Expression Of Reporter Genes In Yeast Research Articles

The C-Terminal Region of SLIM1 Transcription Factor Is Required for Sulfur Deficiency Response.

Sulfur LIMitation1 (SLIM1) transcription factor coordinates gene expression in plants in response to sulfur deficiency (−S). SLIM1 belongs to the family of plant-specific EIL transcription factors with EIN3 and EIL1, which regulate the ethylene-responsive gene expression. The EIL domains consist of DNA binding and dimerization domains highly conserved among EIL family members, while the N- and C-terminal regions are structurally variable and postulated to have regulatory roles in this protein family, such that the EIN3 C-terminal region is essential for its ethylene-responsive activation. In this study, we focused on the roles of the SLIM1 C-terminal region. We examined the transactivation activity of the full-length and the truncated SLIM1 in yeast and Arabidopsis. The full-length SLIM1 and the truncated form of SLIM1 with a deletion of C-terminal 106 amino acids (ΔC105) transactivated the reporter gene expression in yeast when they were fused to the GAL4 DNA binding domain, whereas the deletion of additional 15 amino acids to remove the C-terminal 120 amino acids (ΔC120) eliminated such an activity, identifying the necessity of that 15-amino-acid segment for transactivation. In the Arabidopsis slim1-2 mutant, the transcript levels of SULTR1;2 sulfate transporter and the GFP expression derived from the SULTR1;2 promoter-GFP (PSULTR1;2-GFP) transgene construct were restored under −S by introducing the full-length SLIM1, but not with the C-terminal truncated forms ΔC105 and ΔC57. Furthermore, the transcript levels of −S-responsive genes were restored concomitantly with an increase in glutathione accumulation in the complementing lines with the full-length SLIM1 but not with ΔC57. The C-terminal 57 amino acids of SLIM1 were also shown to be necessary for transactivation of a −S-inducible gene, SHM7/MSA1, in a transient expression system using the SHM7/MSA1 promoter-GUS as a reporter. These findings suggest that the C-terminal region is essential for the SLIM1 activity.

Transcription activation activity of ERD15 protein from Morus indica

Early Responsive to Dehydration (ERD) genes are described as rapid response mediators of dehydration stress. Recently, ERD15 has emerged as a novel stress induced transcription factor which might be involved in mediating distinct stress responses in plants. In order to determine whether mulberry ERD15 can act as functional transcription factor, yeast-based assays were performed. Mulberry ERD15 was found to drive high level reporter gene expression in yeast which suggests it may function as a transcription factor. However, due to lack of an identifiable DNA binding domain, deletion analysis was carried out to determine the putative region of the protein involved in mediating protein-DNA interaction. Our results indicate that the region between 70 and 100 amino acids is critical in conferring transcription activation activity and might harbor the DNA binding region of ERD15.

Ehrlichia chaffeensis TRP32 is a Nucleomodulin that Directly Regulates Expression of Host Genes Governing Differentiation and Proliferation.

Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate numerous host cell processes, including transcription. In a previous study, we reported that E. chaffeensis TRP32, a type 1 secreted effector, interacts with multiple host nucleus-associated proteins and also auto-activates reporter gene expression in yeast. In this study, we demonstrate that TRP32 is a nucleomodulin that binds host DNA and alters host gene transcription. TRP32 enters the host cell nucleus via a noncanonical translocation mechanism that involves phosphorylation of Y179 located in a C-terminal tri-tyrosine motif. Both genistein and mutation of Y179 inhibited TRP32 nuclear entry. An electromobility shift assay (EMSA) demonstrated TRP32 host DNA binding via its tandem repeat domain. TRP32 DNA binding and motif preference were further confirmed by supershift assays, as well as competition and mutant probe analyses. Using ChIP-Seq, we determined that TRP32 binds a G-rich motif primarily within ±500 bp of the gene transcription start site. An ontology analysis identified genes involved in processes such as immune cell differentiation, chromatin remodeling, and RNA transcription and processing, as primary TRP32 targets. TRP32 bound genes (n=1223) were distributed on all chromosomes and included several global regulators of proliferation and inflammation such as FOS and JUN, AKT3 and NRAS, and non-coding RNA genes, miRNA 21 and miRNA 142. TRP32 target genes were differentially regulated during infection, the majority of which were repressed, and direct repression/activation of these genes by TRP32 was confirmed in vitro with a cellular luciferase reporter assay.

The chili pepper CaATL1: an AT‐hook motif‐containing transcription factor implicated in defence responses against pathogens

SUMMARY Using cDNA microarray analysis, we isolated a cDNA clone, CaATL1 (Capsicum annuum L. Bukang AT-hook-Like gene 1), from a chili pepper plant incompatibly interacting with bacterial pathogens. The deduced amino acid sequence has a potential nuclear localization sequence and an AT-hook DNA binding motif which can bind AT-rich sequence elements. Expression of CaATL1 was specifically induced in host- and non-host-resistant responses against bacterial and viral pathogens in pepper plants. In addition, CaATL1 transcripts also increased following salicylic acid and ethephone treatment but were only mildly induced by methyl-jasmonate treatment. CaATL1::smGFP (soluble-modified green fluorescent protein) fusion protein localized to nuclei in tobacco BY2 protoplasts. The C-terminal region of the CaATL1 protein fused to the LexA DNA binding domain was able to activate reporter gene expression in yeast. To analyse further the role of the CaATL1 in pathogen defence response, we generated CaATL1-over-expressing transgenic tomato plants. These transgenic plants showed enhanced disease resistance against bacterial and oomycete pathogens. Taken together, these results provide the first evidence of a role for a plant AT-hook motif-containing transcription factor in pathogen defence response.

Gateway-Compatible Yeast One-Hybrid Screens

INTRODUCTIONProtein-DNA interactions (PDIs) between transcription factors (TFs) and their target genes form the backbone of transcription regulatory networks. Such PDIs can be identified with either a TF or a gene as a starting point. The Gateway-compatible yeast one-hybrid (Y1H) system provides a high-throughput, gene-centered method for the identification of interactions between a "DNA bait" (e.g., cis-regulatory DNA elements or gene promoters) and "protein preys" (e.g., TFs). The Y1H system is a genetic system to detect PDIs based on selection of reporter gene expression in yeast. DNA baits are fused by Gateway cloning to two reporter genes, HIS3 and lacZ, and the resulting DNA bait::reporter constructs are subsequently integrated into the genome of the host yeast strain. After integration, baits are examined for self-activation (i.e., their ability to drive reporter gene expression in the absence of an exogenous prey protein). Subsequently, each DNA bait is screened for interacting proteins by transforming a library of preys into the corresponding Y1H DNA bait yeast strain. Preys are hybrid proteins composed of a protein from the organism of interest and a heterologous transcription activation domain. When a prey protein binds to the DNA bait, the heterologous activation domain activates reporter gene expression. Thus, physical interactions between both repressors and activators and their DNA targets can be identified.

BR22, a novel protein, interacts with thyroid transcription factor-1 and activates the human surfactant protein B promoter.

Surfactant protein (SP)-B expression is restricted to type II pneumocytes and Clara cells in the lung. Previously, a promoter region of human SP-B gene from -64 to -118 has been identified as critical for the tissue-specific expression of this gene. Two cis-elements for thyroid transcription factor (TTF)-1 and hepatocyte nuclear factor (HNF)-3alpha binding were found within this area. Using an oligonucleotide fragment, we incorporated this region sequence into the promoter of a HIS3 reporter gene in yeast. With this modified yeast a human lung complementary DNA (cDNA) library was screened for DNA-binding proteins, other than TTF-1 and HNF-3alpha, that interacted with this promoter segment. A cDNA clone encoding a novel polypeptide, BR22, was identified that activated the reporter gene expression in yeast. This gene is expressed in many tissues and encodes a protein with bipartite nuclear localization signals. Studies using in vivo yeast two-hybrid analysis, in vitro protein-protein interactions, and coimmunoprecipitation analyses demonstrated that BR22 formed a protein complex with TTF-1. In vivo cotransfection studies further indicated that BR22 could act with TTF-1 to synergistically activate the SP-B promoter in mammalian cells. Our data suggest that BR22 is a TTF-1-associated protein. Through a protein-protein interaction with TTF-1, BR22 can form a complex and activate the human SP-B promoter in vivo.

Identification of COUP-TFII as a peroxisome proliferator response element binding factor using genetic selection in yeast: COUP-TFII activates transcription in yeast but anatagonizes PPAR signaling in mammalian cells

Peroxisome proliferator-response elements (PPRE) are cis-acting regulatory elements that confer responsiveness to peroxisome proliferators and various fatty acids by serving as target sites for ligand-activated peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers. Other cellular factors, including additional nuclear hormone receptors, also interact with PPREs and modulate PPAR function. We have developed a positive selection strategy in yeast to identify mammalian factors that functionally interact with PPREs. Saccharomyces cerevisiae containing an integrated copy of the HIS3 gene under transcriptional control of a minimal CYC1 promoter and two copies of the rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase PPRE was constructed and transformed with a rat liver cDNA yeast expression library. Plasmids were isolated from his + transformants. One plasmid contained a cDNA encoding the complete rat chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), an orphan member of the nuclear hormone receptor superfamily. COUP-TFII potently activated PPRE-linked reporter gene expression in yeast, and COUP-TFII synthesized in yeast or in vitro formed specific protein/DNA complexes with this PPRE. Significantly, COUP-TFII did not activate transcription of PPRE-linked reporter genes in mammalian cells but rather strongly inhibited induction mediated by PPAR/RXR. Our findings demonstrate the utility of using genetic screening in yeast to identify sequence-specific DNA binding transcription factors.