scholarly journals Mutational analysis of the DNA binding, dimerization, and transcriptional activation domains of MEF2C.

1996 ◽  
Vol 16 (6) ◽  
pp. 2627-2636 ◽  
Author(s):  
J D Molkentin ◽  
B L Black ◽  
J F Martin ◽  
E N Olson
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Gene Activation ◽  
Positive Control ◽  
Dominant Negative ◽  
Activation Domains ◽  
Muscle Gene Expression ◽  
Transcriptional Activation Domains ◽  
Muscle Gene

There are four members of the myocyte enhancer factor 2 (MEF2) family of transcription factors in vertebrates, MEF2A, -B, -C, and -D, which have homology within a MADS box at their amino termini and an adjacent motif known as the MEF2 domain. These factors activate muscle gene expression by binding as homo- and heterodimers to an A/T-rich DNA sequence in the control regions of muscle-specific genes. To understand the mechanisms of muscle gene activation of MEF2 factors, we generated a series of deletion and site-directed mutants of MEF2C. These mutants demonstrated that the MADS and MEF2 domains mediate DNA binding and dimerization, whereas the carboxyl terminus is required for transcriptional activation. Amino acids that are essential for MEF2 site-dependent transcription but which do not affect DNA binding were also identified in the MEF2 domain. This type of positive-control mutant demonstrates that the transcription activation domain of MEF2C, although separate from the MEF2 domain, is dependent on this domain for transcriptional activation through the MEF2 site. MEF2 mutants that are defective for DNA binding act as dominant negative mutants and can inhibit activation of MEF2-dependent genes by wild-type MEF2C.

scholarly journals A heat shock-responsive domain of human HSF1 that regulates transcription activation domain function.

1995 ◽  
Vol 15 (6) ◽  
pp. 3354-3362 ◽  
Author(s):  
M Green ◽  
T J Schuetz ◽  
E K Sullivan ◽  
R E Kingston
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Chimeric Proteins ◽  
Dna Binding Domain ◽  
Regulatory Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domains

Human heat shock factor 1 (HSF1) stimulates transcription from heat shock protein genes following stress. We have used chimeric proteins containing the GAL4 DNA binding domain to identify the transcriptional activation domains of HSF1 and a separate domain that is capable of regulating activation domain function. This regulatory domain conferred heat shock inducibility to chimeric proteins containing the activation domains. The regulatory domain is located between the transcriptional activation domains and the DNA binding domain of HSF1 and is conserved between mammalian and chicken HSF1 but is not found in HSF2 or HSF3. The regulatory domain was found to be functionally homologous between chicken and human HSF1. This domain does not affect DNA binding by the chimeric proteins and does not contain any of the sequences previously postulated to regulate DNA binding of HSF1. Thus, we suggest that activation of HSF1 by stress in humans is controlled by two regulatory mechanisms that separately confer heat shock-induced DNA binding and transcriptional stimulation.

scholarly journals Mutational Analysis of Conserved Residues in the Putative DNA-Binding Domain of the Response Regulator Spo0A ofBacillus subtilis

2000 ◽  
Vol 182 (24) ◽  
pp. 6975-6982 ◽  
Author(s):  
Janet K. Hatt ◽  
Philip Youngman
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Response Regulator ◽  
Gene Activation ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Specific Gene ◽  
Dna Binding Domain ◽  
Conserved Residues

ABSTRACT The Spo0A protein of Bacillus subtilis is a DNA-binding protein that is required for the expression of genes involved in the initiation of sporulation. Spo0A binds directly to and both activates and represses transcription from the promoters of several genes required during the onset of endospore formation. The C-terminal 113 residues are known to contain the DNA-binding activity of Spo0A. Previous studies identified a region of the C-terminal half of Spo0A that is highly conserved among species of endospore-formingBacillus and Clostridium and which encodes a putative helix-turn-helix DNA-binding domain. To test the functional significance of this region and determine if this motif is involved in DNA binding, we changed three conserved residues, S210, E213, and R214, to Gly and/or Ala by site-directed mutagenesis. We then isolated and analyzed the five substitution-containing Spo0A proteins for DNA binding and sporulation-specific gene activation. The S210A Spo0A mutant exhibited no change from wild-type binding, although it was defective in spoIIA and spoIIE promoter activation. In contrast, both the E213G and E213A Spo0A variants showed decreased binding and completely abolished transcriptional activation of spoIIA and spoIIE, while the R214G and R214A variants completely abolished both DNA binding and transcriptional activation. These data suggest that these conserved residues are important for transcriptional activation and that the E213 residue is involved in DNA binding.

scholarly journals Fungal Mediator Tail Subunits Contain Classical Transcriptional Activation Domains

2015 ◽  
Vol 35 (8) ◽  
pp. 1363-1375 ◽  
Author(s):  
Zhongle Liu ◽  
Lawrence C. Myers
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Weak Interactions ◽  
Primary Role ◽  
Eukaryotic Transcription ◽  
Content Type ◽  
Activation Domains ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Transcriptional Activation Domains

Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits ofSaccharomyces cerevisiae,Candida albicans, andCandida dubliniensisMediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains ofS. cerevisiaeMed2 and Med3, as well asC. dubliniensisTlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen.

scholarly journals Modulation of Smooth Muscle Gene Expression by Association of Histone Acetyltransferases and Deacetylases with Myocardin

2005 ◽  
Vol 25 (1) ◽  
pp. 364-376 ◽  
Author(s):  
Dongsun Cao ◽  
Zhigao Wang ◽  
Chun-Li Zhang ◽  
Jiyeon Oh ◽  
Weibing Xing ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Transcriptional Activation ◽  
Histone Deacetylases ◽  
Serum Response Factor ◽  
Gene Activation ◽  
Muscle Gene Expression ◽  
P300 Binding ◽  
Muscle Genes ◽  
Muscle Gene

ABSTRACT Differentiation of smooth muscle cells is accompanied by the transcriptional activation of an array of muscle-specific genes controlled by serum response factor (SRF). Myocardin is a cardiac and smooth muscle-specific expressed transcriptional coactivator of SRF and is sufficient and necessary for smooth muscle gene expression. Here, we show that myocardin induces the acetylation of nucleosomal histones surrounding SRF-binding sites in the control regions of smooth muscle genes. The promyogenic activity of myocardin is enhanced by p300, a histone acetyltransferase that associates with the transcription activation domain of myocardin. Conversely, class II histone deacetylases interact with a domain of myocardin distinct from the p300-binding domain and suppress smooth muscle gene activation by myocardin. These findings point to myocardin as a nexus for positive and negative regulation of smooth muscle gene expression by changes in chromatin acetylation.

scholarly journals Functional domains of the transcription factor USF2: atypical nuclear localization signals and context-dependent transcriptional activation domains.

1996 ◽  
Vol 16 (4) ◽  
pp. 1367-1375 ◽  
Author(s):  
X Luo ◽  
M Sawadogo
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Transcriptional Activation ◽  
Core Promoter ◽  
Minimal Promoter ◽  
Functional Domains ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domains ◽  
Basic Region

USF is a family of basic helix-loop transcriptional factors that recognizes DNA-binding sites similar to those of the Myc oncoproteins. Here, various functional domains in the mouse USF2 protein were identified and characterized. Indirect immunofluorescence studies with transiently transfected cells revealed that both the basic region and the highly conserved USF-specific region (USR) are involved in the nuclear localization of USF2. Cotransfection assays with deletion mutants containing the DNA-binding domain of either USF2 or GAL4 identified two distinct transcriptional activation domains in USF2, the USR and the exon 5-encoded region. Activity of the exon 5 activation domain was detectable in both assay systems. Within USF2, however, its potency varied with the conformation induced by the surrounding regions, especially that encoded by alternatively spliced exon 4. In contrast, the USR activated transcription only in its natural context upstream of the USF2 basic region and only with reporter constructs containing the adenovirus major late minimal promoter but not the E1b minimal promoter. However, insertion of an initiator element downstream of the TATA box rescued the activity of the USR on the E1b-driven reporters. The USR therefore represents a new type of activation domain whose function depends very strongly on the core promoter context.

scholarly journals Definition of the Transcriptional Activation Domains of Three Human HOX Proteins Depends on the DNA-Binding Context

1998 ◽  
Vol 18 (11) ◽  
pp. 6201-6212 ◽  
Author(s):  
Maria Alessandra Viganò ◽  
Giuliana Di Rocco ◽  
Vincenzo Zappavigna ◽  
Fulvio Mavilio
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Regulatory Elements ◽  
Hox Proteins ◽  
Activation Domains ◽  
Downstream Target ◽  
Transcriptional Machinery ◽  
Transcriptional Activation Domains

ABSTRACT Hox proteins control developmental patterns and cell differentiation in vertebrates by acting as positive or negative regulators of still unidentified downstream target genes. The homeodomain and other small accessory sequences encode the DNA-protein and protein-protein interaction functions which ultimately dictate target recognition and functional specificity in vivo. The effector domains responsible for either positive or negative interactions with the cell transcriptional machinery are unknown for most Hox proteins, largely due to a lack of physiological targets on which to carry out functional analysis. We report the identification of the transcriptional activation domains of three human Hox proteins, HOXB1, HOXB3, and HOXD9, which interact in vivo with the autoregulatory and cross-regulatory enhancers of the murine Hoxb-1 and human HOXD9 genes. Activation domains have been defined both in a homologous context, i.e., within a HOX protein binding as a monomer or as a HOX-PBX heterodimer to the specific target, and in a heterologous context, after translocation to the yeast Gal4 DNA-binding domain. Transfection analysis indicates that activation domains can be identified in different regions of the three HOX proteins depending on the context in which they interact with the DNA target. These results suggest that Hox proteins may be multifunctional transcriptional regulators, interacting with different cofactors and/or components of the transcriptional machinery depending on the structure of their target regulatory elements.

scholarly journals Identification, Mutational Analysis, and Coactivator Requirements of Two Distinct Transcriptional Activation Domains of the Saccharomyces cerevisiae Hap4 Protein

2004 ◽  
Vol 3 (2) ◽  
pp. 339-347 ◽  
Author(s):  
John L. Stebbins ◽  
Steven J. Triezenberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Mutational Analysis ◽  
Transcriptional Coactivator ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Transcriptional Activation Domains

ABSTRACT The Hap4 protein of the budding yeast Saccharomyces cerevisiae activates the transcription of genes that are required for growth on nonfermentable carbon sources. Previous reports suggested the presence of a transcriptional activation domain within the carboxyl-terminal half of Hap4 that can function in the absence of Gcn5, a transcriptional coactivator protein and histone acetyltransferase. The boundaries of this activation domain were further defined to a region encompassing amino acids 359 to 476. Within this region, several clusters of hydrophobic amino acids are critical for transcriptional activity. This activity does not require GCN5 or two other components of the SAGA coactivator complex, SPT3 and SPT8, but it does require SPT7 and SPT20. Contrary to previous reports, a Hap4 fragment comprising amino acids 1 to 330 can support the growth of yeast on lactate medium, and when tethered to lexA, can activate a reporter gene with upstream lexA binding sites, demonstrating the presence of a second transcriptional activation domain. In contrast to the C-terminal activation domain, the transcriptional activity of this N-terminal region depends on GCN5. We conclude that the yeast Hap4 protein has at least two transcriptional activation domains with strikingly different levels of dependence on specific transcriptional coactivator proteins.

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