scholarly journals MondoA, a Novel Basic Helix-Loop-Helix–Leucine Zipper Transcriptional Activator That Constitutes a Positive Branch of a Max-Like Network

2000 ◽  
Vol 20 (23) ◽  
pp. 8845-8854 ◽  
Author(s):  
Andrew N. Billin ◽  
Alanna L. Eilers ◽  
Kathryn L. Coulter ◽  
Jennifer S. Logan ◽  
Donald E. Ayer
Keyword(s):  
Transcriptional Activation ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Functional Characterization ◽  
Nuclear Accumulation ◽  
Cytoplasmic Localization ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix

ABSTRACT Max is a common dimerization partner for a family of transcription factors (Myc, Mad [or Mxi]), and Mnt [or Rox] proteins) that regulate cell growth, proliferation, and apoptosis. We recently characterized a novel Max-like protein, Mlx, which interacts with Mad1 and Mad4. Here we describe the cloning and functional characterization of a new family of basic helix-loop-helix–leucine zipper heterodimeric partners for Mlx termed the Mondo family. MondoA forms homodimers weakly and does not interact with Max or members of the Myc or Mad families. MondoA and Mlx associate in vivo, and surprisingly, they are localized primarily to the cytoplasm of cultured mammalian cells. Treatment of cells with the nuclear export inhibitor leptomycin B results in the nuclear accumulation of MondoA and Mlx, demonstrating that they shuttle between the cytoplasmic and nuclear compartments rather than having exclusively cytoplasmic localization. MondoA preferentially forms heterodimers with Mlx, and this heterocomplex can bind to, and activate transcription from, CACGTG E-boxes when targeted to the nucleus via a heterologous nuclear localization signal. The amino termini of the Mondo proteins are highly conserved among family members and contain separable and autonomous cytoplasmic localization and transcription activation domains. Therefore, Mlx can mediate transcriptional repression in conjunction with the Mad family and can mediate transcriptional activation via the Mondo family. We propose that Mlx, like Max, functions as the center of a transcription factor network.

scholarly journals Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension

1996 ◽  
Vol 271 (4) ◽  
pp. C1172-C1180 ◽  
Author(s):  
B. H. Jiang ◽  
G. L. Semenza ◽  
C. Bauer ◽  
H. H. Marti
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Functional Characterization ◽  
Hypoxia Inducible Factor ◽  
Binding Activity ◽  
Maximal Response ◽  
Hypoxia Inducible Factor 1 ◽  
Dna Binding Activity

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein implicated in the transcriptional activation of genes encoding erythropoietin, glycolytic enzymes, and vascular endothelial growth factor in hypoxic mammalian cells. In this study, we have quantitated HIF-1 DNA-binding activity and protein levels of the HIF-1 alpha and HIF-1 beta subunits in human HeLa cells exposed to O2 concentrations ranging from 0 to 20% in the absence or presence of 1 mM KCN to inhibit oxidative phosphorylation and cellular O2 consumption. HIF-1 DNA-binding activity, HIF-1 alpha protein and HIF-1 beta protein each increased exponentially as cells were subjected to decreasing O2 concentrations, with a half maximal response between 1.5 and 2% O2 and a maximal response at 0.5% O2, both in the presence and absence of KCN. The HIF-1 response was greatest over O2 concentrations associated with ischemic/hypoxic events in vivo. These results provide evidence for the involvement of HIF-1 in O2 homeostasis and represent a functional characterization of the putative O2 sensor that initiates hypoxia signal transduction leading to HIF-1 expression.

scholarly journals Multi-parameter analysis of the kinetics of NF-κB signalling and transcription in single living cells

2002 ◽  
Vol 115 (6) ◽  
pp. 1137-1148 ◽  
Author(s):  
Glyn Nelson ◽  
Luminita Paraoan ◽  
David G. Spiller ◽  
Geraint J. C. Wilde ◽  
Mark A. Browne ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Translocation ◽  
Fluorescent Proteins ◽  
Parameter Analysis ◽  
Nuclear Accumulation ◽  
Fusion Protein Expression ◽  
Kinetics Of

Proteins of the NF-κB transcription factor family normally reside in the cytoplasm of cells in a complex with IκB inhibitor proteins. Stimulation with TNFα leads to proteosomal degradation of the IκB proteins and nuclear translocation of the NF-κB proteins. Expression of p65 and IκBα fused to fluorescent proteins was used to measure the dynamics of these processes in transfected HeLa cells. Simultaneous visualisation of p65-dsRed translocation and IκBα-EGFP degradation indicated that in the presence of dual fluorescent fusion protein expression,the half-time of IκBα-EGFP degradation was reduced and that of p65 translocation was significantly increased when compared with cells expressing the single fluorescent fusion proteins. These results suggest that the ratio of IκBα and p65 determine the kinetics of transcription factor translocation into the nucleus and indicate that the complex of p65 and IκBα is the true substrate for TNFα stimulation in mammalian cells. When cells were treated with the CRM-1-dependent nuclear export inhibitor,leptomycin B (LMB), there was nuclear accumulation of IκBα-EGFP and p65-dsRed, with IκBα-EGFP accumulating more rapidly. No NF-κB-dependent transcriptional activation was seen in response to LMB treatment. Following 1 hour treatment with LMB, significant IκBα-EGFP nuclear accumulation, but low levels of p65-dsRed nuclear accumulation, was observed. When these cells were stimulated with TNFα, degradation of IκBα-EGFP was observed in both the cytoplasm and nucleus. A normal transient transcription response was observed in the same cells using luminescence imaging of NF-κB-dependent transcription. These observations suggest that both normal activation and post-induction repression of NF-κB-dependent transcription occur even when nuclear export of NF-κB is inhibited. The results provide functional evidence that other factors, such as modification of p65 by phosphorylation, or interaction with other proteins such as transcriptional co-activators/co-repressors, may critically modulate the kinetics of transcription through this signalling pathway.

scholarly journals Visualization of Myc/Max/Mad Family Dimers and the Competition for Dimerization in Living Cells

2004 ◽  
Vol 24 (10) ◽  
pp. 4294-4308 ◽  
Author(s):  
Asya V. Grinberg ◽  
Chang-Deng Hu ◽  
Tom K. Kerppola
Keyword(s):  
Nuclear Export ◽  
Leucine Zipper ◽  
Nuclear Export Signal ◽  
Living Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Cytoplasmic Localization ◽  
Amino Acid Residues ◽  
Helix Loop Helix ◽  
Cell Growth And Proliferation ◽  
Export Signal

ABSTRACT Myc and Mad family proteins play opposing roles in the control of cell growth and proliferation. We have visualized the subcellular locations of complexes formed by Myc/Max/Mad family proteins using bimolecular fluorescence complementation (BiFC) analysis. Max was recruited to different subnuclear locations by interactions with Myc versus Mad family members. Complexes formed by Max with Mxi1, Mad3, or Mad4 were enriched in nuclear foci, whereas complexes formed with Myc were more uniformly distributed in the nucleoplasm. Mad4 was localized to the cytoplasm when it was expressed separately, and Mad4 was recruited to the nucleus through dimerization with Max. The cytoplasmic localization of Mad4 was determined by a CRM1-dependent nuclear export signal located near the amino terminus. We compared the relative efficiencies of complex formation among Myc, Max, and Mad family proteins in living cells using multicolor BiFC analysis. Max formed heterodimers with the basic helix-loop-helix leucine zipper (bHLHZIP) domain of Myc (bMyc) more efficiently than it formed homodimers. Replacement of two amino acid residues in the leucine zipper of Max reversed the relative efficiencies of homo- and heterodimerization in cells. Surprisingly, Mad3 formed complexes with Max less efficiently than bMyc, whereas Mad4 formed complexes with Max more efficiently than bMyc. The distinct subcellular locations and the differences between the efficiencies of dimerization with Max indicate that Mad3 and Mad4 are likely to modulate transcription activation by Myc at least in part through distinct mechanisms.

scholarly journals Triptolide Downregulates the Expression of NRF2 Target Genes by Increasing Cytoplasmic Localization of NRF2 in A549 Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Le Ba Nam ◽  
Won Jun Choi ◽  
Young-Sam Keum
Keyword(s):  
Nuclear Export ◽  
Target Genes ◽  
Luciferase Activity ◽  
A549 Cells ◽  
Nuclear Accumulation ◽  
Cytoplasmic Localization ◽  
Oxidative Damages ◽  
Induced Apoptosis ◽  
Intracellular Oxidative Stress

We have identified triptolide as a novel NRF2 inhibitor, which significantly attenuates ARE-luciferase activity at nanomolar concentrations. Triptolide did not affect the level of NRF2, but significantly inhibited the expression of NRF2 target genes in A549 cells. We found that NRF2 possesses a previously unrecognized NES in the Neh2 domain, and that triptolide promotes an interaction between NRF2 and CRM1. Triptolide also decreased nuclear accumulation of NRF2, suggesting that it promotes nuclear export of NRF2. In addition, we show that triptolide decreased the expression of NRF2 target genes and increased intracellular oxidative stress, suppressing invasion and promoting cisplatin-induced apoptosis in A549 cells. Finally, oral administration of triptolide suppressed the growth of A549 xenografts in athymic mice by decreasing the expression of NRF2 target genes and promoting oxidative damages via the nuclear export of NRF2 and CRM1 in vivo. To the best of our knowledge, triptolide is the first type of compound to inhibit NRF2 by increasing cytoplasmic localization of NRF2.

scholarly journals The Drosophila Basic Helix-Loop-Helix Protein DIMMED Directly Activates PHM, a Gene Encoding a Neuropeptide-Amidating Enzyme

2007 ◽  
Vol 28 (1) ◽  
pp. 410-421 ◽  
Author(s):  
Dongkook Park ◽  
Orie T. Shafer ◽  
Stacie P. Shepherd ◽  
Hyunsuk Suh ◽  
Jennifer S. Trigg ◽  
...  
Keyword(s):  
Cell Fate ◽  
Transcriptional Activation ◽  
Regulatory Region ◽  
Total Activity ◽  
Bhlh Protein ◽  
Hek 293 ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
E Boxes

ABSTRACT The basic helix-loop-helix (bHLH) protein DIMMED (DIMM) supports the differentiation of secretory properties in numerous peptidergic cells of Drosophila melanogaster. DIMM is coexpressed with diverse amidated neuropeptides and with the amidating enzyme peptidylglycine α-hydroxylating monooxygenase (PHM) in approximately 300 cells of the late embryo. Here we confirm that DIMM has transcription factor activity in transfected HEK 293 cells and that the PHM gene is a direct target. The mammalian DIMM orthologue MIST1 also transactivated the PHM gene. DIMM activity was dependent on the basic region of the protein and on the sequences of three E-box sites within PHM's first intron; the sites make different contributions to the total activity. These data suggest a model whereby the three E boxes interact cooperatively and independently to produce high PHM transcriptional activation. This DIMM-controlled PHM regulatory region displayed similar properties in vivo. Spatially, its expression mirrored that of the DIMM protein, and its activity was largely dependent on dimm. Further, in vivo expression was highly dependent on the sequences of the same three E boxes. This study supports the hypothesis that DIMM is a master regulator of a peptidergic cell fate in Drosophila and provides a detailed transcriptional mechanism of DIMM action on a defined target gene.

scholarly journals Nuclear Export of the Transcription Factor NirA Is a Regulatory Checkpoint for Nitrate Induction in Aspergillus nidulans

2006 ◽  
Vol 27 (3) ◽  
pp. 791-802 ◽  
Author(s):  
Andreas Bernreiter ◽  
Ana Ramon ◽  
Javier Fernández-Martínez ◽  
Harald Berger ◽  
Lidia Araújo-Bazan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Aspergillus Nidulans ◽  
Transcriptional Activation ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Constitutive Expression ◽  
Nuclear Accumulation ◽  
Nuclear Retention ◽  
Nitrate Induction

ABSTRACT NirA, the specific transcription factor of the nitrate assimilation pathway of Aspergillus nidulans, accumulates in the nucleus upon induction by nitrate. NirA interacts with the nuclear export factor KapK, which bridges an interaction with a protein of the nucleoporin-like family (NplA). Nitrate induction disrupts the NirA-KapK interaction in vivo, whereas KapK associates with NirA when this protein is exported from the nucleus. A KpaK leptomycin-sensitive mutation leads to inducer-independent NirA nuclear accumulation in the presence of the drug. However, this does not lead to constitutive expression of the genes controlled by NirA. A nirA c 1 mutation leads to constitutive nuclear localization and activity, remodeling of chromatin, and in vivo binding to a NirA upstream activation sequence. The nirA c 1 mutation maps in the nuclear export signal (NES) of the NirA protein. The NirA-KapK interaction is nearly abolished in NirAc1 and NirA proteins mutated in canonical leucine residues in the NirA NES. The latter do not result in constitutively active NirA protein, which implies that nuclear retention is necessary but not sufficient for NirA activity. The results are consistent with a model in which activation of NirA by nitrate disrupts the interaction of NirA with the NplA/KapK nuclear export complex, thus resulting in nuclear retention, leading to AreA-facilitated DNA binding of the NirA protein and subsequent chromatin remodeling and transcriptional activation.

scholarly journals Transcriptional Activation by ETS and Leucine Zipper-Containing Basic Helix-Loop-Helix Proteins

1999 ◽  
Vol 19 (4) ◽  
pp. 2946-2957 ◽  
Author(s):  
Gang Tian ◽  
Batu Erman ◽  
Haruhiko Ishii ◽  
Samudra S. Gangopadhyay ◽  
Ranjan Sen
Keyword(s):  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Transcription Activation ◽  
Chain Gene ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Gene Enhancer ◽  
Novel Interactions ◽  
Heavy Chain Gene Enhancer

ABSTRACT The immunoglobulin μ heavy-chain gene enhancer contains closely juxtaposed binding sites for ETS and leucine zipper-containing basic helix-loop-helix (bHLH-zip) proteins. To understand the μ enhancer function, we have investigated transcription activation by the combination of ETS and bHLH-zip proteins. The bHLH-zip protein TFE3, but not USF, cooperated with the ETS domain proteins PU.1 and Ets-1 to activate a tripartite domain of this enhancer. Deletion mutants were used to identify the domains of the proteins involved. Both TFE3 and USF enhanced Ets-1 DNA binding in vitro by relieving the influence of an autoinhibitory domain in Ets-1 by direct protein-protein associations. Several regions of Ets-1 were found to be necessary, whereas the bHLH-zip domain was sufficient for this effect. Our studies define novel interactions between ETS and bHLH-zip proteins that may regulate combinatorial transcription activation by these protein families.

scholarly journals A Novel Heterodimerization Domain, CRM1, and 14-3-3 Control Subcellular Localization of the MondoA-Mlx Heterocomplex

2002 ◽  
Vol 22 (24) ◽  
pp. 8514-8526 ◽  
Author(s):  
Alanna L. Eilers ◽  
Eleanor Sundwall ◽  
Monica Lin ◽  
April A. Sullivan ◽  
Donald E. Ayer
Keyword(s):  
Subcellular Localization ◽  
Nuclear Export ◽  
Leucine Zipper ◽  
Sequence Similarity ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Cytoplasmic Localization ◽  
Terminal Domain ◽  
N Terminus ◽  
Dimerization Interface

ABSTRACT Among members of the bHLHZip family of transcriptional regulators, MondoA and Mlx have the unique property of cytoplasmic localization. We have proposed that MondoA-Mlx heterodimers accumulate in the nucleus in response to extracellular cues. Our previous work implicated heterodimerization between MondoA and Mlx and a conserved domain in the N terminus of MondoA as important determinants of MondoA-Mlx subcellular localization. MondoA and Mlx share sequence similarity in their bHLHZip domains and C termini. Here we show that for both MondoA and Mlx, this C-terminal domain has cytoplasmic localization activity that is required by the protein monomers to accumulate in the cytoplasm. This C-terminal domain is also a novel dimerization interface that functions independently of the leucine zipper to mediate heterotypic interactions between MondoA and Mlx. Dimerization between MondoA and Mlx inactivates the cytoplasmic localization activity of their C termini and is necessary for the heterocomplex to accumulate in the nucleus. MondoA-Mlx heterodimers, while poised for nuclear entry, are retained in the cytoplasm by conserved domains in the N terminus of MondoA. Mondo conserved regions (MCRs) II and III contribute to cytoplasmic localization of MondoA-Mlx by functioning as a CRM1-dependent nuclear export signal and as a novel binding site for 14-3-3 family members, respectively. We propose that the nuclear accumulation of MondoA and Mlx is a two-step process. First, heterodimerization abolishes the cytoplasmic localization activity of their C termini. Second, an extracellular signal(s) must overcome the cytoplasmic localization function imparted by CRM1 and 14-3-3 binding to the N terminus of MondoA.

scholarly journals Targeting the Microphthalmia Basic Helix-Loop-Helix–Leucine Zipper Transcription Factor to a Subset of E-Box Elements In Vitro and In Vivo

1998 ◽  
Vol 18 (12) ◽  
pp. 6930-6938 ◽  
Author(s):  
I. Aksan ◽  
C. R. Goding
Keyword(s):  
Transcription Factor ◽  
Leucine Zipper ◽  
Pigment Cells ◽  
Specific Gene ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
P Gene ◽  
E Box

ABSTRACT The development of melanocytes, which are pigment-producing cells responsible for skin, hair, and eye color, is absolutely dependent on the action of the microphthalmia basic helix-loop-helix–leucine zipper (bHLH-LZ) transcription factor (Mi); mice lacking a functional Mi protein are entirely devoid of pigment cells. Mi has been shown to activate transcription of the tyrosinase,TRP-1, TRP-2, and QNR-71 genes through specific E-box elements, most notably the highly conserved M box. We investigated the mechanism which enables Mi to be recruited specifically to a restricted subset of E boxes in target promoters while being prevented from binding E-box elements in other promoters. We show both in vitro and in vivo that the presence of a T residue flanking a CATGTG E box is an essential determinant of the ability of Mi to bind DNA, and we successfully predict that the CATGTG E box from the P gene would not bind Mi. In contrast, no specific requirement for the sequences flanking a CACGTG E box was observed, and no binding to an atypical E box in the c-Kit promoter was detected. The relevance of these observations to the control of melanocyte-specific gene expression was highlighted by the fact that the E-box elements located in thetyrosinase, TRP-1, TRP-2, andQNR-71 promoters without exception possess a 5′ flanking T residue which is entirely conserved between species as diverse as man and turtle. The ability of Mi to discriminate between different E-box motifs provides a mechanism to restrict the repertoire of genes which are likely to be regulated by Mi and provides insight into the ability of bHLH-LZ transcription factors to achieve the specificity required for the precise coordination of transcription during development.

scholarly journals A basic helix-loop-helix-leucine zipper transcription complex in yeast functions in a signaling pathway from mitochondria to the nucleus.

1997 ◽  
Vol 17 (3) ◽  
pp. 1110-1117 ◽  
Author(s):  
Y Jia ◽  
B Rothermel ◽  
J Thornton ◽  
R A Butow
Keyword(s):  
Leucine Zipper ◽  
Citrate Synthase ◽  
Glyoxylate Cycle ◽  
Gene Encoding ◽  
Sequence Element ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Bhlh Proteins ◽  
Basic Region

The expression of some nuclear genes in Saccharomyces cerevisiae, such as the CIT2 gene, which encodes a glyoxylate cycle isoform of citrate synthase, is responsive to the functional state of mitochondria. Previous studies identified a basic helix-loop-helix-leucine zipper (bHLH/Zip) transcription factor encoded by the RTG1 gene that is required for both basal expression of the CIT2 gene and its increased expression in respiratory-deficient cells. Here, we describe the cloning and characterization of RTG3, a gene encoding a 54-kDa bHLH/Zip protein that is also required for CIT2 expression. Rtg3p binds together with Rtg1p to two identical sites oriented as inverted repeats 28 bp apart in a regulatory upstream activation sequence element (UASr) in the CIT2 promoter. The core binding site for the Rtg1p-Rtg3p heterodimer is 5'-GGTCAC-3', which differs from the canonical E-box site, CANNTG, to which most other bHLH proteins bind. We demonstrate that both of the Rtg1p-Rtg3p binding sites in the UAS(r) element are required in vivo and act synergistically for CIT2 expression. The basic region of Rtg3p conforms well to the basic region of most bHLH proteins, whereas the basic region of Rtg1p does not. These findings suggest that the Rtg1p-Rtg3p complex interacts in a novel way with its DNA target sites.

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