scholarly journals Polyglutamine tracts as modulators of transcriptional activation from yeast to mammals

2012 ◽  
Vol 393 (1-2) ◽  
pp. 63-70 ◽  
Author(s):  
Lilit Atanesyan ◽  
Viola Günther ◽  
Bernhard Dichtl ◽  
Oleg Georgiev ◽  
Walter Schaffner
Keyword(s):  
Transcription Factors ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Coding Region ◽  
Triplet Repeats ◽  
Widespread Occurrence ◽  
Activation Domains ◽  
Linker Effect ◽  
Two Hybrid ◽  
Polyq Tract

Abstract Microsatellite repeats are genetically unstable and subject to expansion and shrinkage. A subset of them, triplet repeats, can occur within the coding region and specify homomeric tracts of amino acids. Polyglutamine (polyQ) tracts are enriched in eukaryotic regulatory proteins, notably transcription factors, and we had shown before that they can contribute to transcriptional activation in mammalian cells. Here we generalize this finding by also including evolutionarily divergent organisms, namely, Drosophila and baker’s yeast. In all three systems, Gal4-based model transcription factors were more active if they harbored a polyQ tract, and the activity depended on the length of the tract. By contrast, a polyserine tract was inactive. PolyQs acted from either an internal or a C-terminal position, thus ruling out a merely structural ‘linker’ effect. Finally, a two-hybrid assay in mammalian cells showed that polyQ tracts can interact with each other, supporting the concept that a polyQ-containing transcription factor can recruit other factors with polyQ tracts or glutamine-rich activation domains. The widespread occurrence of polyQ repeats in regu­latory proteins suggests a beneficial role; in addition to the contribution to transcriptional activity, their genetic instability might help a species to adapt to changing environmental conditions in a potentially reversible manner.

scholarly journals Identification and Characterization of ART-27, a Novel Coactivator for the Androgen Receptor N Terminus

2002 ◽  
Vol 13 (2) ◽  
pp. 670-682 ◽  
Author(s):  
Steven M. Markus ◽  
Samir S. Taneja ◽  
Susan K. Logan ◽  
Wenhui Li ◽  
Susan Ha ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Hybrid Approach ◽  
Independent Manner ◽  
Activation Domains ◽  
Cell Library ◽  
Nuclear Extracts ◽  
N Terminus ◽  
Transcriptional Activation Domains

The androgen receptor (AR) is a ligand-regulated transcription factor that stimulates cell growth and differentiation in androgen-responsive tissues. The AR N terminus contains two activation functions (AF-1a and AF-1b) that are necessary for maximal transcriptional enhancement by the receptor; however, the mechanisms and components regulating AR transcriptional activation are not fully understood. We sought to identify novel factors that interact with the AR N terminus from an androgen-stimulated human prostate cancer cell library using a yeast two-hybrid approach designed to identify proteins that interact with transcriptional activation domains. A 157-amino acid protein termed ART-27 was cloned and shown to interact predominantly with the AR153–336, containing AF-1a and a part of AF-1b, localize to the nucleus and increase the transcriptional activity of AR when overexpressed in cultured mammalian cells. ART-27 also enhanced the transcriptional activation by AR153–336 fused to the LexA DNA-binding domain but not other AR N-terminal subdomains, suggesting that ART-27 exerts its effect via an interaction with a defined region of the AR N terminus. ART-27 interacts with AR in nuclear extracts from LNCaP cells in a ligand-independent manner. Interestingly, velocity gradient sedimentation of HeLa nuclear extracts suggests that native ART-27 is part of a multiprotein complex. ART-27 is expressed in a variety of human tissues, including sites of androgen action such as prostate and skeletal muscle, and is conserved throughout evolution. Thus, ART-27 is a novel cofactor that interacts with the AR N terminus and plays a role in facilitating receptor-induced transcriptional activation.

scholarly journals Conservation of transcriptional activation functions of the NF-kappa B p50 and p65 subunits in mammalian cells and Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (3) ◽  
pp. 1666-1674 ◽  
Author(s):  
P A Moore ◽  
S M Ruben ◽  
C A Rosen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Transcriptional Activator ◽  
Genetic System ◽  
Nf Kappa B ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activation Domains ◽  
Inhibitory Molecule

The NF-kappa B transcription factor complex is composed of a 50-kDa (p50) and a 65-kDa (p65) subunit. Both subunits bind to similar DNA motifs and elicit transcriptional activation as either homo- or heterodimers. By using chimeric proteins that contain the DNA binding domain of the yeast transcriptional activator GAL4 and subdomains of p65, three distinct transcriptional activation domains were identified. One domain was localized to a region of 42 amino acids containing a potential leucin zipper structure, consistent with earlier reports. Two other domains, both acidic and rich in prolines, were also identified. Of perhaps more significance, the same minimal activation domains that were functional in mammalian cells were also functional in the yeast Saccharomyces cerevisiae. Coexpression of the NF-kappa B inhibitory molecule, I kappa B, reduced the transcriptional activity of p65 significantly, suggesting the ability of I kappa B to function in a similar manner in S. cerevisiae. Surprisingly, while the conserved rel homology domain of p65 demonstrated no transcriptional activity in either mammalian cells or S. cerevisiae, the corresponding domain in p50 was a strong transcriptional activator in S. cerevisiae. The observation that similar domains elicit transcriptional activation in mammalian cells and S. cerevisiae demonstrates strong conservation of the transcriptional machinery required for NF-kappa B function and provides a powerful genetic system to study the transcriptional mechanisms of these proteins.

scholarly journals Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCN5.

1996 ◽  
Vol 16 (2) ◽  
pp. 593-602 ◽  
Author(s):  
R Candau ◽  
P A Moore ◽  
L Wang ◽  
N Barlev ◽  
C Y Ying ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Sequence Similarity ◽  
Adaptor Proteins ◽  
Yeast Two Hybrid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activation Domains ◽  
Evolutionarily Conserved ◽  
Human Proteins ◽  
Two Hybrid

Transcriptional adaptor proteins are required for full function of higher eukaryotic acidic activators in the yeast Saccharomyces cerevisiae, suggesting that this pathway of activation is evolutionarily conserved. Consistent with this view, we have identified possible human homologs of yeast ADA2 (yADA2) and yeast GCN5 (yGCN5), components of a putative adaptor complex. While there is overall sequence similarity between the yeast and human proteins, perhaps more significant is conservation of key sequence features with other known adaptors. We show several functional similarities between the human and yeast adaptors. First, as shown for yADA2 and yGCN5, human ADA2 (hADA2) and human GCN5 (hGCN5) interacted in vivo in a yeast two-hybrid assay. Moreover, hGCN5 interacted with yADA2 in this assay, suggesting that the human proteins form similar complexes. Second, both yADA2 and hADA2 contain cryptic activation domains. Third, hGCN5 and yGCN5 had similar stabilizing effects on yADA2 in vivo. Furthermore, the region of yADA2 that interacted with yGCN5 mapped to the amino terminus of yADA2, which is highly conserved in hADA2. Most striking, is the behavior of the human proteins in human cells. First, GAL4-hADA2 activated transcription in HeLa cells, and second, either hADA2 or hGCN5 augmented GAL4-VP16 activation. These data indicated that the human proteins correspond to functional homologs of the yeast adaptors, suggesting that these cofactors play a key role in transcriptional activation.

scholarly journals Artificial Recruitment of TFIID, but Not RNA Polymerase II Holoenzyme, Activates Transcription in Mammalian Cells

2000 ◽  
Vol 20 (12) ◽  
pp. 4350-4358 ◽  
Author(s):  
David R. Dorris ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Pol Ii ◽  
Activation Domains ◽  
Synergistic Activation ◽  
The One ◽  
Rna Polymerase Ii Holoenzyme

ABSTRACT In yeast cells, transcriptional activation occurs when the RNA polymerase II (Pol II) machinery is artificially recruited to a promoter by fusing individual components of this machinery to a DNA-binding domain. Here, we show that artificial recruitment of components of the TFIID complex can activate transcription in mammalian cells. Surprisingly, artificial recruitment of TATA-binding protein (TBP) activates transiently transfected and chromosomally integrated promoters with equal efficiency, whereas artificial recruitment of TBP-associated factors activates only chromosomal reporters. In contrast, artificial recruitment of various components of the mammalian Pol II holoenzyme does not confer transcriptional activation, nor does it result in synergistic activation in combination with natural activation domains. In the one case examined in more detail, the Srb7 fusion failed to activate despite being associated with the Pol II holoenzyme and being directly recruited to the promoter. Interestingly, some acidic activation domains are less effective when the promoter is chromosomally integrated rather than transiently transfected, whereas the Sp1 glutamine-rich activation domain is more effective on integrated reporters. Thus, yeast and mammalian cells differ with respect to transcriptional activation by artificial recruitment of the Pol II holoenzyme.

Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53

1994 ◽  
Vol 14 (10) ◽  
pp. 7013-7024 ◽  
Author(s):  
H Xiao ◽  
A Pearson ◽  
B Coulombe ◽  
R Truant ◽  
S Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Point Mutations ◽  
Factor B ◽  
Activation Domains ◽  
Binding Domains ◽  
General Transcription Factors ◽  
Transcriptional Activation Domains ◽  
Simplex Virus

Acidic transcriptional activation domains function well in both yeast and mammalian cells, and some have been shown to bind the general transcription factors TFIID and TFIIB. We now show that two acidic transactivators, herpes simplex virus VP16 and human p53, directly interact with the multisubunit human general transcription factor TFIIH and its Saccharomyces cerevisiae counterpart, factor b. The VP16- and p53-binding domains in these factors lie in the p62 subunit of TFIIH and in the homologous subunit, TFB1, of factor b. Point mutations in VP16 that reduce its transactivation activity in both yeast and mammalian cells weaken its binding to both yeast and human TFIIH. This suggests that binding of activation domains to TFIIH is an important aspect of transcriptional activation.

scholarly journals A Family of LIM-Only Transcriptional Coactivators: Tissue-Specific Expression and Selective Activation of CREB and CREM

2000 ◽  
Vol 20 (22) ◽  
pp. 8613-8622 ◽  
Author(s):  
Gian Maria Fimia ◽  
Dario De Cesare ◽  
Paolo Sassone-Corsi
Keyword(s):  
Transcription Factors ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Target Genes ◽  
Activation Function ◽  
Creb Binding Protein ◽  
Selective Activation ◽  
Specific Expression ◽  
Tissue Specific ◽  
Family Control

ABSTRACT Transcription factors of the CREB family control the expression of a large number of genes in response to various signaling pathways. Regulation mediated by members of the CREB family has been linked to various physiological functions. Classically, activation by CREB is known to occur upon phosphorylation at an essential regulatory site (Ser133 in CREB) and the subsequent interaction with the ubiquitous coactivator CREB-binding protein (CBP). However, the mechanism by which selectivity is achieved in the identification of target genes, as well as the routes adopted to ensure tissue-specific activation, remains unrecognized. We have recently described the first tissue-specific coactivator of CREB family transcription factors, ACT (activator of CREM in testis). ACT is a LIM-only protein which associates with CREM in male germ cells and provides an activation function which is independent of phosphorylation and CBP. Here we characterize a family of LIM-only proteins which share common structural organization with ACT. These are referred to as four-and-a-half-LIM-domain (FHL) proteins and display tissue-specific and developmentally regulated expression. FHL proteins display different degrees of intrinsic activation potential. They provide powerful activation function to both CREB and CREM when coexpressed either in yeast or in mammalian cells, specific combinations eliciting selective activation. Deletion analysis of the ACT protein shows that the activation function depends on specific arrangements of the LIM domains, which are essential for both transactivation and interaction properties. This study uncovers the existence of a family of tissue-specific coactivators that operate through novel, CBP-independent routes to elicit transcriptional activation by CREB and CREM. The future identification of additional partners of FHL proteins is likely to reveal unappreciated aspects of tissue-specific transcriptional regulation.

scholarly journals Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

1994 ◽  
Vol 14 (10) ◽  
pp. 7013-7024 ◽  
Author(s):  
H Xiao ◽  
A Pearson ◽  
B Coulombe ◽  
R Truant ◽  
S Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Point Mutations ◽  
Factor B ◽  
Activation Domains ◽  
Binding Domains ◽  
General Transcription Factors ◽  
Transcriptional Activation Domains ◽  
Simplex Virus

Acidic transcriptional activation domains function well in both yeast and mammalian cells, and some have been shown to bind the general transcription factors TFIID and TFIIB. We now show that two acidic transactivators, herpes simplex virus VP16 and human p53, directly interact with the multisubunit human general transcription factor TFIIH and its Saccharomyces cerevisiae counterpart, factor b. The VP16- and p53-binding domains in these factors lie in the p62 subunit of TFIIH and in the homologous subunit, TFB1, of factor b. Point mutations in VP16 that reduce its transactivation activity in both yeast and mammalian cells weaken its binding to both yeast and human TFIIH. This suggests that binding of activation domains to TFIIH is an important aspect of transcriptional activation.

scholarly journals Conservation of Glutamine-Rich Transactivation Function between Yeast and Humans

2000 ◽  
Vol 20 (8) ◽  
pp. 2774-2782 ◽  
Author(s):  
Dominik Escher ◽  
Morana Bodmer-Glavas ◽  
Alcide Barberis ◽  
Walter Schaffner
Keyword(s):  
Transcription Factors ◽  
Transcriptional Activation ◽  
Budding Yeast ◽  
Human Cells ◽  
Yeast Genome ◽  
Eukaryotic Transcription ◽  
Activation Domains ◽  
Promoter Architecture ◽  
Rich Domain ◽  
General Transcription Factor

ABSTRACT Several eukaryotic transcription factors such as Sp1 or Oct1 contain glutamine-rich domains that mediate transcriptional activation. In human cells, promoter-proximally bound glutamine-rich activation domains activate transcription poorly in the absence of acidic type activators bound at distal enhancers, but synergistically stimulate transcription with these remote activators. Glutamine-rich activation domains were previously reported to also function in the fission yeastSchizosaccharomyces pombe but not in the budding yeastSaccharomyces cerevisiae, suggesting that budding yeast lacks this pathway of transcriptional activation. The strong interaction of an Sp1 glutamine-rich domain with the general transcription factor TAFII110 (TAFII130), and the absence of any obvious TAFII110 homologue in the budding yeast genome, seemed to confirm this notion. We reinvestigated the phenomenon by reconstituting in the budding yeast an enhancer-promoter architecture that is prevalent in higher eukaryotes but less common in yeast. Under these conditions, we observed that glutamine-rich activation domains derived from both mammalian and yeast transcription factors activated only poorly on their own but strongly synergized with acidic activators bound at the remote enhancer position. The level of activation by the glutamine-rich activation domains of Sp1 and Oct1 in combination with a remote enhancer was similar in yeast and human cells. We also found that mutations in a glutamine-rich domain had similar phenotypes in budding yeast and human cells. Our results show that glutamine-rich activation domains behave very similarly in yeast and mammals and that their activity in budding yeast does not depend on the presence of a TAFII110 homologue.

scholarly journals A highly conserved domain of RNA polymerase II shares a functional element with acidic activation domains of upstream transcription factors

1994 ◽  
Vol 14 (11) ◽  
pp. 7507-7516
Author(s):  
H Xiao ◽  
J D Friesen ◽  
J T Lis
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Normal Function ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activation Domains ◽  
Acidic Domain ◽  
General Transcription Factors ◽  
Derivatives Of

We report here that the largest subunit of yeast RNA polymerase II contains an acidic domain that is similar to acidic activators of transcription. This domain includes the highly conserved homology box H. A hybrid protein containing this acidic domain fused to the DNA-binding domain of GAL4 is a potent activator of transcription in the yeast Saccharomyces cerevisiae. Interestingly, mutations that reduce the upstream activating activity of this acidic domain also abolish the normal function of RNA polymerase II. Such functional defects can be rescued by the acidic activation domains of VP16 and GAL4 when inserted into the mutant derivatives of RNA polymerase II. We further show that this acidic domain of RNA polymerase II interacts directly with two general transcription factors, the TATA-binding protein and TFIIB, and that the acidic activation domain of VP16 can compete specifically with the acidic domain of the RNA polymerase for these interactions. We discuss the implications of this finding for the mechanisms of transcriptional activation in eucaryotes.

Conservation of transcriptional activation functions of the NF-kappa B p50 and p65 subunits in mammalian cells and Saccharomyces cerevisiae

1993 ◽  
Vol 13 (3) ◽  
pp. 1666-1674
Author(s):  
P A Moore ◽  
S M Ruben ◽  
C A Rosen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Transcriptional Activator ◽  
Genetic System ◽  
Nf Kappa B ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activation Domains ◽  
Inhibitory Molecule

The NF-kappa B transcription factor complex is composed of a 50-kDa (p50) and a 65-kDa (p65) subunit. Both subunits bind to similar DNA motifs and elicit transcriptional activation as either homo- or heterodimers. By using chimeric proteins that contain the DNA binding domain of the yeast transcriptional activator GAL4 and subdomains of p65, three distinct transcriptional activation domains were identified. One domain was localized to a region of 42 amino acids containing a potential leucin zipper structure, consistent with earlier reports. Two other domains, both acidic and rich in prolines, were also identified. Of perhaps more significance, the same minimal activation domains that were functional in mammalian cells were also functional in the yeast Saccharomyces cerevisiae. Coexpression of the NF-kappa B inhibitory molecule, I kappa B, reduced the transcriptional activity of p65 significantly, suggesting the ability of I kappa B to function in a similar manner in S. cerevisiae. Surprisingly, while the conserved rel homology domain of p65 demonstrated no transcriptional activity in either mammalian cells or S. cerevisiae, the corresponding domain in p50 was a strong transcriptional activator in S. cerevisiae. The observation that similar domains elicit transcriptional activation in mammalian cells and S. cerevisiae demonstrates strong conservation of the transcriptional machinery required for NF-kappa B function and provides a powerful genetic system to study the transcriptional mechanisms of these proteins.

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