scholarly journals Functional analysis of Met4, a yeast transcriptional activator responsive to S-adenosylmethionine.

1995 ◽  
Vol 15 (1) ◽  
pp. 208-216 ◽  
Author(s):  
L Kuras ◽  
D Thomas
Keyword(s):  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Transcriptional Activator ◽  
Activation Function ◽  
Functional Domain ◽  
Distinct Region ◽  
Activation Domain ◽  
Inhibitory Region ◽  
Leucine Zipper Protein ◽  
Basic Region

Transcription of the genes necessary for sulfur amino acid biosynthesis in Saccharomyces cerevisiae is dependent on Met4, a transcriptional activator that belongs to the basic region-leucine zipper protein family. In this report, we show that one mechanism permitting the repression of the sulfur network by S-adenosylmethionine (AdoMet) involves inhibition of the transcriptional activation function of Met4. Using a wide array of deleted LexA-Met4 fusion proteins as well as various Gal4-Met4 hybrids, we identify the functional domains of Met4 and characterize their relationship. Met4 appears to contain only one activation domain, located in its N-terminal part. We demonstrate that this activation domain functions in a constitutive manner and that AdoMet responsiveness requires a distinct region of Met4. Furthermore, we show that when fused to a heterologous activation domain, this inhibitory region confers inhibition by AdoMet. Met4 contains another distinct functional domain that appears to function as an antagonist of the inhibitory region when intracellular AdoMet is low. On the basis of the presented results, a model for intramolecular regulation of Met4 is proposed.

scholarly journals Proto-oncogene FosB: the amino terminus encodes a regulatory function required for transformation.

1993 ◽  
Vol 13 (5) ◽  
pp. 2635-2643 ◽  
Author(s):  
R Wisdom ◽  
I M Verma
Keyword(s):  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Regulatory Function ◽  
Amino Terminus ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Constitutive Activation ◽  
Functional Conservation ◽  
N Terminus ◽  
Basic Region

Overexpression of some members of the Fos gene family, including FosB, leads to transformation of established rodent fibroblasts. We have previously shown that transformation by FosB requires the presence of a C-terminal transcriptional activation domain. We now report that transformation by FosB also requires an intact DNA-binding domain composed of the functionally bipartite basic region and leucine zipper as well as sequences present in the N terminus that serve a regulatory function. Deletion of the N-terminal sequences results in proteins impaired in transcriptional activation and transformation. This region does not itself function as a transcriptional activation domain but instead regulates the transactivation functions present in the FosB-Jun complex. The requirement for this N-terminal region can be abolished by the presence of a strong constitutive activation domain. The primary sequence of the region that we have defined is highly conserved in the Fos family of proteins, suggesting functional conservation.

Transcriptional regulation by Fos and Jun in vitro: interaction among multiple activator and regulatory domains

1991 ◽  
Vol 11 (7) ◽  
pp. 3624-3632
Author(s):  
C Abate ◽  
D Luk ◽  
T Curran
Keyword(s):  
Transcriptional Regulation ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Regulatory Function ◽  
Activation Domain ◽  
Regulatory Domains ◽  
Heterodimeric Complex ◽  
Transcriptional Stimulation

The proteins encoded by the proto-oncogenes c-fos and c-jun (Fos and Jun, respectively) form a heterodimeric complex that regulates transcription by interacting with the DNA-regulatory element known as the activator protein 1 (AP-1) binding site. Fos and Jun are members of a family of related transcription factors that dimerize via a leucine zipper structure and interact with DNA through a bipartite domain formed between regions of each protein that are rich in basic amino acids. Here we have defined other domains in the Fos-Jun heterodimer that contribute to transcriptional function in vitro. Although DNA-binding specificity is mediated by the leucine zipper and basic regions, Jun also contains a proline- and glutamine-rich region that functions as an ancillary DNA-binding domain but does not contribute directly to transcriptional activation. Transcriptional stimulation in vitro was associated with two regions in Fos and a single N-terminal activation domain in Jun. These activator regions were capable of operating independently; however, they appear to function cooperatively in the heterodimeric complex. The activity of these domains was modulated by inhibitory regions in Fos and Jun that repressed transcription in vitro. In the context of the heterodimer, the Jun activation domain was the major contributor to transcriptional stimulation and the inhibitory regions in Fos were the major contributors to transcriptional repression in vitro. Potentially, the inhibitory domains could serve a regulatory function in vivo. Thus, transcriptional regulation by the Fos-Jun heterodimer results from a complex integration of multiple activator and regulatory domains.

scholarly journals Multiple Signal Input and Output Domains of the 160-Kilodalton Nuclear Receptor Coactivator Proteins

1999 ◽  
Vol 19 (9) ◽  
pp. 6164-6173 ◽  
Author(s):  
Han Ma ◽  
Heng Hong ◽  
Shih-Ming Huang ◽  
Ryan A. Irvine ◽  
Paul Webb ◽  
...  
Keyword(s):  
Nuclear Receptor ◽  
Transcriptional Activation ◽  
Thyroid Hormone Receptor ◽  
Activation Function ◽  
Creb Binding Protein ◽  
Activation Domain ◽  
Nuclear Receptor Coactivator ◽  
Interacting Protein ◽  
Binding Domains ◽  
Signal Input

ABSTRACT Members of the 160-kDa nuclear receptor coactivator family (p160 coactivators) bind to the conserved AF-2 activation function found in the hormone binding domains of nuclear receptors (NR) and are potent transcriptional coactivators for NRs. Here we report that the C-terminal region of p160 coactivators glucocorticoid receptor interacting protein 1 (GRIP1), steroid receptor coactivator 1 (SRC-1a), and SRC-1e binds the N-terminal AF-1 activation function of the androgen receptor (AR), and p160 coactivators can thereby enhance transcriptional activation by AR. While they all interact efficiently with AR AF-1, these same coactivators have vastly different binding strengths with and coactivator effects on AR AF-2. p160 activation domain AD1, which binds secondary coactivators CREB binding protein (CBP) and p300, was previously implicated as the principal domain for transmitting the activating signal to the transcription machinery. We identified a new highly conserved motif in the AD1 region which is important for CBP/p300 binding. Deletion of AD1 only partially reduced p160 coactivator function, due to signaling through AD2, another activation domain located at the C-terminal end of p160 coactivators. C-terminal coactivator fragments lacking AD1 but containing AD2 and the AR AF-1 binding site served as efficient coactivators for full-length AR and AR AF-1. The two signal input domains (one that binds NR AF-2 domains and one that binds AF-1 domains of some but not all NRs) and the two signal output domains (AD1 and AD2) of p160 coactivators played different relative roles for two different NRs: AR and thyroid hormone receptor.

scholarly journals A Dominant-Negative Inhibitor of CREB Reveals that It Is a General Mediator of Stimulus-Dependent Transcription of c-fos

1998 ◽  
Vol 18 (2) ◽  
pp. 967-977 ◽  
Author(s):  
Sohyun Ahn ◽  
Michelle Olive ◽  
Seema Aggarwal ◽  
Dmitry Krylov ◽  
David D. Ginty ◽  
...  
Keyword(s):  
Dna Binding ◽  
Pc12 Cells ◽  
Transcriptional Activation ◽  
Cortical Neurons ◽  
Leucine Zipper ◽  
Uv Light ◽  
Regulatory Region ◽  
Dominant Negative ◽  
Intracellular Camp ◽  
Basic Region

ABSTRACT Several studies have characterized the upstream regulatory region of c-fos, and identified cis-acting elements termed the cyclic AMP (cAMP) response elements (CREs) that are critical for c-fos transcription in response to a variety of extracellular stimuli. Although several transcription factors can bind to CREs in vitro, the identity of the transcription factor(s) that activates the c-fos promoter via the CRE in vivo remains unclear. To help identify the trans-acting factors that regulate stimulus-dependent transcription of c-fos via the CREs, dominant-negative (D-N) inhibitor proteins that function by preventing DNA binding of B-ZIP proteins in a dimerization domain-dependent fashion were developed. A D-N inhibitor of CREB, termed A-CREB, was constructed by fusing a designed acidic amphipathic extension onto the N terminus of the CREB leucine zipper domain. The acidic extension of A-CREB interacts with the basic region of CREB forming a coiled-coil extension of the leucine zipper and thus prevents the basic region of wild-type CREB from binding to DNA. Other D-N inhibitors generated in a similar manner with the dimerization domains of Fos, Jun, C/EBP, ATF-2, or VBP did not block CREB DNA binding activity, nor did they inhibit transcriptional activation of a minimal promoter containing a single CRE in PC12 cells. A-CREB inhibited activation of CRE-mediated transcription evoked by three distinct stimuli: forskolin, which increases intracellular cAMP; membrane depolarization, which promotes Ca2+ influx; and nerve growth factor (NGF). A-CREB completely inhibited cAMP-mediated, but only partially inhibited Ca2+- and NGF-mediated, transcription of a reporter gene containing 750 bp of the native c-fos promoter. Moreover, glutamate induction of c-fos expression in primary cortical neurons was dependent on CREB. In contrast, induction of c-fos transcription by UV light was not inhibited by A-CREB. Lastly, A-CREB attenuated NGF induction of morphological differentiation in PC12 cells. These results suggest that CREB or its closely related family members are general mediators of stimulus-dependent transcription of c-fos and are required for at least some of the long-term actions of NGF.

MET4, a leucine zipper protein, and centromere-binding factor 1 are both required for transcriptional activation of sulfur metabolism in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (4) ◽  
pp. 1719-1727
Author(s):  
D Thomas ◽  
I Jacquemin ◽  
Y Surdin-Kerjan
Keyword(s):  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Leucine Zipper ◽  
Fusion Gene ◽  
Sulfur Metabolism ◽  
Upstream Region ◽  
Binding Factor ◽  
The Family ◽  
Sulfate Assimilation Pathway ◽  
Leucine Zipper Protein

Inactivation of the centromere-binding factor 1 (CBF1) gene results in yeast strains that require methionine for growth. This auxotrophy is due to the inability of such strains to concentrate and assimilate sulfate from the medium. Northern (RNA) blot experiments reveal that the CBF1 protein is required for full induction of MET25 and MET16 gene transcription. However, we show that induction of the sulfate assimilation pathway is not achieved solely by CBF1. This induction also requires the integrity of a positive trans-acting factor, encoded by the MET4 gene. The MET4 gene was cloned, and its sequence reveals that it encodes a protein related to the family of the bZIP transcriptional activators. Evidence that MET4 is a transcriptional activator was provided by demonstrating that DNA-bound LexA-MET4 fusion proteins stimulate expression of a nearby promoter. The use of LexA-MET4 fusion proteins also reveals that the leucine zipper of MET4 is required for the recognition of the MET25 promoter. Moreover, an 18-bp fragment of the MET25 5' upstream region was found to confer S-adenosylmethionine-dependent regulation of a fusion gene. This regulation was shown to depend on both MET4 and CBF1. The obtained results suggest that the binding of CBF1 to its cognate sequences increases the ability of MET4 to stimulate transcription of the MET genes.

Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization

1991 ◽  
Vol 11 (2) ◽  
pp. 954-962
Author(s):  
C V Dang ◽  
J Barrett ◽  
M Villa-Garcia ◽  
L M Resar ◽  
G J Kato ◽  
...  
Keyword(s):  
Dna Binding ◽  
Reporter Gene ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Chimeric Protein ◽  
Dna Binding Domain ◽  
Cytoplasmic Protein ◽  
Activation Domain ◽  
In Cells

The physiological significance of in vitro leucine zipper interactions was studied by the use of two strategies which detect specific protein-protein interactions in mammalian cells. Fusion genes were constructed which produce chimeric proteins containing leucine zipper domains from several proteins fused either to the DNA-binding domain of the Saccharomyces cerevisiae GAL4 protein or to the transcriptional activation domain of the herpes simplex virus VP16 protein. Previous studies in mammalian cells have demonstrated that a single chimeric polypeptide containing these two domains will activate transcription of a reporter gene present downstream of the GAL4 DNA-binding site. Similarly, if the GAL4 DNA-binding domain of a chimeric protein could be complexed through leucine zipper interactions with the VP16 activation domain of another chimeric protein, then transcriptional activation of the reporter gene would be detected. Using this strategy for detecting leucine zipper interactions, we observed homo-oligomerization between leucine zipper domains of the yeast protein GCN4 and hetero-oligomerization between leucine zipper regions from the mammalian transcriptional regulating proteins c-Jun and c-Fos. In contrast, homo-oligomerization of the leucine zipper domain from c-Myc was not detectable in cells. The inability of the c-Myc leucine zipper to homo-oligomerize strongly in cells was confirmed independently. The second strategy to detect leucine zipper interactions takes advantage of the observation that the addition of nuclear localization sequences to a cytoplasmic protein will allow the cytoplasmic protein to be transported to and retained in the nucleus. Chimeric genes encoding proteins with sequences from a cytoplasmic protein fused either to the GCN4 or c-Myc leucine zipper domains were constructed. Experiments with the c-Myc chimeric protein failed to demonstrate transport of the cytoplasmic marker protein to the nucleus in cells expressing the wild-type c-Myc protein. In contrast, the cytoplasmic marker was translocated into the nucleus when the GCN4 leucine zippers were present on both the cytoplasmic marker and a nuclear protein, presumably as a result of leucine zipper interaction. These results suggest that c-Myc function requires hetero-oligomerization to an as yet undefined factor.

scholarly journals The basic region of myogenin cooperates with two transcription activation domains to induce muscle-specific transcription.

1992 ◽  
Vol 12 (1) ◽  
pp. 266-275 ◽  
Author(s):  
J J Schwarz ◽  
T Chakraborty ◽  
J Martin ◽  
J M Zhou ◽  
E N Olson
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcription Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
E Box ◽  
Myogenic Program ◽  
Basic Region

Myogenin is a skeletal muscle-specific transcription factor that can activate myogenesis when introduced into a variety of nonmuscle cell types. Activation of the myogenic program by myogenin is dependent on its binding to a DNA sequence known as an E box, which is associated with numerous muscle-specific genes. Myogenin shares homology with MyoD and other myogenic regulatory factors within a basic region and a helix-loop-helix (HLH) motif that mediate DNA binding and dimerization, respectively. Here we show that the basic region-HLH motif of myogenin alone lacks transcriptional activity and is dependent on domains in the amino and carboxyl termini to activate transcription. Analysis of these N- and C-terminal domains through creation of chimeras with the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that they act as strong transcriptional activators. These transcription activation domains are dependent for activity on a specific amino acid sequence within the basic region, referred to as the myogenic recognition motif (MRM), when an E box is the target for DNA binding. However, the activation domains function independent of the MRM when DNA binding is mediated through a heterologous DNA-binding domain. The activation domain of the acidic coactivator VP16 can substitute for the myogenin activation domains and restore strong myogenic activity to the basic region-HLH motif. Within a myogenin-VP16 chimera, however, the VP16 activation domain also relies on the MRM for activation of the myogenic program. These findings reveal that DNA binding and transcriptional activation are separable functions, encoded by different domains of myogenin, but that the activity of the transcriptional activation domains is influenced by the DNA-binding domain. Activation of muscle-specific transcription requires collaboration between the DNA-binding and activation domains of myogenin and is dependent on events in addition to DNA binding.

scholarly journals LIP19, a Basic Region Leucine Zipper Protein, is a Fos-like Molecular Switch in the Cold Signaling of Rice Plants

2005 ◽  
Vol 46 (10) ◽  
pp. 1623-1634 ◽  
Author(s):  
Hidekazu Shimizu ◽  
Kazuhito Sato ◽  
Thomas Berberich ◽  
Atsushi Miyazaki ◽  
Rei Ozaki ◽  
...  
Keyword(s):  
Leucine Zipper ◽  
Molecular Switch ◽  
Rice Plants ◽  
Leucine Zipper Protein ◽  
Basic Region

scholarly journals Intramolecular Regulation of MyoD Activation Domain Conformation and Function

1998 ◽  
Vol 18 (9) ◽  
pp. 5478-5484 ◽  
Author(s):  
Jing Huang ◽  
Hal Weintraub ◽  
Larry Kedes
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Helix Loop Helix ◽  
Dna Binding Specificity ◽  
E Box ◽  
And Function ◽  
Bhlh Proteins ◽  
Basic Region

ABSTRACT The MyoD family of basic helix-loop-helix (bHLH) proteins is required for myogenic determination and differentiation. The basic region carries the myogenic code and DNA binding specificity, while the N terminus contains a potent transcriptional activation domain. Myogenic activation is abolished when the basic region, bound to a myogenic E box, carries a mutation of Ala-114. It has been proposed that DNA binding of the MyoD basic region leads to recruitment of a recognition factor that unmasks the activation domain. Here we demonstrate that an A114N mutant exhibits an altered conformation in the basic region and that this local conformational difference can lead to a more global change affecting the conformation of the activation domain. This suggests that the deleterious effects of this class of mutations may result directly from defective conformation. Thus, the activation domain is unmasked only upon DNA binding by the correct basic region. Such a coupled conformational relationship may have evolved to restrict myogenic specificity to a small number of bHLH proteins among many with diverse functions yet with DNA binding specificities known to be similar.

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