Yeast One-hybrid Screening Research Articles

Enhanced Y1H assays for Arabidopsis

We present an Arabidopsis thaliana full-length transcription factor resource of 92% of root stele-expressed transcription factors and 74.5% of root-expressed transcription factors. We demonstrate its use with enhanced yeast one-hybrid (eY1H) screening for rapid, systematic mapping of plant transcription factor-promoter interactions. We identified 158 interactions with 13 stele-expressed promoters, many of which occur physically or are regulatory in planta.

Characterising the role of the Eucalyptus grandis SND2promoter in secondary cell wall biosynthesis

Background NAC and MYB transcription factors (TFs) have been shown to play prominent roles in the regulation of plant developmental processes. Two Arabidopsis thaliana NAC domain TFs (AtSND2, AtSND3) and one MYB domain TF (AtMYB103) were shown to be downstream targets of two master regulators of xylem fibre cell development, NST1 and SND1 [1,2]. These TFs were able to induce the expression of a GUS reporter gene under the control of the AtCesA8 promoter [3], indicating that they may be involved in the regulation of cellulose biosynthesis in the secondary cell walls of A. thaliana xylem fibres. It is hypothesized that putative orthologs of these TFs will also play important roles in regulating fibre secondary cell wall biosynthesis in woody plants such as Eucalyptus grandis. The transcriptional network regulating wood fibre development is uncharacterized in E. grandis, therefore it would be beneficial to identify upstream components of this transcriptional network in E. grandis. In this ongoing study we aim to identify TFs which bind to the promoter of the putative ortholog of AtSND2 in E. grandis (EgSND2) and possibly also to the promoters of the orthologs of AtSND3 and AtMYB103. In parallel, we are characterizing the detailed heterologous expression patterns of the EgSND2, EgSND3 and MYB103 promoter regions in A. thaliana plants. This work forms part of an effort to elucidate the transcriptional network regulating wood fibre development in E. grandis. Methods A reverse BLAST approach was used to identify candidate orthologs of AtSND2, AtSND3 and AtMYB103 in Eucalyptus, Populus and Vitis. In silico cis-element analysis was performed on the promoter regions of the putative orthologs to identify previously characterised and novel cis-elements. The 1.5 kb regions upstream of the translational start site (TSS) of EgSND2, EgSND3 and EgMYB103 were isolated from E. grandis genomic DNA. The amplified fragments were cloned into pMDC162, a GUS reporter vector, and introduced into A. thaliana Col-O plants for heterologous GUS expression analysis. The same reporter constructs were also transformed directly into the vascular cambium of potted E. grandis plants to determine endogenous promoter activity by Induced Somatic Sector Analysis (ISSA, [4]). Qualitative GUS reporter analyses were performed on the EgSND2promoter::GUS, EgSND3promoter::GUS and EgMYB103promoter::GUS constructs in Arabidopsis plants at 1, 3 and 6 weeks. A 500 bp truncation upstream of the TSS of EgSND2 was generated and cloned into the pHIS2.1 vector along with the 1.5 kb EgSND2 promoter sequence for use in yeast onehybrid (Y1-H) screening. Candidate proteins which bind to the cloned promoter sequences will be identified using Y1-H analysis and further characterised.

Krüppel-like factor 15 activates hepatitis B virus gene expression and replication

Hepatitis B virus (HBV) is a small DNA virus that requires cellular transcription factors for the expression of its genes. To understand the molecular mechanisms that regulate HBV gene expression, we conducted a yeast one-hybrid screen to identify novel cellular transcription factors that may control HBV gene expression. Here, we demonstrate that Krüppel-like factor 15 (KLF15), a liver-enriched transcription factor, can robustly activate HBV surface and core promoters. Mutations in the putative KLF15 binding site in the HBV core promoter abolished the ability of KLF15 to activate the core promoter in luciferase assays. Furthermore, the overexpression of KLF15 stimulated the expression of HBV surface antigen (HBsAg) and the core protein and enhanced viral replication. Conversely, small interfering RNA knockdown of the endogenous KLF15 in Huh7 cells resulted in a reduction in HBV surface- and core-promoter activities. In electrophoretic mobility shift and chromatin immunoprecipitation assays, KLF15 binds to DNA probes derived from the core promoter and the surface promoter. Introduction of an expression vector for KLF15 short hairpin RNA, together with the HBV genome into the mouse liver using hydrodynamic injection, resulted in a significant reduction in viral gene expression and DNA replication. Additionally, mutations in the KLF15 response element in the HBV core promoter significantly reduced viral DNA levels in the mouse serum. KLF15 is a novel transcriptional activator for HBV core and surface promoters. It is possible that KLF15 may serve as a potential therapeutic target to reduce HBV gene expression and viral replication.

Isolation and functional characterization of CE1 binding proteins

BackgroundAbscisic acid (ABA) is a plant hormone that controls seed germination, protective responses to various abiotic stresses and seed maturation. The ABA-dependent processes entail changes in gene expression. Numerous genes are regulated by ABA, and promoter analyses of the genes revealed that cis-elements sharing the ACGTGGC consensus sequence are ubiquitous among ABA-regulated gene promoters. The importance of the core sequence, which is generally known as ABA response element (ABRE), has been demonstrated by various experiments, and its cognate transcription factors known as ABFs/AREBs have been identified. Although necessary, ABRE alone is not sufficient, and another cis-element known as "coupling element (CE)" is required for full range ABA-regulation of gene expression. Several CEs are known. However, despite their importance, the cognate transcription factors mediating ABA response via CEs have not been reported to date. Here, we report the isolation of transcription factors that bind one of the coupling elements, CE1.ResultsTo isolate CE1 binding proteins, we carried out yeast one-hybrid screens. Reporter genes containing a trimer of the CE1 element were prepared and introduced into a yeast strain. The yeast was transformed with library DNA that represents RNA isolated from ABA-treated Arabidopsis seedlings. From the screen of 3.6 million yeast transformants, we isolated 78 positive clones. Analysis of the clones revealed that a group of AP2/ERF domain proteins binds the CE1 element. We investigated their expression patterns and analyzed their overexpression lines to investigate the in vivo functions of the CE element binding factors (CEBFs). Here, we show that one of the CEBFs, AtERF13, confers ABA hypersensitivity in Arabidopsis, whereas two other CEBFs enhance sugar sensitivity.ConclusionsOur results indicate that a group of AP2/ERF superfamily proteins interacts with CE1. Several CEBFs are known to mediate defense or abiotic stress response, but the physiological functions of other CEBFs remain to be determined. Our in vivo functional analysis of several CEBFs suggests that they are likely to be involved in ABA and/or sugar response. Together with previous results reported by others, our current data raise an interesting possibility that the coupling element CE1 may function not only as an ABRE but also as an element mediating biotic and abiotic stress responses.

Efficient Yeast One-/Two-Hybrid Screening Using a Library Composed Only of Transcription Factors in Arabidopsis thaliana

Yeast one-hybrid screening is widely used for the identification of transcription factors (TFs) that interact with specific DNA sequences. However, screening a whole cDNA library is not efficient for the identification of TFs because TF genes represent only a small percentage of clones in a cDNA library. Here, we present the development of an efficient yeast one-hybrid screening system using a prey library composed only of approximately 1,500 TF cDNAs of Arabidopsis thaliana. This library enabled us to isolate a TF that binds to a specific promoter sequence with high efficiency, even when the promoter region of the gene of interest was directly employed as bait. Furthermore, this library was also successfully applied as a yeast two-hybrid library to find TFs that interact with specific proteins. This efficient system will contribute to the elucidation of gene regulatory networks in plants.

SOX9, through Interaction with Microphthalmia-associated Transcription Factor (MITF) and OTX2, Regulates BEST1 Expression in the Retinal Pigment Epithelium

BEST1 is highly and preferentially expressed in the retinal pigment epithelium (RPE) and causes Best macular dystrophy when mutated. We previously demonstrated that the human BEST1 upstream region -154 to +38 bp is sufficient to direct expression in the RPE of transgenic mice, and microphthalmia-associated transcription factor (MITF) and OTX2 regulate this BEST1 promoter. However, a number of questions remained. Here, we show that yeast one-hybrid screen with bait corresponding to BEST1 -120 to -88 bp identified the SOX-E factors, SOX8, SOX9, and SOX10. A paired SOX site was found in this bait, and mutation of either of the paired sites significantly decreased BEST1 promoter activity in RPE primary cultures. Among the SOX-E genes, SOX9 is highly and preferentially expressed in the RPE, and chromatin immunoprecipitation with fresh RPE cells revealed binding of SOX9, but not SOX10, to the BEST1 region where the paired SOX site is located. BEST1 promoter activity was increased by SOX9 overexpression and decreased by siRNA-mediated SOX9 knockdown. Importantly, SOX9 physically interacted with MITF and OTX2 and orchestrated synergistic activation of the BEST1 promoter with the paired SOX site playing essential roles. A combination of the expression patterns of SOX9, MITF, and OTX2 yielded tissue distribution remarkably similar to that of BEST1. Lastly, the BEST1 promoter was also active in Sertoli cells of the testis in transgenic mice where SOX9 is highly expressed. These results define SOX9 as a key regulator of BEST1 expression and demonstrate for the first time its functional role in the RPE.

Identification of Glucose Transporter 4 Knockdown-dependent Transcriptional Activation Element on the Retinol Binding Protein 4 Gene Promoter and Requirement of the 20 S Proteasome Subunit for Transcriptional Activity

Retinol binding protein 4 (RBP4) is the transport protein that carries retinol in blood. RBP4 was described recently as a new adipokine that reduced insulin sensitivity. Mice lacking glucose transporter 4 (GLUT4) in adipocytes have enhanced Rbp4 gene expression; however, the molecular mechanism is unknown. We found a G4KA (GLUT4 knockdown-dependent transcriptional activation) element located approximately 1.3 kb upstream of the Rbp4 promoter. Mutations within the G4KA sequence significantly reduced expression of the Rbp4 promoter-reporter construct in G4KD-L1 (GLUT4 knockdown 3T3-L1) adipocyte cells. In a yeast one-hybrid screen of a G4KD-L1 cell cDNA library, using the G4KA element as bait, we identified subunits of the 20 S proteasome, PSMB1 and PSMA4, as binding partners. In chromatin immunoprecipitation assays, both subunits bound to the G4KA element; however, only PSMB1 was tightly bound in the GLUT4 knockdown model. PSMB1 RNA interference, but not PSMA4, significantly inhibited Rbp4 transcription. Nuclear transportation of PSMB1 was increased in G4KD-L1 cells. These results provide evidence for an exclusive proteasome subunit-related mechanism for transcriptional activation of RBP4 within a GLUT4 knockdown model.

Polypyrimidine tract-binding protein is required for the repression of gene expression by all-trans retinoic acid

All-trans retinoic acid is a key regulator of early development. High concentrations of retinoic acid interfere with differentiation and migration of neural crest cells. Here we report that a dinucleotide repeat in the cis-element of Snail2 (previously known as Slug) gene plays a role in repression by all-trans retinoic acid. We analyzed the cis-acting regulatory regions of the Xenopus Snail2 gene, whose expression is repressed by all-trans retinoic acid. The analysis identified a TG/CA repeat as a necessary element for the repression. By performing a yeast one-hybrid screen, we found that a polypyrimidine tract-binding protein (PTB), which is known to be a regulator of the alternative splicing of pre-messenger RNA, binds to the TG/CA repeat. Overexpression and knockdown experiments for PTB in HEK293 cells and Xenopus embryos indicated that PTB is required for repression by retinoic acid. The green fluorescent protein-PTB fusion protein was localized in the nucleus of 293T cells. In situ hybridization for PTB in Xenopus embryos showed that PTB is expressed at the regions including neural crest at the early stages. Our results indicate that PTB plays a role in the repression of gene expression by retinoic acid through binding to the TG/CA repeats.

G-Box Binding Factor1 Reduces CATALASE2 Expression and Regulates the Onset of Leaf Senescence in Arabidopsis

Hydrogen peroxide (H(2)O(2)) is discussed as being a signaling molecule in Arabidopsis (Arabidopsis thaliana) leaf senescence. Intracellular H(2)O(2) levels are controlled by the H(2)O(2)-scavenging enzyme catalase in concert with other scavenging and producing systems. Catalases are encoded by a small gene family, and the expression of all three Arabidopsis catalase genes is regulated in a senescence-associated manner. CATALASE2 (CAT2) expression is down-regulated during bolting time at the onset of leaf senescence and appears to be involved in the elevation of the H(2)O(2) level at this time point. To understand the role of CAT2 in senescence regulation in more detail, we used CAT2 promoter fragments in a yeast one-hybrid screen to isolate upstream regulatory factors. Among others, we could identify G-Box Binding Factor1 (GBF1) as a DNA-binding protein of the CAT2 promoter. Transient overexpression of GBF1 together with a CAT2:beta-glucuronidase construct in tobacco (Nicotiana benthamiana) plants and Arabidopsis protoplasts revealed a negative effect of GBF1 on CAT2 expression. In gbf1 mutant plants, the CAT2 decrease in expression and activity at bolting time and the increase in H(2)O(2) could no longer be observed. Consequently, the onset of leaf senescence and the expression of senescence-associated genes were delayed in gbf1 plants, clearly indicating a regulatory function of GBF1 in leaf senescence, most likely via regulation of the intracellular H(2)O(2) content.

Retinoic acid receptor gamma 2 interactions with vitamin D response elements

The vitamin D receptor (VDR) typically binds DNA in a heterodimer complex with the retinoid X receptor (RXR) to direct repeat sequences separated by three base pairs, or vitamin D response elements (VDREs). A modified yeast one-hybrid screen was utilized to search for partner proteins capable of associating with the VDR on a repressor VDRE. Screening of a HeLa cell cDNA library revealed that retinoic acid receptor gamma 2 (RARγ2) could specifically interact with VDREs, either in the presence or absence of the VDR. Importantly, the A-domain of RARγ2 appeared to be crucial for this interaction as evidenced by the inability of RARγ1 to affect reporter gene activity. Transfection data in COS-7 cells revealed the combination of both receptor ligands strongly attenuated transcriptional activation from an enhancer VDRE when RARγ2 was co-transfected into these cells with the VDR. Furthermore, a VDR/RARγ2 complex was detected in the mobility shift assay from nuclear extracts of transfected cells. Thus, the data highlight the novel ability of RARγ2 to interact with VDREs and impact vitamin D activity, which would allow for additional fine-tuning of a transcriptional response depending on ligand availability and expression profile of these nuclear receptors in a given cell type.

AmGAI-like interacts with ROSINA, a putative transcriptional regulator of DEFICIENS in Antirrhinum majus

In Antirrhinum, development of petals and stamens in the second and third whorl is controlled by the homeotic B-function genes DEFICIENS and GLOBOSA, which belong to the MADS-box family of transcription factors. The encoded proteins form heterodimers and control petal and stamen organogenesis. A first step for a better understanding of the molecular control mechanisms was the isolation of ROSINA ( RSI), a putative regulator of DEF, in a yeast one-hybrid screen using a DEF promoter fragment. RSI is a member of the b-ZIP family of transcription. Since such factors often need partners for exerting their regulatory function as heterodimers or multidimers, RSI was used as ‘bait’ in a yeast two-hybrid system to search for such potential partners. The most interesting candidate, named AmGAI-like, showed strong similarity to members of the GRAS family (GAI and RGA) of Arabidopsis. Expression of AmGAI-like revealed great similarity to the expression patterns of GAI and RGA of Arabidopsis. The protein–protein interaction between AmGAI-like and RSI was confirmed also biochemically by GST-pull down experiments.

Characterization of MYB proteins acting as transcriptional regulatory factors for carrot phenylalanine ammonia-lyase gene (DcPAL3)

In suspension-cultured carrot cells, a phenylalanine ammonia-lyase gene, DcPAL3, plays an important role in regulation of anthocyanin synthesis. In the DcPAL3 promoter region, a putative cis-element, box-L, which is committed to the upregulation of promoter activity, has been identified. Here, we isolated DcMYB5 cDNA using yeast one-hybrid screening with box-L as a bait from a cDNA library prepared from cells of a variant cultured cell line constitutively synthesizing anthocyanin. Although expression of DcMYB2, 3, and 4 was observed, all of which were previously isolated by plaque hybridization from a subtracted cDNA library of anthocyanin-synthesizing cells of a normal cultured cell line cultured in medium lacking 2,4-dichlorophenoxyacetic acid, DcMYB5 expression was not observed in cells of the normal cultured cell line; high expression of DcMYB5 was observed in cells of the variant cultured cell line. Although not only DcMYB5 but also DcMYB2, 3, and 4 could bind to the box-L sequence in yeast, DcMYB3 and 5 showed strong transcriptional activation activity for DcPAL3 promoter in carrot protoplasts. These results suggest that DcMYB3 and 5 might play an important role in the upregulation of DcPAL3 promoter activity in the different regulatory paths between the normal and variant cultured cell lines.

The Phytochrome-Interacting Factor PIF7 Negatively RegulatesDREB1Expression under Circadian Control in Arabidopsis

Transcription factors of the DRE-Binding1 (DREB1)/C-repeat binding factor family specifically interact with a cis-acting dehydration-responsive element/C-repeat involved in low-temperature stress-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Expression of DREB1s is induced by low temperatures and is regulated by the circadian clock under unstressed conditions. Promoter sequences of DREB1s contain six conserved motifs, boxes I to VI. We analyzed the promoter region of DREB1C using transgenic plants and found that box V with the G-box sequence negatively regulates DREB1C expression under circadian control. The region around box VI contains positive regulatory elements for low-temperature-induced expression of DREB1C. Using yeast one-hybrid screens, we isolated cDNA encoding the transcriptional factor Phytochrome-Interacting Factor7 (PIF7), which specifically binds to the G-box of the DREB1C promoter. The PIF7 gene was expressed in rosette leaves, and the PIF7 protein was localized in the nuclei of the cells. Transactivation experiments using Arabidopsis protoplasts indicated that PIF7 functions as a transcriptional repressor for DREB1C expression and that its activity is regulated by PIF7-interacting factors TIMING OF CAB EXPRESSION1 and Phytochrome B, which are components of the circadian oscillator and the red light photoreceptor, respectively. Moreover, in the pif7 mutant, expression of DREB1B and DREB1C was not repressed under light conditions, indicating that PIF7 functions as a transcriptional repressor for the expression of DREB1B and DREB1C under circadian control. This negative regulation of DREB1 expression may be important for avoiding plant growth retardation by the accumulation of DREB1 proteins under unstressed conditions.

Identification and characterization of bZIP‐type transcription factors involved in carrot (Daucus carota L.) somatic embryogenesis

Seed dormancy is an important adaptive trait that enables seeds of many species to remain quiescent until conditions become favorable for germination. Abscisic acid (ABA) plays an important role in these developmental processes. Like dormancy and germination, the elongation of carrot somatic embryo radicles is retarded by sucrose concentrations at or above 6%, and normal growth resumes at sucrose concentrations below 3%. Using a yeast one-hybrid screening system, we isolated two bZIP-type transcription factors, CAREB1 and CAREB2, from a cDNA library prepared from carrot somatic embryos cultured in a high-sucrose medium. Both CAREB1 and CAREB2 were localized to the nucleus, and specifically bound to the ABA response element (ABRE) in the Dc3 promoter. Expression of CAREB2 was induced in seedlings by drought and exogenous ABA application; whereas expression of CAREB1 increased during late embryogenesis, and reduced dramatically when somatic embryos were treated with fluridone, an inhibitor of ABA synthesis. Overexpression of CAREB1 caused somatic embryos to develop slowly when cultured in low-sucrose medium, and retarded the elongation of the radicles. These results indicate that CAREB1 and CAREB2 have similar DNA-binding activities, but play different roles during carrot development. Our results indicate that CAREB1 functions as an important trans-acting factor in the ABA signal transduction pathway during carrot somatic embryogenesis.

Regulation of sperm gene expression by the GATA factor ELT-1

Cell fate specification is mediated primarily through the expression of cell-type-specific genes. The regulatory pathway that governs the sperm/egg decision in the hermaphrodite germ line of Caenorhabditis elegans has been well characterized, but the transcription factors that drive these developmental programs remain unknown. We report the identification of ELT-1, a GATA transcription factor that specifies hypodermal fate in the embryo, as a regulator of sperm-specific transcription in the germ line. Computational analysis identified a conserved bipartite sequence element that is found almost exclusively in the promoters of a number of sperm genes. ELT-1 was recovered in a yeast one-hybrid screen for factors that bind to that sperm consensus site. In vitro assays defined the sperm consensus sequence as an optimal binding site for ELT-1. We determined that expression of elt-1 is elevated in the sperm-producing germ line, and that ELT-1 is required for sperm function. Deletion of the ELT-1 binding site from a sperm promoter abrogates sperm-specific expression of a reporter transgene. This work demonstrates a role for the ELT-1 transcription factor in sperm, and provides a critical link between the germ line sex determination program and gamete-specific gene expression.

Tissue-specific expression of the PNZIP promoter is mediated by combinatorial interaction of different cis-elements and a novel transcriptional factor

Recent studies demonstrated that PNZIP and its homologs encode a special cyclase and play an important role in chlorophyll biosynthesis in higher plants. To investigate the molecular mechanism governing the PNZIP gene, the PNZIP promoter was isolated and analyzed. Deletion analysis indicated that G-box is an important element in the regulation of the reporter gene expression. Further mutation assay demonstrated that G-box and GATACT elements are necessary and sufficient for the high and tissue-specific expression of the GUS gene. Using yeast one-hybrid screening, we have isolated a novel tobacco bZIP protein, NtbZIP, which can specifically recognize the G-box of the PNZIP promoter. The NtbZIP protein shares a limited amino acid homology to Arabidopsis ABI5 and AtAREB1 and very low homology to other bZIP proteins. Northern blot analysis showed that the NtbZIP gene is not induced by exogenous ABA and is expressed in different tobacco organs. Cotransformation assays showed that the NtbZIP protein could activate the transcription of the GUS gene driven by the PNZIP promoter. Transgenic tobaccos analysis demonstrated that constitutively expressing antisense NtbZIP gene resulted in a lower NTZIP synthesis and reduced chlorophyll levels. We suggest that NTZIP is a target gene of NtbZIP, which is involved in the regulation of chlorophyll biosynthesis.

Gastrin activates paracrine networks leading to induction of PAI-2 via MAZ and ASC-1

The gastric hormone gastrin regulates the expression of a variety of genes involved in control of acid secretion and also in the growth and organization of the gastric mucosa. One putative target is plasminogen activator inhibitor-2 (PAI-2), which is a component of the urokinase activator system that acts extracellularly to inhibit urokinase plasminogen activator (uPA) and intracellularly to suppress apoptosis. Previous studies have demonstrated that gastrin induces PAI-2 both in gastric epithelial cells expressing the gastrin (CCK-2) receptor and, via activation of paracrine networks, in adjacent cells that do not express the receptor. We have now sought to identify the response element(s) in the PAI-2 promoter targeted by paracrine mediators initiated by gastrin. Mutational analysis identified two putative response elements in the PAI-2 promoter that were downstream of gastrin-activated paracrine signals. One was identified as a putative MAZ site, mutation of which dramatically reduced both basal and gastrin-stimulated responses of the PAI-2 promoter by a mechanism involving PGE(2) and the small GTPase RhoA. Yeast one-hybrid screening identified the other as binding the activating signal cointegrator-1 (ASC-1) complex, which was shown to be the target of IL-8 released by gastrin. RNA interference (RNAi) knockdown of two subunits of the ASC-1 complex (p50 and p65) inhibited induction of PAI-2 expression by gastrin. The data reveal previously unsuspected transcriptional mechanisms activated as a consequence of gastrin-triggered paracrine networks and emphasize the elaborate and complex cellular control mechanisms required for a key component of tissue responses to damage and infection.

A Novel Function of RING Finger Protein 10 in Transcriptional Regulation of the Myelin-Associated Glycoprotein Gene and Myelin Formation in Schwann Cells

Myelin-associated glycoprotein (MAG) has been detected in Schwann cells prior to the onset of myelination, suggesting its functions in the initiation of myelination. However, transcriptional regulatory mechanisms of MAG remain to be elucidated. Here, we analyzed the promoter of the MAG gene by using luciferase reporter systems in the primary rat Schwann cells. We identified a novel cis-acting element located 160 bp upstream from the MAG transcription initiation site. Using the identified cis-element as a bait, we performed yeast one-hybrid screening and isolated a cDNA encoding a RNF10 as a putative trans-acting protein. When overexpressed in Schwann cells, RNF10 enhanced the activity of the MAG promoter. When RNF10 expression in Schwann cells was knocked down by siRNA, endogenous MAG mRNA and protein expression decreased. Furthermore, we evaluated myelin synthesis using Schwann cell-DRG neuron cocultures. When Schwann cells were infected with retrovirus expressing RNF10 siRNA, myelin formation was inhibited. These data suggest that RNF10 regulates MAG expression and is required for myelin formation.

Identification of potential protein regulators bound to the tissue‐specific positive and negative cis‐acting elements of a green tissue‐specific promoter in rice

Although some tissue-specific cis-acting elements have been identified, the molecular mechanisms of tissue-specific gene expression remain elusive. Here, we report the identification by a yeast one-hybrid screen of five proteins, Os10g31330/glycine-rich, Os01g10400/metallothionein-like, Os05g51180/nucleic acid-binding, Os05g37930/unknown and Os01g01689/phosphatidylinositol kinase that bound to either the positive or negative tissue-specific cis elements of a rice promoter from the green tissue-specific D54O gene. These proteins are localised in the nucleus and the genes encoding them are differentially expressed in different tissues, further suggesting their putative roles in regulating gene expression. These results suggest that the green tissue-specific expression of the D54O gene may be regulated by the interaction of multiple proteins with cis elements in the promoter region.

Distinct Functions of CCAAT Enhancer-binding Protein Isoforms in the Regulation of Manganese Superoxide Dismutase during Interleukin-1β Stimulation

The mitochondrial antioxidant enzyme manganese superoxide dismutase (Mn-SOD) is crucial in maintaining cellular and organismal homeostasis. Mn-SOD expression is tightly regulated in a manner that synchronizes its cytoprotective functions during inflammatory challenges. Induction of Mn-SOD gene expression by the proinflammatory cytokine IL-1beta is mediated through a complex intronic enhancer element. To identify and characterize the transcription factors required for Mn-SOD enhancer function, a yeast one-hybrid assay was utilized, and two CCAAT enhancer-binding protein (C/EBP) members, C/EBP beta and C/EBP delta, were identified. These two transcription factors responded to IL-1beta treatment with distinct expression profiles, different temporal yet inducible interactions with the endogenous Mn-SOD enhancer, and also opposite effects on Mn-SOD transcription. C/EBP beta is expressed as three isoforms, LAP* (liver-activating protein), LAP, and LIP (liver-inhibitory protein). Our functional analysis demonstrated that only the full-length C/EBP beta/LAP* served as a true activator for Mn-SOD, whereas LAP, LIP, and C/EBP delta functioned as potential repressors. Finally, our systematic mutagenesis of the unique N-terminal 21 amino acids further solidified the importance of LAP* in the induction of Mn-SOD and emphasized the crucial role of this isoform. Our data demonstrating the physiological relevance of the N-terminal peptide also provide a rationale for revisiting the role of LAP* in the regulation of other genes and in pathways such as lipogenesis and development.