GAL4 Protein Research Articles

Enhancing β-galactosidase production via GAL80 gene knockout in Kluyveromyces marxianus; an in-vitro and in-silico study on GAL/LAC system proteins

Lactose is a disaccharide composed of galactose and glucose which naturally exist only in the milk. β-galactosidase is one of the crucial industrial enzymes that hydrolyzes lactose. Kluyveromyces marxianus can grow on lactose as the only source of carbon and energy allowing this species to use whey as a cheap source. So far, no structural and comprehensive study has been done on its GAL/LAC system. Therefore, in this study, the proteins involved in the GAL/LAC system of K. marxianus were identified, and their secondary and tertiary structures and functions were determined for the first time. Also, the GAL80 inhibitory gene was deleted from the chromosome of this species. Its effect on β-Galactosidase enzyme production and its expression was investigated, as well as the expression of other genes involved in the production system of this enzyme. Lac9 and Lac12 proteins do not have beta sheets in their structures, in the Gal1, Gal7, and Gal80 proteins, the amount of helix is more than beta sheets, while in the Gal10 protein, the amount of beta sheets is more. Also, in the Lac12 protein, 46% of the total protein comprises transmembrane helices. Gal7 and Gal80 form homodimers in the cell. We showed that by deletion of the GAL80 gene from K. marxianus chromosome, the expression of genes involved in the GAL system, including LAC4 (β-galactosidase), LAC12 (permease), LAC9 (transcriptional activator), GAL1 (galactokinase), GAL7 (transferase), and GAL10 (epimerase), and the production of β-galactosidase, increased significantly in the culture medium containing different sugars.

Transcriptome analysis reveals the effect of cold storage time on the expression of genes related to oxidative metabolism in Chinese black truffle.

Chinese black truffle (Tuber indicum) is a hypogenous fungus of great value due to its distinctive aroma. In this study, both transcriptome and physicochemical analyses were performed to investigate the changes of nutrients and gene expression in truffle fruiting bodies during cold storage. The results of physicochemical analysis revealed the active metabolism of fruiting bodies in cold storage, showing the decreased contents of protein and soluble sugar, the variations in both polyphenol oxidase activity and total phenol content, and the detrimental effect of reactive oxygen species production caused by heavy metals (cadmium and lead) in truffles. Transcriptome analysis identified a total of 139,489 unigenes. Down-regulated expression of genes encoding the catalase-like domain-containing protein (katE), glutaredoxin protein (GRX), a copper/zinc superoxide dismutase (Sod_Cu), and aspartate aminotransferase (AAT) affected the degradation metabolism of intracellular oxides. Ribulose-5-phosphate-3-epimerase (RPE) was a key enzyme in response to oxidative stress in truffle cells through the pentose phosphate pathway (PPP). A total of 51,612 simple sequence repeats were identified, providing valuable resources for further genetic diversity analysis, molecular breeding, and genetic map-ping in T. indicum. Transcription factors GAL4 and SUF4-like protein were involved in glucose metabolism and histone methylation processes, respectively. Our study provided a fundamental characterization of the physicochemical and molecular variations in T. indicum during the cold storage at 4°C, providing strong experimental evidence to support the improvement of storage quality of T. indicum.

The cyclic peptide G4CP2 enables the modulation of galactose metabolism in yeast by interfering with GAL4 transcriptional activity.

Genetically-encoded combinatorial peptide libraries are convenient tools to identify peptides to be used as therapeutics, antimicrobials and functional synthetic biology modules. Here, we report the identification and characterization of a cyclic peptide, G4CP2, that interferes with the GAL4 protein, a transcription factor responsible for the activation of galactose catabolism in yeast and widely exploited in molecular biology. G4CP2 was identified by screening CYCLIC, a Yeast Two-Hybrid-based combinatorial library of cyclic peptides developed in our laboratory. G4CP2 interferes with GAL4-mediated activation of galactose metabolic enzymes both when expressed intracellularly, as a recombinant peptide, and when provided exogenously, as a chemically-synthesized cyclic peptide. Our results support the application of G4CP2 in microbial biotechnology and, additionally, demonstrate that CYCLIC can be used as a tool for the rapid identification of peptides, virtually without any limitations with respect to the target protein. The possible biotechnological applications of cyclic peptides are also discussed.

Exploiting the Gal4/UAS System as Plant Orthogonal Molecular Toolbox to Control Reporter Expression in Arabidopsis Protoplasts.

The ability of protein domains to fold independently from the rest of the polypeptide is the principle governing the generation of fusion proteins with customized functions. A clear example is the split transcription factor system based on the yeast GAL4 protein and its cognate UAS enhancer. The rare occurrence of the UAS element in the transcriptionally sensitive regions of the Arabidopsis genome makes this transcription factor an ideal orthogonal platform to control reporter induction. Moreover, heterodimeric transcriptional complexes can be generated by exploiting posttranslational modifications hampering or promoting the interaction between GAL4-fused transcriptional partners, whenever this leads to the reconstitution of a fully functional GAL4 factor.The assembly of multiple engineered proteins into a synthetic transcriptional complex requires preliminary testing, before its components can be stably introduced into the plant genome. Mesophyll protoplast transformation represents a fast and reliable technique to test and optimize synthetic regulatory modules. Remarkable properties are the possibility to transform different combinations of plasmids (co-transformation) and the physiological resemblance of these isolated cells with the original tissue.Here we describe an extensive protocol to produce and exploit Arabidopsis mesophyll protoplasts to investigate the transcriptional output of GAL4/UAS-based complexes that are sensitive to posttranslational protein modifications.

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Announcement of the 2022 winners of the Spychala and Women in Science Awards

A double role of the Gal80 N terminus in activation of transcription by Gal4p

The yeast galactose switch operated by the Gal4p-Gal80p-Gal3p regulatory module is a textbook model of transcription regulation in eukaryotes. The Gal80 protein inhibits Gal4p-mediated transcription activation by binding to the transcription activation domain. In Saccharomyces cerevisiae, inhibition is relieved by formation of an alternative Gal80-Gal3 complex. In yeasts lacking a Gal3p ortholog, such as Kluyveromyces lactis, the Gal1 protein (KlGal1p) combines regulatory and enzymatic activity. The data presented here reveal a yet unknown role of the KlGal80 N terminus in the mechanism of Gal4p activation. The N terminus contains an NLS, which is responsible for nuclear accumulation of KlGal80p and KlGal1p and for KlGal80p-mediated galactokinase inhibition. Herein, we present a model where the N terminus of KlGal80p reaches the catalytic center of KlGal1p causing enzyme inhibition in the nucleus and stabilization of the KlGal1-KlGal80p complex. We corroborate this model by genetic analyses and structural modelling and provide a rationale for the divergent evolution of the mechanism activating Gal4p.

Development of a Gateway-compatible two-component expression vector system for plants.

Genetic transformation of plants offers the possibility of functional characterization of individual genes and the improvement of plant traits. Development of novel transformation vectors is essential to improve plant genetic transformation technologies for various applications. Here, we present the development of a Gateway-compatible two-component expression vector system for Agrobacterium-mediated plant transformation. The expression system contains two independent plasmid vector sets, the activator vector and the reporter vector, based on the concept of the GAL4/UAS trans-activation system. The activator vector expresses a modified GAL4 protein (GAL4-VP16) under the control of specific promoter. The GAL4-VP16 protein targets the UAS in the reporter vector and subsequently activates reporter gene expression. Both the activator and reporter vectors contain the Gateway recombination cassette, which can be rapidly and efficiently replaced by any specific promoter and reporter gene of interest, to facilitate gene cloning procedures. The efficiency of the activator-reporter expression system has been assessed using agroinfiltration mediated transient expression assay in Nicotiana benthamiana and stable transgenic expression in Arabidopsis thaliana. The reporter genes were highly expressed with precise tissue-specific and subcellular localization. This Gateway-compatible two-component expression vector system will be a useful tool for advancing plant gene engineering.

Modeling transcriptional activation changes to Gal4 variants via structure-based computational mutagenesis.

As a DNA binding transcriptional activator, Gal4 promotes the expression of genes responsible for galactose metabolism. The Gal4 protein from Saccharomyces cerevisiae (baker’s yeast) has become a model for studying eukaryotic transcriptional activation in general because its regulatory properties mirror those of several eukaryotic organisms, including mammals. Given the availability of a crystallographic structure for Gal4, here we implement an in silico mutagenesis technique that makes use of a four-body knowledge-based energy function, in order to empirically quantify the structural impacts associated with single residue substitutions on the Gal4 protein. These results were used to examine the structure-function relationship in Gal4 based on a recently published experimental mutagenesis study, whereby functional changes to a uniformly distributed set of 1,068 single residue Gal4 variants were obtained by measuring their transcriptional activation levels relative to wild-type. A significant correlation was observed between computed (scalar) structural effect data and measured activity values for this collection of single residue Gal4 variants. Additionally, attribute vectors quantifying position-specific environmental impacts were generated for each of the Gal4 variants via computational mutagenesis, and we implemented supervised classification and regression statistical machine learning algorithms to train predictive models of variant Gal4 activity based on these structural changes. All models performed well under cross-validation testing, with balanced accuracy reaching 91% among the classification models, and with the actual and predicted activity values displaying a correlation as high as r = 0.80 for the regression models. Reliable predictions of transcriptional activation levels for Gal4 variants that have yet to be studied can be instantly generated by submitting their respective structure-based feature vectors to the trained models for testing. Such a computational pre-screening of Gal4 variants may potentially reduce costs associated with running large-scale mutagenesis experiments.

The 9aaTAD Is Exclusive Activation Domain in Gal4.

The Gal4 protein is a well-known prototypic acidic activator that has multiple activation domains. We have previously identified a new activation domain called the nine amino acid transactivation domain (9aaTAD) in Gal4 protein. The family of the 9aaTAD activators currently comprises over 40 members including p53, MLL, E2A and other members of the Gal4 family; Oaf1, Pip2, Pdr1 and Pdr3. In this study, we revised function of all reported Gal4 activation domains. Surprisingly, we found that beside of the activation domain 9aaTAD none of the previously reported activation domains had considerable transactivation potential and were not involved in the activation of transcription. Our results demonstrated that the 9aaTAD domain is the only decisive activation domain in the Gal4 protein. We found that the artificial peptides included in the original Gal4 constructs were results of an unintended consequence of cloning that were responsible for the artificial transcriptional activity. Importantly, the activation domain 9aaTAD, which is the exclusive activation domain in Gal4, is also the central part of a conserved sequence recognized by the inhibitory protein Gal80. We propose a revision of the Gal4 regulation, in which the activation domain 9aaTAD is directly linked to both activation function and Gal80 mediated inhibition.

Analyzing negative feedback using a synthetic gene network expressed in the Drosophila melanogaster embryo.

BackgroundA complex network of gene interactions controls gene regulation throughout development and the life of the organisms. Insights can be made into these processes by studying the functional interactions (or “motifs”) which make up these networks.ResultsWe sought to understand the functionality of one of these network motifs, negative feedback, in a multi-cellular system. This was accomplished using a synthetic network expressed in the Drosophila melanogaster embryo using the yeast proteins Gal4 (a transcriptional activator) and Gal80 (an inhibitor of Gal4 activity). This network is able to produce an attenuation or shuttling phenotype depending on the Gal80/Gal4 ratio. This shuttling behavior was validated by expressing Gal3, which inhibits Gal80, to produce a localized increase in free Gal4 and therefore signaling. Mathematical modeling was used to demonstrate the capacity for negative feedback to produce these varying outputs.ConclusionsThe capacity of a network motif to exhibit different phenotypes due to minor changes to the network in multi-cellular systems was shown. This work demonstrates the importance of studying network motifs in multi-cellular systems.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0330-z) contains supplementary material, which is available to authorized users.

Practical Recommendations for the Use of the GeneSwitch Gal4 System to Knock-Down Genes in Drosophila melanogaster.

Drosophila melanogaster is a popular research model organism thanks to its’ powerful genetic tools that allow spatial and temporal control of gene expression. The inducible GeneSwitch Gal4 system (GS) system is a modified version of the classic UAS/GAL4 system which allows inducible regulation of gene expression and eliminates background effects. It is widely acknowledged that the GS system is leaky, with low level expression of UAS transgenes in absence of the inducer RU-486 (the progesterone analog that activates the modified GAL4 protein). However, in the course of our experiments, we have observed that the extent of this leak depends on the nature of the transgene being expressed. In the absence of RU-486, when strong drivers are used to express protein coding transgenes, leaky expression is low or negligible, however expression of RNA interference (RNAi) transgenes results in complete depletion of protein levels. The majority of published studies, using the GS system and RNAi transgenes validate knock-down efficiency by comparing target gene mRNA levels between induced and non-induced groups. Here, we demonstrate that this approach is lacking and that both additional control groups and further validation is required at the protein level. Unfortunately, this experimental limitation of the GS system eliminates “the background advantage”, but does offer the possibility of performing more complex experiments (e.g. studying depletion and overexpression of different proteins in the same genetic background). The limitations and new possible applications of the GS system are discussed in detail.

Zinc finger domain of Su(Hw) protein is required for the formation of functional Su(Hw)-dependent insulator complex

This study is devoted to clarifying the role of Mod(mdg4)-67.2 and Su(Hw) proteins in the interaction between Su(Hw)-dependent insulator complexes and identifying the specific domains of the Su(Hw) protein required for insulation or mutual neutralization of insulators. Using genetic techniques and experiments in yeast two-hybrid system, we have demonstrated that the zinc finger domain of the Su(Hw) protein is involved in forming a functional insulator complex and cannot be replaced with the DNA-binding domain of the GAL4 protein.

Galectin-4 interacts with the drug transporter human concentrative nucleoside transporter 3 to regulate its function.

The intracellular N-terminal domain of the nucleoside and drug transporter human concentrative nucleoside transporter (hCNT)3 was used as bait in a glutathione S-transferase pull-down approach, to identify hCNT3 protein partners, using human colon homogenates as a prey source. Galectin (Gal)-4 was identified as a potential hCNT3 partner in the colon. The biochemical validation of the Gal-4-hCNT3 interaction was verified by targeted pull-down assays and coimmunoprecipitation experiments in HT-29 cells, which endogenously express hCNT3 and Gal-4. Furthermore, Gal-4 was shown to colocalize with hCNT3 in HT-29 cells. The biologic significance of this interaction was obtained from experiments in which Gal-4 was knocked down, showing that this protein is a regulator of hCNT3 trafficking and retention at the cell membrane, reducing its plasma membrane location by 70%. Conversely, the addition of Gal-4 increased hCNT3 location at the plasma membrane by 77%, thereby demonstrating that this lectin modulates hCNT3 function in colonic cells. The integrity of this partnership may be clinically relevant, because hCNT3 may be responsible for the translocation of thiopurines, such as 6-mercaptopurine, a front-line treatment in inflammatory bowel disease. The expression of Gal-4 and hCNT3 proteins is not impaired in inflamed colon from patients with Crohn's disease, thereby anticipating the integrity of this system for drug targeting.

Abstract 3165: Galectin-4, a key regulator of inflammation and cancer of the intestine

Abstract The inflammatory bowel diseases including ulcerative colitis and Crohn's disease are chronic inflammatory disorders of the intestine. It is well recognized that a serious long-term complication of chronic inflammation is the development of colorectal cancer (CRC). Although various mechanisms underlying different chronic inflammation conditions are known, it is increasing clear that proinflammatory cytokines produced during immune responses are the key contributing factors to the above disease states. On the other hand, genetic alterations inducing CRC also promote inflammatory responses in the gastrointestinal microenvironment. This interdependent relationship between oncogenesis and inflammation suggests that it is possible to identify a regulatory molecule that links these two cellular processes. The long-term interest of our efforts is to determine the mechanisms of galectin-4 (gal-4) in the normal and disease physiology of the human gastrointestinal tract. Gal-4 is expressed predominantly by the gastrointestinal epithelia. It is secreted and binds to specific oligosaccharide moiety in glycoproteins on the cell surface. We have recently demonstrated that gal-4 down-regulates cell proliferation and its loss of expression coincides with CRC onset and progression, and suggested that gal-4 functions as a tumor suppressor. The purpose of this study is to determine whether gal-4 regulates inflammatory responses in colorectal cells. In this study, we used gal-4 +ve CRC cells, HT-29 and LS-180 and neutralized their surface-bound gal-4 with gal-4 antibodies. Such cells proliferated profusely with concomitant up-regulation of transcription of 47 genes of which 17 are known partners/gene targets of multiple inflammation signaling pathways. Extracellular (spent) media obtained from these experiments were found to contain unique cytokines that are known to be pro-tumorigenic. In converse experiments, exposure of gal-4 -ve HCT116 cells to purified gal-4 resulted in decreased cell proliferation. Concomitantly, there was increased expression of p27, decreased expressions of cyclin D1 and c-Myc. These data suggested that binding of gal-4 to cell surface arrests cell cycle. Finally, analysis of rat colon tissue sections obtained from dextran sulfate-sodium (DSS) induced chronic colitis (kindly given by Dr. Daniel W. Rosenberg, Univ. of Connecticut CT) suggested that the epithelial cells at the colitis regions exhibited loss of gal-4 expression. These data together suggested that surface gal-4 down-regulates inflammatory response pathways, cell proliferation and oncogenic events. These data also suggested that gal-4 protein therapy and re-expression of gal-4 in lesions of chronic inflammation and CRC are potentially attractive methods of controlling these diseases. *A part of this study was carried out by the authors while working at the Texas Tech University Health Sciences Center Note: This abstract was not presented at the meeting. Citation Format: U.S. Rao, Prema S. Rao. Galectin-4, a key regulator of inflammation and cancer of the intestine. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3165. doi:10.1158/1538-7445.AM2015-3165

On the Mechanism of Gene Silencing in Saccharomyces cerevisiae.

Multiple mechanisms have been proposed for gene silencing in Saccharomyces cerevisiae, ranging from steric occlusion of DNA binding proteins from their recognition sequences in silenced chromatin to a specific block in the formation of the preinitiation complex to a block in transcriptional elongation. This study provided strong support for the steric occlusion mechanism by the discovery that RNA polymerase of bacteriophage T7 could be substantially blocked from transcribing from its cognate promoter when embedded in silenced chromatin. Moreover, unlike previous suggestions, we found no evidence for stalled RNA polymerase II within silenced chromatin. The effectiveness of the Sir protein–based silencing mechanism to block transcription activated by Gal4 at promoters in the domain of silenced chromatin was marginal, yet it improved when tested against mutant forms of the Gal4 protein, highlighting a role for specific activators in their sensitivity to gene silencing.

A purified truncated form of yeast Gal4 expressed in Escherichia coli and used to functionalize poly(lactic acid) nanoparticle surface is transcriptionally active in cellulo

Gal4/UAS system is a powerful tool for the analysis of numerous biological processes. Gal4 is a large yeast transcription factor that activates genes including UAS sequences in their promoter. Here, we have synthesized a minimal form of Gal4 DNA sequence coding for the binding and dimerization regions, but also part of the transcriptional activation domain. This truncated Gal4 protein was expressed as inclusion bodies in Escherichia coli. A structured and active form of this recombinant protein was purified and used to cover poly(lactic acid) (PLA) nanoparticles. In cellulo, these Gal4-vehicles were able to activate the expression of a Green Fluorescent Protein (GFP) gene under the control of UAS sequences, demonstrating that the decorated Gal4 variant can be delivery into cells where it still retains its transcription factor capacities. Thus, we have produced in E. coli and purified a short active form of Gal4 that retains its functions at the surface of PLA-nanoparticles in cellular assay. These decorated Gal4-nanoparticles will be useful to decipher their tissue distribution and their potential after ingestion or injection in UAS-GFP recombinant animal models.

Identification of putative interactions between swine and human influenza A virus nucleoprotein and human host proteins

BackgroundInfluenza A viruses (IAVs) are important pathogens that affect the health of humans and many additional animal species. IAVs are enveloped, negative single-stranded RNA viruses whose genome encodes at least ten proteins. The IAV nucleoprotein (NP) is a structural protein that associates with the viral RNA and is essential for virus replication. Understanding how IAVs interact with host proteins is essential for elucidating all of the required processes for viral replication, restrictions in species host range, and potential targets for antiviral therapies.MethodsIn this study, the NP from a swine IAV was cloned into a yeast two-hybrid “bait” vector for expression of a yeast Gal4 binding domain (BD)-NP fusion protein. This “bait” was used to screen a Y2H human HeLa cell “prey” library which consisted of human proteins fused to the Gal4 protein’s activation domain (AD). The interaction of “bait” and “prey” proteins resulted in activation of reporter genes.ResultsSeventeen positive bait-prey interactions were isolated in yeast. All of the “prey” isolated also interact in yeast with a NP “bait” cloned from a human IAV strain. Isolation and sequence analysis of the cDNAs encoding the human prey proteins revealed ten different human proteins. These host proteins are involved in various host cell processes and structures, including purine biosynthesis (PAICS), metabolism (ACOT13), proteasome (PA28B), DNA-binding (MSANTD3), cytoskeleton (CKAP5), potassium channel formation (KCTD9), zinc transporter function (SLC30A9), Na+/K+ ATPase function (ATP1B1), and RNA splicing (TRA2B).ConclusionsTen human proteins were identified as interacting with IAV NP in a Y2H screen. Some of these human proteins were reported in previous screens aimed at elucidating host proteins relevant to specific viral life cycle processes such as replication. This study extends previous findings by suggesting a mechanism by which these host proteins associate with the IAV, i.e., physical interaction with NP. Furthermore, this study revealed novel host protein-NP interactions in yeast.

An ancient oxidoreductase making differential use of its cofactors

Abstract Many transcription factors contribute to cellular homeostasis by integrating multiple signals. Signaling via the yeast Gal80 protein, a negative regulator of the prototypic transcription activator Gal4, is primarily regulated by galactose. ScGal80 from Saccharomyces cerevisiae has been reported to bind NAD(P). Here, we show that the ability to bind these ligands is conserved in KlGal80, a Gal80 homolog from the distantly related yeast Kluyveromyces lactis. However, the homologs apparently have diverged with respect to response to the dinucleotide. Strikingly, ScGal80 binds NAD(P) and NAD(P)H with more than 50-fold higher affinity than KlGal80. In contrast to ScGal80, where NAD is neutral, NAD and NADP have a negative effect in KlGal80 on its interaction with a KlGal4-peptide in vitro. Swapping a loop in the NAD(P) binding Rossmann-fold of ScGal80 into KlGal80 increases the affinity for NAD(P) and has a significant impact on KlGal4 regulation in vivo. Apparently, dinucleotide binding allows coupling of the metabolic state of the cell to regulation of the GAL/LAC genes. The particular sequences involved in binding determine how exactly the metabolic state is sensed and integrated by Gal80 to regulate Gal4.

Phosphorylation of Shox2 is required for its function to control sinoatrial node formation.

BackgroundInactivation of Shox2, a member of the short‐stature homeobox gene family, leads to defective development of multiple organs and embryonic lethality as a result of cardiovascular defects, including bradycardia and severe hypoplastic sinoatrial node (SAN) and sinus valves, in mice. It has been demonstrated that Shox2 regulates a genetic network through the repression of Nkx2.5 to maintain the fate of the SAN cells. However, the functional mechanism of Shox2 protein as a transcriptional repressor on Nkx2.5 expression remains completely unknown.Methods and ResultsA specific interaction between the B56δ regulatory subunit of PP2A and Shox2a, the isoform that is expressed in the developing heart, was demonstrated by yeast 2‐hybrid screen and coimmunoprecipitation. Western blotting and immunohistochemical assays further confirmed the presence of phosphorylated Shox2a (p‐Shox2a) in cell culture as well as in the developing mouse and human SAN. Site‐directed mutagenesis and in vitro kinase assays identified Ser92 and Ser110 as true phosphorylation sites and substrates of extracellular signal‐regulated kinase 1 and 2. Despite that Shox2a and its phosphorylation mutants possessed similar transcriptional repressive activities in cell cultures when fused with Gal4 protein, the mutant forms exhibited a compromised repressive effect on the activity of the mouse Nkx2.5 promoter in cell cultures, indicating that phosphorylation is required for Shox2a to repress Nkx2.5 expression specifically. Transgenic expression of Shox2a, but not Shox2a‐S92AS110A, mutant in the developing heart resulted in down‐regulation of Nkx2.5 in wild‐type mice and rescued the SAN defects in the Shox2 mutant background. Last, we demonstrated that elimination of both phosphorylation sites on Shox2a did not alter its nuclear location and dimerization, but depleted its capability to bind to the consensus sequences within the Nkx2.5 promoter region.ConclusionsOur studies reveal that phosphorylation is essential for Shox2a to repress Nkx2.5 expression during SAN development and differentiation.

JAB1/CSN5 inhibits the activity of Luman/CREB3 by promoting its degradation

Luman/CREB3 (also called LZIP) is an endoplasmic reticulum (ER)-bound transcription factor that has been implicated in the ER stress response. In this study, we used the region of Luman containing the basic DNA-binding domain as bait in a yeast two-hybrid screen and identified the Jun activation domain-binding protein 1 (JAB1) or the COP9 signalosome complex unit 5 (CSN5) as an interacting protein. We confirmed their direct binding by glutathione S-transferase pull-down assays, and verified the existence of such interaction in the cellular environment by mammalian two-hybrid and co-immunoprecipitation assays. Deletion mapping studies revealed that the MPN domain in JAB1 was essential and sufficient for the binding. JAB1 also colocalized with Luman in transfected cells. More interestingly, the nuclear form of Luman was shown to promote the translocation of JAB1 into the nucleus. We found that overexpression of JAB1 shortened the half-life of Luman by 67%, and repressed its transactivation function on GAL4 and unfolded protein response element (UPRE)-containing promoters. We therefore propose that JAB1 is a novel binding partner of Luman, which negatively regulates the activity of Luman by promoting its degradation.