Basic Region-leucine Zipper Research Articles

Phosphorylation of the Conserved Transcription Factor ATF-7 by PMK-1 p38 MAPK Regulates Innate Immunity in Caenorhabditis elegans

Innate immunity in Caenorhabditis elegans requires a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway that regulates the basal and pathogen-induced expression of immune effectors. The mechanisms by which PMK-1 p38 MAPK regulates the transcriptional activation of the C. elegans immune response have not been identified. Furthermore, in mammalian systems the genetic analysis of physiological targets of p38 MAPK in immunity has been limited. Here, we show that C. elegans ATF-7, a member of the conserved cyclic AMP–responsive element binding (CREB)/activating transcription factor (ATF) family of basic-region leucine zipper (bZIP) transcription factors and an ortholog of mammalian ATF2/ATF7, has a pivotal role in the regulation of PMK-1–mediated innate immunity. Genetic analysis of loss-of-function alleles and a gain-of-function allele of atf-7, combined with expression analysis of PMK-1–regulated genes and biochemical characterization of the interaction between ATF-7 and PMK-1, suggest that ATF-7 functions as a repressor of PMK-1–regulated genes that undergoes a switch to an activator upon phosphorylation by PMK-1. Whereas loss-of-function mutations in atf-7 can restore basal expression of PMK-1–regulated genes observed in the pmk-1 null mutant, the induction of PMK-1–regulated genes by pathogenic Pseudomonas aeruginosa PA14 is abrogated. The switching modes of ATF-7 activity, from repressor to activator in response to activated PMK-1 p38 MAPK, are reminiscent of the mechanism of regulation mediated by the corresponding ancestral Sko1p and Hog1p proteins in the yeast response to osmotic stress. Our data point to the regulation of the ATF2/ATF7/CREB5 family of transcriptional regulators by p38 MAPK as an ancient conserved mechanism for the control of innate immunity in metazoans, and suggest that ATF2/ATF7 may function in a similar manner in the regulation of mammalian innate immunity.

Induced ATF-2 represses CDK4 transcription through dimerization with JunD inhibiting intestinal epithelial cell growth after polyamine depletion.

Intestinal epithelium is a rapidly self-renewing tissue in the body, and its homeostasis is tightly regulated by numerous factors including polyamines. Decreased levels of cellular polyamines increase activating transcription factor (ATF)-2, but the exact role and mechanism of induced ATF-2 in the regulation of intestinal epithelial cell (IEC) growth remain elusive. Cyclin-dependent kinase (CDK) 4 is necessary for the G1-to-S phase transition during the cell cycle, and its expression is predominantly controlled at the transcription level. Here, we reported that induced ATF-2 following polyamine depletion repressed CDK4 gene transcription in IECs by increasing formation of the ATF-2/JunD heterodimers. ATF-2 formed complexes with JunD as measured by immunoprecipitation using the ATF-2 and JunD antibodies and by glutathione S-transferase (GST) pull-down assays using GST-ATF-2 fusion proteins. Studies using various mutants of GST-ATF-2 revealed that formation of the ATF-2/JunD dimers depended on the COOH-terminal basic region-leucine zipper domain of ATF-2. Polyamine depletion increased ATF-2/JunD complex and inhibited CDK4 transcription as indicated by a decrease in the levels of CDK4-promoter activity and its mRNA. ATF-2 silencing not only prevented inhibition of CDK4 transcription in polyamine-deficient cells but also abolished repression of CDK4 expression induced by ectopic JunD overexpression. ATF-2 silencing also promoted IEC growth in polyamine-depleted cells. These results indicate that induced ATF-2/JunD association following polyamine depletion represses CDK4 transcription, thus contributing to the inhibition of IEC growth.

Antioxidant-induced Phosphorylation of Tyrosine 486 Leads to Rapid Nuclear Export of Bach1 That Allows Nrf2 to Bind to the Antioxidant Response Element and Activate Defensive Gene Expression

Antioxidants cause stabilization and nuclear translocation of NF-E2-related factor 2 (Nrf2), where it binds to the antioxidant response element (ARE) and induces up-regulation of defensive genes that protect cells against oxidative and electrophilic stress. Bach1, the negative regulator of Nrf2, competes with Nrf2 for binding to the ARE in the human NQO1 promoter. In this study, we demonstrate that Bach1 exits the nucleus within 1-2 h upon antioxidant treatment. Genistein, an inhibitor of tyrosine kinases, blocked nuclear export of Bach1. Site-directed mutagenesis and immunoprecipitation assays identified tyrosine 486 that was phosphorylated in response to the antioxidant and was essential for nuclear export of Bach1. Chromatin immunoprecipitation assays revealed a competitive interplay between Bach1 and Nrf2 at 1-2 and 4 h for binding to the human NQO1 ARE. Luciferase and real time PCR assays showed a significant decrease in antioxidant induction of reporter activity and mRNA levels in cells transfected with mutant Bach1 compared with wild type. This decrease was due to the absence of nuclear export of the mutant protein. Bach1 levels inside the nucleus returned to normal at 4 h after antioxidant treatment in the absence but not in the presence of protein synthesis inhibitor cycloheximide. In addition, antioxidant treatment increased the transcription of Bach1 as shown by pulse chase and real time PCR experiments. Taken together, these results indicate that increased synthesis of Bach1 restored its nuclear levels to normal at 4 h. In conclusion, antioxidant-induced tyrosine 486 phosphorylation leads to nuclear exit of Bach1, thus allowing Nrf2 access to the ARE.

Emerging Roles for XBP1, a sUPeR Transcription Factor

X-box binding protein 1 (XBP1) is a unique basic region leucine zipper (bZIP) transcription factor whose active form is generated by a nonconventional splicing reaction upon disruption of homeostasis in the endoplasmic reticulum (ER) and activation of the unfolded protein response (UPR). XBP1, first identified as a key regulator of major histocompatibility complex (MHC) class II gene expression in B cells, represents the most conserved signaling component of UPR and is critical for cell fate determination in response to ER stress. Here we review recent advances in our understanding of this multifaceted transcription factor in health and diseases.

ATF3, a Hub of the Cellular Adaptive-Response Network, in the Pathogenesis of Diseases: Is Modulation of Inflammation a Unifying Component?

Activating transcription factor 3 (ATF3) gene encodes a member of the ATF family of transcription factors and is induced by various stress signals. All members of this family share the basic region-leucine zipper (bZip) DNA binding motif and bind to the consensus sequence TGACGTCA in vitro. Previous reviews and an Internet source have covered the following topics: the nomenclature of ATF proteins, the history of their discovery, the potential interplays between ATFs and other bZip proteins, ATF3-interacting proteins, ATF3 target genes, and the emerging roles of ATF3 in cancer and immunity (see footnote 1). In this review, we present evidence and clues that prompted us to put forth the idea that ATF3 functions as a "hub" of the cellular adaptive-response network. We will then focus on the roles of ATF3 in modulating inflammatory response. Inflammation is increasingly recognized to play an important role for the development of many diseases. Putting this in the context of the hub idea, we propose that modulation of inflammation by ATF3 is a unifying theme for the potential involvement of ATF3 in various diseases.

Amino acid deprivation induces CREBZF/Zhangfei expression via an AARE-like element in the promoter

CREBZF (also called ZF or Zhangfei) is a basic region–leucine zipper transcription factor that has been implicated in the herpesvirus infection cycle and related cellular processes. Since ATF4 is known to play a key role in cellular responses to various ER stresses as well as amino acid deprivation, we sought to examine the potential involvement of CREBZF in the amino acid response (AAR). We found that the CREBZF protein was induced by amino acid deprivation in the canine MDCK cells. We subsequently cloned a canine CREBZF promoter region (−1767 bp to +1 bp) that responds to amino acid limitation. Using deletion mapping and site-directed mutagenesis, we identified a 9-bp sequence 5′-ATTCACTCA-3′ in the promoter (−1227 to −1219), deletion of which resulted in a complete loss of inducibility by amino acid deprivation. This sequence is similar to the known amino acid response elements (AAREs) found in other AAR-inducible genes, such as CHOP (C/EBP homologous protein, also known as GADD153). These results suggest that CREBZF may be an amino acid stress sensor. Considering the AARE-like sequence found in CREBZF and other similarities between CREBZF and CHOP, we postulate that CREBZF and CHOP may be two sensors that regulate different yet related signaling pathways governing the AAR.

Interaction betweenDrosophilabZIP proteins Atf3 and Jun prevents replacement of epithelial cells during metamorphosis

Epithelial sheet spreading and fusion underlie important developmental processes. Well-characterized examples of such epithelial morphogenetic events have been provided by studies in Drosophila, and include embryonic dorsal closure, formation of the adult thorax and wound healing. All of these processes require the basic region-leucine zipper (bZIP) transcription factors Jun and Fos. Much less is known about morphogenesis of the fly abdomen, which involves replacement of larval epidermal cells (LECs) with adult histoblasts that divide, migrate and finally fuse to form the adult epidermis during metamorphosis. Here, we implicate Drosophila Activating transcription factor 3 (Atf3), the single ortholog of human ATF3 and JDP2 bZIP proteins, in abdominal morphogenesis. During the process of the epithelial cell replacement, transcription of the atf3 gene declines. When this downregulation is experimentally prevented, the affected LECs accumulate cell-adhesion proteins and their extrusion and replacement with histoblasts are blocked. The abnormally adhering LECs consequently obstruct the closure of the adult abdominal epithelium. This closure defect can be either mimicked and further enhanced by knockdown of the small GTPase Rho1 or, conversely, alleviated by stimulating ecdysone steroid hormone signaling. Both Rho and ecdysone pathways have been previously identified as effectors of the LEC replacement. To elicit the gain-of-function effect, Atf3 specifically requires its binding partner Jun. Our data thus identify Atf3 as a new functional partner of Drosophila Jun during development.

Nfil3/E4bp4 is required for the development and maturation of NK cells in vivo

Nuclear factor interleukin-3 (Nfil3; also known as E4-binding protein 4) is a basic region leucine zipper transcription factor that has antiapoptotic activity in vitro under conditions of growth factor withdrawal. To study the role of Nfil3 in vivo, we generated gene-targeted Nfil3-deficient (Nfil3−/−) mice. Nfil3−/− mice were born at normal Mendelian frequency and were grossly normal and fertile. Although numbers of T cells, B cells, and natural killer (NK) T cells were normal in Nfil3−/− mice, a specific disruption in NK cell development resulted in severely reduced numbers of mature NK cells in the periphery. This defect was NK cell intrinsic in nature, leading to a failure to reject MHC class I–deficient cells in vivo and reductions in both interferon γ production and cytolytic activity in vitro. Our results confirm the specific and essential requirement of Nfil3 for the development of cells of the NK lineage.

Structural Basis of Alternative DNA Recognition by Maf Transcription Factors

Maf transcription factors constitute a family of the basic region-leucine zipper (bZip) factors and recognize unusually long DNA motifs (13 or 14 bp), termed the Maf recognition element (MARE). The MARE harbors extended GC sequences on each side of its core motif, which is similar to TRE or CRE (7 or 8 bp) recognized by the AP1 and CREB/ATF families, respectively. To ascertain the structural basis governing the acquirement of such unique DNA recognition, we determined the crystal structure of the MafG-DNA complex. Each MafG monomer consists of three helices in which the carboxyl-terminal long helix organizes one DNA-contacting element and one coiled-coil dimer formation element. To our surprise, two well-conserved residues, Arg57 and Asn61 in the basic region, play critical roles in Maf-specific DNA recognition. These two residues show unique side-chain orientations and interact directly with the extended GC bases. Maf-specific residues in the amino-terminal and basic regions appear to indirectly stabilize MARE recognition through DNA backbone phosphate interactions. This study revealed an alternative DNA recognition mechanism of the bZip factors that bestows specific target gene profiles upon Maf homodimers or Maf-containing heterodimers.

C/EBPα and β couple interfollicular keratinocyte proliferation arrest to commitment and terminal differentiation

The transcriptional regulators that couple interfollicular basal keratinocyte proliferation arrest to commitment and differentiation are yet to be identified. Here we report that the basic region leucine zipper transcription factors C/EBPalpha and C/EBPbeta are co-expressed in basal keratinocytes, and are coordinately upregulated as keratinocytes exit the basal layer and undergo terminal differentiation. Mice lacking both C/EBPalpha and beta in the epidermis showed increased proliferation of basal keratinocytes and impaired commitment to differentiation. This led to ectopic expression of keratin 14 (K14) and DeltaNp63 in suprabasal cells, decreased expression of spinous and granular layer proteins, parakeratosis and defective epidermal water barrier function. Knock-in mutagenesis revealed that C/EBP-E2F interaction was required for control of interfollicular epidermis (IFE) keratinocyte proliferation, but not for induction of spinous and granular layer markers, whereas C/EBP DNA binding was required for DeltaNp63 downregulation and K1/K10 induction. Finally, loss of C/EBPalpha/beta induced stem cell gene expression signatures in the epidermis. C/EBPs, therefore, couple basal keratinocyte cell cycle exit to commitment to differentiation through E2F repression and DNA binding, respectively, and may act to restrict the epidermal stem cell compartment.

1-Cyano-2,3-epithiopropane is a novel plant-derived chemopreventive agent which induces cytoprotective genes that afford resistance against the genotoxic , -unsaturated aldehyde acrolein

Epithionitriles represent a previously unrecognized class of cancer chemopreventive phytochemical generated from alkenyl glucosinolates in cruciferous vegetables. In rat liver RL-34 epithelial cells, 1-cyano-2,3-epithiopropane (CETP), 1-cyano-3,4-epithiobutane (CETB) and 1-cyano-4,5-epithiopentane (CETPent) were shown to induce cytoprotective enzymes including NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione (GSH) S-transferase A3 and the glutamate-cysteine ligase modifier subunit; CETP was more potent in this regard than were either CETB or CETPent, with 50 microM CETP eliciting a remarkable approximately 10-fold induction of NQO1. Furthermore, 50 microM CETP stimulated a 2.0-fold overproduction of GSH in RL-34 cells. Transfection experiments demonstrated that epithionitriles induced gene expression through an antioxidant response element (ARE) and that transactivation of an Nqo1-luciferase reporter plasmid was dependent on NF-E2 p45-related factor 2 (Nrf2), a cap'n'collar basic region leucine zipper transcription factor. Evidence is presented that CETP affected Nrf2-mediated induction of ARE-driven transcription by inhibiting Kelch-like ECH-associated protein 1 (Keap1), a ubiquitin ligase substrate adaptor that negatively regulates Nrf2. We found that Nqo1 was expressed constitutively at high levels in Keap1(-/-) mouse embryonic fibroblasts (MEFs) and it was not further induced by CETP. However, knock-in of mouse Keap1 or zebrafish Keap1a into Keap1(-/-) MEFs repressed Nqo1-luciferase reporter gene activity, but repression by the murine or zebrafish proteins was antagonized by CETP. Pre-treatment of Nrf2(+/+) MEFs, but not Nrf2(-/-) MEFs, with 15 microM CETP for 24 h conferred 2.4-fold resistance against subsequent exposure to the alpha,beta-unsaturated aldehyde acrolein, indicating that the phytochemical exerts chemopreventive properties against genotoxic xenobiotics.

C-Jun DNAzymes Inhibit Myocardial Inflammation, ROS Generation, Infarct Size, and Improve Cardiac Function After Ischemia-Reperfusion Injury

Coronary reperfusion has been the mainstay therapy for reduced infarct size after a heart attack. However, this intervention also results in myocardial injury by initiating a marked inflammatory reaction, and new treatments are keenly sought. The basic-region leucine zipper protein, c-Jun is poorly expressed in the normal myocardium and is induced within 24 hours after myocardial ischemia-reperfusion injury. Synthetic catalytic DNA molecules (DNAzymes) targeting c-Jun (Dz13) reduce infarct size in the area-at-risk (AAR) regardless of whether it is delivered intramyocardially at the initiation of ischemia or at the time of reperfusion. Dz13 attenuates neutrophil infiltration, c-Jun and ICAM-1 expression in vascular endothelium, cardiomyocyte apoptosis, and the generation of reactive oxygen species in the reperfused myocardium. It inhibits infiltration into the AAR of complement 3 (C3), C3a receptor (C3aR), membrane attack complex-1 (Mac-1), or matrix metalloproteinase-2 (MMP-2) positive inflammatory cells. Dz13 also improves cardiac function without influencing myocardial vascularity or fibrosis. These findings demonstrate the regulatory role of c-Jun in the pathogenesis of myocardial inflammation and infarction following ischemia-reperfusion injury, and inhibition of this process using catalytic DNA.

The Importance of Being Flexible: The Case of Basic Region Leucine Zipper Transcriptional Regulators

Large volumes of protein sequence and structure data acquired by proteomic studies led to the development of computational bioinformatic techniques that made possible the functional annotation and structural characterization of proteins based on their primary structure. It has become evident from genome-wide analyses that many proteins in eukaryotic cells are either completely disordered or contain long unstructured regions that are crucial for their biological functions. The content of disorder increases with evolution indicating a possibly important role of disorder in the regulation of cellular systems. Transcription factors are no exception and several proteins of this class have recently been characterized as premolten/molten globules. Yet, mammalian cells rely on these proteins to control expression of their 30,000 or so genes. Basic region:leucine zipper (bZIP) DNA-binding proteins constitute a major class of eukaryotic transcriptional regulators. This review discusses how conformational flexibility "built" into the amino acid sequence allows bZIP proteins to interact with a large number of diverse molecular partners and to accomplish their manifold cellular tasks in a strictly regulated and coordinated manner.

Identification of the Candida albicans Cap1p Regulon

Cap1p, a transcription factor of the basic region leucine zipper family, regulates the oxidative stress response (OSR) in Candida albicans. Alteration of its C-terminal cysteine-rich domain (CRD) results in Cap1p nuclear retention and transcriptional activation. To better understand the function of Cap1p in C. albicans, we used genome-wide location profiling (chromatin immunoprecipitation-on-chip) to identify its transcriptional targets in vivo. A triple-hemagglutinin (HA(3)) epitope was introduced at the C terminus of wild-type Cap1p (Cap1p-HA(3)) or hyperactive Cap1p with an altered CRD (Cap1p-CSE-HA(3)). Location profiling using whole-genome oligonucleotide tiling microarrays identified 89 targets bound by Cap1p-HA(3) or Cap1p-CSE-HA(3) (the binding ratio was at least twofold; P < or = 0.01). Strikingly, Cap1p binding was detected not only at the promoter region of its target genes but also at their 3' ends and within their open reading frames, suggesting that Cap1p may associate with the transcriptional or chromatin remodeling machinery to exert its activity. Overrepresented functional groups of the Cap1p targets (P < or = 0.02) included 11 genes involved in the OSR (CAP1, GLR1, TRX1, SOD1, CAT1, and others), 13 genes involved in response to drugs (PDR16, MDR1, FLU1, YCF1, FCR1, and others), 4 genes involved in phospholipid transport (PDR16, GIT1, RTA2, and orf19.932), and 3 genes involved in the regulation of nitrogen utilization (GST3, orf19.2693, and orf19.3121), suggesting that Cap1p has other cellular functions in addition to the OSR. Bioinformatic analyses of the bound sequences suggest that Cap1p recognizes the DNA motif 5'-MTKASTMA. Finally, transcriptome analyses showed that increased expression generally accompanies Cap1p binding at its targets, indicating that Cap1p functions as a transcriptional activator.

Design of protein-interaction specificity gives selective bZIP-binding peptides

Interaction specificity is a required feature of biological networks and a necessary characteristic of protein or small-molecule reagents and therapeutics. The ability to alter or inhibit protein interactions selectively would advance basic and applied molecular science. Assessing or modelling interaction specificity requires treating multiple competing complexes, which presents computational and experimental challenges. Here we present a computational framework for designing protein interaction specificity and use it to identify specific peptide partners for human bZIP transcription factors. Protein microarrays were used to characterize designed, synthetic ligands for all but one of 20 bZIP families. The bZIP proteins share strong sequence and structural similarities and thus are challenging targets to bind specifically. Yet many of the designs, including examples that bind the oncoproteins cJun, cFos and cMaf, were selective for their targets over all 19 other families. Collectively, the designs exhibit a wide range of novel interaction profiles, demonstrating that human bZIPs have only sparsely sampled the possible interaction space accessible to them. Our computational method provides a way to systematically analyze tradeoffs between stability and specificity and is suitable for use with many types of structure-scoring functions; thus it may prove broadly useful as a tool for protein design.

MCRS2 represses the transactivation activities of Nrf1

BackgroundNrf1 [p45 nuclear factor-erythroid 2 (p45 NF-E2)-related factor 1], a member of the CNC-bZIP (CNC basic region leucine zipper) family, is known to be a transcriptional activator by dimerization with distinct partners, such as Maf, FosB, c-Jun, JunD, etc. The transcriptional roles of CNC-bZIP family are demonstrated to be involved in globin gene expression as well as the antioxidant response. For example, CNC-bZIP factors can regulate the expression of detoxification proteins through AREs, such as expression of human gamma-glutamylcysteine synthetases (GCS), glutathione S-transferases (GST), UDP-glucuronosyl transferase (UDP-GT), NADP (H) quinone oxidoreductase (NQOs), etc. To further explore other factor(s) in cells related to the function of Nrf1, we performed a yeast two-hybrid screening assay to identify any Nrf1-interacting proteins. In this study, we isolated a cDNA encoding residues 126–475 of MCRS2 from the HeLa cell cDNA library. Some functions of MCRS1 and its splice variant-MSP58 and MCRS2 have been previously identified, such as transforming, nucleolar sequestration, ribosomal gene regulation, telomerase inhibition activities, etc. Here, we demonstrated MCRS2 can function as a repressor on the Nrf1-mediated transactivation using both in vitro and in vivo systems.ResultsTo find other proteins interacting with the CNC bZIP domain of Nrf1, the CNC-bZIP region of Nrf1 was used as a bait in a yeast two-hybrid screening assay. MCRS2, a splicing variant of p78/MCRS1, was isolated as the Nrf1-interacting partner from the screenings. The interaction between Nrf1 and MCRS2 was confirmed in vitro by GST pull-down assays and in vivo by co-immunoprecipitation. Further, the Nrf1-MCRS2 interaction domains were mapped to the residues 354–447 of Nrf1 as well as the residues 314–475 of MCRS2 respectively, by yeast two-hybrid and GST pull-down assays. By immunofluorescence, MCRS2-FLAG was shown to colocalize with HA-Nrf1 in the nucleus and didn't result in the redistribution of Nrf1. This suggested the existence of Nrf1-MCRS2 complex in vivo. To further confirm the biological function, a reporter driven by CNC-bZIP protein binding sites was also shown to be repressed by MCRS2 in a transient transfection assay. An artificial reporter gene activated by LexA-Nrf1 was also specifically repressed by MCRS2.ConclusionFrom the results, we showed MCRS2, a new Nrf1-interacting protein, has a repression effect on Nrf1-mediated transcriptional activation. This was the first ever identified repressor protein related to Nrf1 transactivation.

Bach1 Deficiency Ameliorates Hepatic Injury in a Mouse Model

Bach1 is a basic region-leucine zipper (bZip) protein that forms heterodimers with the small Maf proteins and functions as a repressor of gene expression. One of the target genes of Bach1 is Hmox-1 that encodes heme oxygenase-1 (HO-1). HO-1 degrades heme into carbon monoxide (CO), biliverdin, and iron. HO-1 is strongly induced by various stresses as well as its substrate heme, and protects cells and tissues against insults through diverse cytoprotective functions of the reaction products CO and biliverdin. Bach1-deficiency in mice leads to higher expression of Hmox-1 in various tissues. Here we investigated the effects of Bach1-deficiency in mice on tissue injuries: hepatic injury induced by D-galactosamine (GalN) and lipopolysaccharide (LPS), and mouse paw edema induced by carrageenin, polysaccharide derived from various seaweeds. Bach1-deficiency suppressed induction of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in response to the GalN/LPS-treatment. However, production of tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO), both being cytotoxic mediators in LPS-induced hepatic injury, in Bach1-deficient mice and their peritoneal macrophages was similar to wild type controls. In contrast, Bach1-deficiency did not affect extent of mouse paw edema induced by carrageenin, which enhances vascular permeability by activating kinin release. These results indicate that Bach1 plays an inhibitory role in the cytoprotection of LPS-induced liver injury but not in the kinin-mediated inflammatory edema. The inhibitory role for Bach1 may stem from its activity to repress gene expression including HO-1.

A dimer-specific function of the transcription factor NFATp

The transcription factor NFATp integrates multiple signal transduction pathways through coordinate binding with basic-region leucine zipper (bZIP) proteins and other transcription factors. The NFATp monomer, even in the absence of its activation domains, recruits bZIP proteins to canonical NFAT-bZIP composite DNA elements. By contrast, the NFATp dimer and its bZIP partner bind noncooperatively to the NFAT-bZIP element of the tumor necrosis factor (TNF) gene promoter. This observation raises the possibility that the function of the activation domains of NFATp is dimer-specific. Here, we determine the consensus DNA binding site of the NFATp dimer, describe monomer- and dimer-specific NFATp-DNA contact patterns, and demonstrate that NFATp dimerization and dimer-specific activation subdomains are required for transcriptional activation from the TNF NFAT-bZIP element. We also show that these NFATp subdomains interact with the coactivator CBP (CREB-binding protein), which is required for NFATp-dependent TNF gene transcription. Thus, the context-specific function of the activation domains of NFAT can be potentiated by DNA-directed dimerization.

PIN1 Blocks Granulocytic Differentiation Via C-Jun in Acute Myeloid Leukemia with CEBPA Mutations.

Transcription factor CCAAT enhancer binding protein α (C/EBPα) is the master regulator of myeloid differentiation programme. Tumor suppressor function of C/EBPα is shown by the deregulation of C/EBPα in AML by various mechanisms. Mutations in the CEBPA gene are reported in around 9% of patients with acute myeloid leukemia (AML). The CEBPA mutations reported are frame shift mutation at N-terminal domain, which results in C/EBPα-p30 and point mutations at the basic region Leucine zipper. The mutant form of C/EBPα (C/EBPα-p30) displays dominant negative function over the wild type protein and induces AML in mouse model. The mechanism for the dominant negative action of C/EBPα-p30 in AML is poorly understood. Peptidyl-prolyl cis/trans isomerase, PIN1 binds to and isomerizes specific pSer/Thr-Pro motifs. Recent studies show that PIN1 is overexpressed in many types of cancers and it acts as a crucial catalyst for oncogenesis. Here we investigated the role of PIN1 in AML with CEBPA mutations. We report C/EBPα-p30 could induce PIN1 mRNA and protein levels as revealed by quantitative Real-Time RT-PCR analysis and western blot analysis, respectively. Affymetrix mRNA expression analysis shows that PIN1 mRNA is upregulated in patients with AML. Interestingly, the wild type C/EBPα down regulates PIN1 mRNA and protein levels. Chromatin immunoprecipitation assays demonstrate that induction of C/EBPα-p30 results in recruitment of E2F1 in the PIN1 promoter. Silencing PIN1 with inhibitor against PIN1 (PiB) in AML blast cells with CEBPA mutation and in Kasumi-6 cells leads to myeloid differentiation as assessed by myeloid markers by FACS analysis and quantitative Real-Time RT-PCR analysis. Furthermore, PIN1 inhibition by PiB leads to upregulation of wild type C/EBPα protein level. Next, we investigated the mechanism through which the C/EBPα-p30 blocks the wild type protein. c-Jun expression has been shown to be high in AML patients with CEBPA mutation and c-Jun blocks C/EBPα function by proteinprotein interaction. In vivo ubiquitination assay demonstrates that PIN1 increases the stability of the c-Jun protein by inhibiting c-Jun ubiquitination. We also show that c-Jun blocks granulocytic differentiation mediated by C/EBPα as assessed by myeloid specific markers. In summary, C/EBPα-p30 induces PIN1 expression and increases the stability of c-Jun and c-Jun blocks the wild type C/EBPα. Our data suggest the inhibition of PIN1 could be a potential strategy in the treatment of AML patients with CEBPA mutation.

LebZIP2 induced by salt and drought stress and transient overexpression by Agrobacterium

The full-length cDNA of LebZIP2 (Lycopersicon esculentum bZIP2) encodes a protein of 164 amino acids and contains a N-terminal basic-region leucine zipper domain. Analysis of the deduced tomato LebZIP2 amino acid sequence revealed that it shares 85% sequence identity with both tobacco bZIP and pepper CcbZIP. LebZIP2 mRNA is expressed at a high level exclusively in flowers. Presently, LebZIP2 was strongly increased also following NaCl and mannitol treatments. No significant LebZIP2 expression was evident following cold treatment. Transient LebZIP2 overexpression resulted in increased NbNOA1 and NbNR transcript levels in Nicotiana benthamiana leaves. Our results indicate that LebZIP2 might play roles as an abiotic stress-signaling pathway and as a transcriptional regulator of the NbNOA1 or NbNR genes.