is an important horticultural crop and a popular ornamental plant, but because the abundant pollen pollutes the flowers and surroundings, its use is restricted. To solve this problem, the mechanism of pollen development in lily needs to be analyzed. However, the complex and delicate process of anther development in lily remains largely unknown. In this study, LoUDT1, a bHLH transcription factor (TF), was isolated and identified in lily. LoUDT1 was closely related to OsUDT1 of Oryza sativa and AtDYT1 of Arabidopsis. It was localized in the cytoplasm and nucleus and showed no transcriptional activation in yeast cells. LoUDT1 interacted with another bHLH TF, LoAMS, and the interaction depended on their BIF domains. LoUDT1 and LoAMS were both expressed in the anthers but showed different expression patterns. LoUDT1 was continuously expressed during the entire development of anthers, whereas LoAMS was only highly expressed early in anther development. With overexpression of LoUDT1 in Arabidopsis, normal anther development was affected and defective pollens were produced, which caused partial male sterility of transgenic plants. These defects depended on the level of LoUDT1 accumulation. By contrast, with the appropriate expression of LoUDT1 in a dyt1-3 mutant, normal pollen grains were produced, showing partial fertility. Thus, LoUDT1 might be a key regulator of anther development in lily. By further increasing the understanding of anther development, the results of this study can provide a theoretical basis for the molecular breeding of pollen-free lilies.

Cloning and cold-resistance analyses of CfICE1 gene in Cryptomeria fortunei

Cryptomeria fortunei is a conifer species that can attain a height of ~70 m and is cultivated for its timber as well as its ornamental value. It is a subtropical plant that prefers a warm and humid environment. Therefore, low temperature (LT) affects its growth, development, productivity and ecological distribution. Inducer of C-repeat binding factor (CBF) expression 1 (ICE1) plays an important role in the response to cold/freezing stress in plants through the CBF regulation pathway. To date, there is no research on homologue of ICE1 in C. fortunei. In this study, we first isolated the CfICE1 transcript from C. fortunei. The CfICE1 coding sequence was 1728 nucleotides encoding a 575-aa protein and contained a serine-rich motif, a basic helix-loop-helix-Zipper (bHLH-Zip), an ACT domain and a nuclear localization signal (NLS), which were conserved in ICE1 homologous genes. Phylogenetic analysis showed that CfICE1 and all dicots ICE1 proteins were clustered together. CfICE1 had transcriptional activity in yeast cells, was predominantly located in the nucleus and highly expressed in tender needles and roots. 35S::CfICE1 transgenic Arabidopsis thaliana could increase antioxidant enzyme activities and photosynthesis and reduce the malondialdehyde content compared to the wild-type to better cope with LT. Under LT, CfICE1 expression was higher; the C. fortunei clone with stronger cold resistance (CR) significantly upregulated the expression of CfICE1 compared to the weaker clone. In conclusion, these results suggest that CfICE1 plays an active role in CR, which provides a theoretical basis for breeding for CR in C. fortunei.

NbWRKY40 Positively Regulates the Response of Nicotiana benthamiana to Tomato Mosaic Virus via Salicylic Acid Signaling.

WRKY transcription factors play important roles in plants, including responses to stress; however, our understanding of the function of WRKY genes in plant responses to viral infection remains limited. In this study, we investigate the role of NbWRKY40 in Nicotiana benthamiana resistance to tomato mosaic virus (ToMV). NbWRKY40 is significantly downregulated by ToMV infection, and subcellular localization analysis indicates that NbWRKY40 is targeted to the nucleus. In addition, NbWRKY40 activates W-box-dependent transcription in plants and shows transcriptional activation in yeast cells. Overexpressing NbWRKY40 (OEWRKY40) inhibits ToMV infection, whereas NbWRKY40 silencing confers susceptibility. The level of salicylic acid (SA) is significantly higher in OEWRKY40 plants compared with that of wild-type plants. In addition, transcript levels of the SA-biosynthesis gene (ICS1) and SA-signaling genes (PR1b and PR2) are dramatically higher in OEWRKY40 plants than in the control but lower in NbWRKY40-silenced plants than in the control. Furthermore, electrophoretic mobility shift assays show that NbWRKY40 can bind the W-box element of ICS1. Callose staining reveals that the plasmodesmata is decreased in OEWRKY40 plants but increased in NbWRKY40-silenced plants. Exogenous application of SA also reduces viral accumulation in NbWRKY40-silenced plants infected with ToMV. RT-qPCR indicates that NbWRKY40 does not affect the replication of ToMV in protoplasts. Collectively, our findings suggest that NbWRKY40 likely regulates anti-ToMV resistance by regulating the expression of SA, resulting in the deposition of callose at the neck of plasmodesmata, which inhibits viral movement.

Chrysanthemum (Chrysanthemum morifolium) CmICE2 conferred freezing tolerance in Arabidopsis

Genes of the ICE (Inducer of CBF Expression) family play a key role in cold and freezing stresses response via the CBF regulatory pathway. In this work, we identified the ICE family gene, CmICE2, from Chrysanthemum morifolium ‘Jinba’. CmICE2 encodes a 451-amino acid protein with a conserved nuclear localization domain, a bHLH domain and ACT domain. CmICE2 is expressed in abundance in leaves and flowers, and the expression of CmICE2 is induced by freezing and drought stresses. CmICE2 localized to the nucleus, and has transcriptional activity in yeast cells. After a 24-hour 4 °C acclimation, Arabidopsis plants overexpressing CmICE2 were more tolerant to freezing stress (−9 °C for 6 h) than the Col-0. When exposed to −9 °C for 6 h, the expression levels of genes such as AtCBF1, AtCBF2, AtCBF4, AtCOR 6.6A, AtCOR 414 and AtKIN1 were up-regulated significantly in CmICE2 overexpression plant lines compared to wild type. The proline contents, activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were also increased in plants overexpressing CmICE2. In summary, CmICE2 confers to plant response to freezing stress.

Overexpression of a Novel Apple NAC Transcription Factor Gene, MdNAC1, Confers the Dwarf Phenotype in Transgenic Apple (Malus domestica).

Plant height is an important trait for fruit trees. The dwarf characteristic is commonly associated with highly efficient fruit production, a major objective when breeding for apple (Malus domestica). We studied the function of MdNAC1, a novel NAC transcription factor (TF) gene in apple related to plant dwarfing. Localized primarily to the nucleus, MdNAC1 has transcriptional activity in yeast cells. Overexpression of the gene results in a dwarf phenotype in transgenic apple plants. Their reduction in size is manifested by shorter, thinner stems and roots, and a smaller leaf area. The transgenics also have shorter internodes and fewer cells in the stems. Levels of endogenous abscisic acid (ABA) and brassinosteroid (BR) are lower in the transgenic plants, and expression is decreased for genes involved in the biosynthesis of those phytohormones. All of these findings demonstrate that MdNAC1 has a role in plants dwarfism, probably by regulating ABA and BR production.

OsERF71 confers drought tolerance via modulating ABA signaling and proline biosynthesis

Plants have evolved multiple protective strategies to adapt to adverse environmental conditions. Upland rice (UR) has evolved as a “drought-resistant type”. However, little is known about genes or mechanisms in UR that underlying drought tolerance at the molecular level. Here we report isolation and functional characterization of the ERF gene, OsERF71, from the UR variety, IRAT109. The expression of OsERF71 was induced by abscisic acid (ABA) and various abiotic stresses preferentially in IRAT109 under ABA, dehydration, and polyethyleneglycol (PEG) treatments. OsERF71 was verified as a nuclear-localized protein and had transcriptional activity in yeast cells. Overexpression of the OsERF71 in Nipponbare demonstrated a significant increase in tolerance to drought stress and a reduced rate of water loss. In contrast, OsERF71 interference lines were sensitive to drought stress and exhibited a higher rate of water loss. OsERF71-overexpressing lines also showed enhanced tolerance to high salinity. Moreover, OsERF71 regulated the expression of several ABA- responsive and proline biosynthesis genes under drought stress, resulting in enhanced sensitivity to exogenous ABA treatment and proline accumulation. Accordingly, we suggest that OsERF71 plays a positive role in drought stress tolerance by increasing the expression of genes associated with ABA signaling and proline biosynthesis under stress.

Prioritized Expression of BTN2 of Saccharomyces cerevisiae under Pronounced Translation Repression Induced by Severe Ethanol Stress.

Severe ethanol stress (>9% ethanol, v/v) as well as glucose deprivation rapidly induces a pronounced repression of overall protein synthesis in budding yeast Saccharomyces cerevisiae. Therefore, transcriptional activation in yeast cells under severe ethanol stress does not always indicate the production of expected protein levels. Messenger RNAs of genes containing heat shock elements can be intensively translated under glucose deprivation, suggesting that some mRNAs are preferentially translated even under severe ethanol stress. In the present study, we tried to identify the mRNA that can be preferentially translated under severe ethanol stress. BTN2 encodes a v-SNARE binding protein, and its null mutant shows hypersensitivity to ethanol. We found that BTN2 mRNA was efficiently translated under severe ethanol stress but not under mild ethanol stress. Moreover, the increased Btn2 protein levels caused by severe ethanol stress were smoothly decreased with the elimination of ethanol stress. These findings suggested that severe ethanol stress extensively induced BTN2 expression. Further, the BTN2 promoter induced protein synthesis of non-native genes such as CUR1, GIC2, and YUR1 in the presence of high ethanol concentrations, indicating that this promoter overcame severe ethanol stress-induced translation repression. Thus, our findings provide an important clue about yeast response to severe ethanol stress and suggest that the BTN2 promoter can be used to improve the efficiency of ethanol production and stress tolerance of yeast cells by modifying gene expression in the presence of high ethanol concentration.

Involvement of CmWRKY10 in Drought Tolerance of Chrysanthemum through the ABA-Signaling Pathway.

Drought is one of the important abiotic factors that adversely affects plant growth and production. The WRKY transcription factor plays a pivotal role in plant growth and development, as well as in the elevation of many abiotic stresses. Among three major groups of the WRKY family, the group IIe WRKY has been the least studied in floral crops. Here, we report functional aspects of group IIe WRKY member, i.e., CmWRKY10 in chrysanthemum involved in drought tolerance. The transactivation assay showed that CmWRKY10 had transcriptional activity in yeast cells and subcellular localization demonstrated that it was localized in nucleus. Our previous study showed that CmWRKY10 could be induced by drought in chrysanthemum. Moreover, the overexpression of CmWRKY10 in transgenic chrysanthemum plants improved tolerance to drought stress compared to wild-type (WT). High expression of DREB1A, DREB2A, CuZnSOD, NCED3A, and NCED3B transcripts in overexpressed plants provided strong evidence that drought tolerance mechanism was associated with abscisic acid (ABA) pathway. In addition, lower accumulation of reactive oxygen species (ROS) and higher enzymatic activity of peroxidase, superoxide dismutase and catalase in CmWRKY10 overexpressed lines than that of WT demonstrates its role in drought tolerance. Together, these findings reveal that CmWRKY10 works as a positive regulator in drought stress by regulating stress-related genes.

CmWRKY1 Enhances the Dehydration Tolerance of Chrysanthemum through the Regulation of ABA-Associated Genes.

WRKY transcription factors serve as antagonistic or synergistic regulators in a variety of abiotic stress responses in plants. Here, we show that CmWRKY1, a member of the group IIb WRKY family isolated from Chrysanthemum morifolium, exhibits no transcriptional activation in yeast cells. The subcellular localization examination showed that CmWRKY1 localizes to the nucleus in vivo. Furthermore, CmWRKY1-overexpressing transgenic lines exhibit enhanced dehydration tolerance in response to polyethylene glycol (PEG) treatment compared with wild-type plants. We further confirmed that the transgenic plants exhibit suppressed expression levels of genes negatively regulated by ABA, such as PP2C, ABI1 and ABI2, and activated expression levels of genes positively regulated by ABA, such as PYL2, SnRK2.2, ABF4, MYB2, RAB18, and DREB1A. Taken together, our results indicate that CmWRKY1 plays an important role in the response to drought in chrysanthemum through an ABA-mediated pathway.

Human heart cell proteins interacting with a C-terminally truncated 2A protein of coxsackie B3 virus: identification by the yeast two-hybrid system.

Protein 2A is a non-structural protein of coxsackievirus B3 (CVB3), an important human pathogen that can cause a variety of human diseases. Protein 2A not only participates in viral life cycle, but also regulates host cell functions; however, the underlying mechanisms remain poorly understood. In order to better understand the molecular mechanisms of CVB3 2A's function, the yeast two-hybrid (Y2H) system was adopted to screen for CVB3 2A interactive proteins in the human heart cDNA library. Full-length 2A shows strong transcriptional activity in yeast cells, which interferes with the application of Y2H system; therefore, a series of 2A deletion mutants were constructed. Analysis of transcriptional self-activation revealed that 2A lost its transcriptional activity after truncation of 60 amino acids (aa) at the N-terminus or deletion of 17 aa at the C-terminus. Choosing the 2A mutant with 17 aa deletion at the C-terminus as the bait protein, four interactive cellular proteins were identified, including TIMP4, MYL2, COX7C, and ENO1. These proteins are mostly related to protein degradation and metabolism. Although the interactions detected by the Y2H system should be considered as preliminary results, the finding of proteins translated from a human heart cDNA library that interacts with the CVB3 2A will stimulate experiments testing the reactivity of a translational mixture derived from that library with full-length 2A protein, followed by co-immunoprecipitation studies.

A NAC transcription factor, EjNAC1, affects lignification of loquat fruit by regulating lignin

Lignin is an important component of secondary cell walls, and its content influences the utilization of plant products for food and fiber. Loquat is a typical fruit accumulating lignin during postharvest low temperature storage. In this research, two EjNAC genes were isolated and designated as EjNAC1 and EjNAC2. Both EjNAC1 and EjNAC2 had transcriptional activity in yeast cells. The expression of EjNAC1 and EjNAC2 in lignin-rich stems was higher than in pulp. However, only EjNAC1 was induced in fruit by low temperature and inhibited by heat treatment (HT), which can alleviate lignification, while EjNAC2 expression was not correlated with fruit lignification. Further analysis indicated that EjNAC1 could trans-activate the gene promoters in loquat and Arabidopsis for genes in the lignin biosynthesis pathway. Transient over-expression of EjNAC1 in tobacco leaves resulted in accumulation of lignin and induction of expression of endogenous lignin biosynthesis genes. In conclusion, EjNAC1 was associated with fruit lignification by activating genes involved in lignin biosynthesis.

Characterization of MxFIT, an iron deficiency induced transcriptional factor in Malus xiaojinensis

Iron deficiency often results in nutritional disorder in fruit trees. Transcription factors play an important role in the regulation of iron uptake. In this study, we isolated an iron deficiency response transcription factor gene, MxFIT, from an iron-efficient apple genotype of Malus xiaojinensis. MxFIT encoded a basic helix-loop-helix protein and contained a 966 bp open reading frame. MxFIT protein was targeted to the nucleus in onion epidermal cells and showed strong transcriptional activation in yeast cells. Spatiotemporal expression analysis revealed that MxFIT was up-regulated in roots under iron deficiency at both mRNA and protein levels, while almost no expression was detected in leaves irrespective of iron supply. Ectopic expression of MxFIT resulted in enhanced iron deficiency responses in Arabidopsis under iron deficiency and stronger resistance to iron deficiency. Thus, MxFIT might be involved in iron uptake and plays an important role in iron deficiency response.

Enterovirus type 71 2A protease functions as a transcriptional activator in yeast

Enterovirus type 71 (EV71) 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME) in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis.

The Amino-terminal domain of tntegrin β3 functions as a transcriptional activator in yeast

A deletion mutant encoding the integrin beta3(4I-F56) with an additional Gln at the carboxyl terminus was found occassionally when we were constructing a counterselection yeast two-hybrid assay modle. This mutant exhibited strong transcriptional activation in yeast cells, bearing the Escherichia coli lacZ reporter gene encoding the beta-galactosidase under the transcriptional control of GAL4 promoter and TATA box. Further analysis revealed that the region between the amino acid residues 23C to S77, an acidic domain involved in the function of several transcriptional activators were critical for optimal level of transactivation.

Reverse recruitment: the Nup84 nuclear pore subcomplex mediates Rap1/Gcr1/Gcr2 transcriptional activation.

The recruitment model for gene activation presumes that DNA is a platform on which the requisite components of the transcriptional machinery are assembled. In contrast to this idea, we show here that Rap1/Gcr1/Gcr2 transcriptional activation in yeast cells occurs through a large anchored protein platform, the Nup84 nuclear pore subcomplex. Surprisingly, Nup84 and associated subcomplex components activate transcription themselves in vivo when fused to a heterologous DNA-binding domain. The Rap1 coactivators Gcr1 and Gcr2 form an important bridge between the yeast nuclear pore complex and the transcriptional machinery. Nucleoporin activation may be a widespread eukaryotic phenomenon, because it was first detected as a consequence of oncogenic rearrangements in acute myeloid leukemia and related syndromes in humans. These chromosomal translocations fuse a homeobox DNA-binding domain to the human homolog (hNup98) of a transcriptionally active component of the yeast Nup84 subcomplex. We conclude that Rap1 target genes are activated by moving to contact compartmentalized nuclear assemblages, rather than through recruitment of the requisite factors to chromatin by means of diffusion. We term this previously undescribed mechanism "reverse recruitment" and discuss the possibility that it is a central feature of eukaryotic gene regulation. Reverse recruitment stipulates that activators work by bringing the DNA to an nuclear pore complex-tethered platform of assembled transcriptional machine components.