Proline-rich Activation Domain Research Articles

Mammalian transcription activation domains of VP16, AP2 and CTF activate transcription in a whole cell extract from Schizosaccharomyces pombe through the SRB/mediator

The acidic-rich activation domain of VP16 and the proline-rich activation domains of human AP2 and human CTF are able to activate transcription in a whole cell extract from Schizosaccharomyces pombe, whereas the glutamine-rich domains of Sp1 and Oct2 are unable to activate transcription in this system. Immunodepletion experiments of the whole cell extracts using specific antibodies against pombe TAF110, pombe TAF 72, pombe TBP and Srb4 shows that the activation of transcription by VP16, AP2 and CTF is through the mediator, since depletion of Srb4 inhibits activated transcription but does not inhibit basal transcription. Immunodepletion of TBP causes inhibition of both activated and basal transcription. On the other hand, immunodepletion of TAFs does not have an effect on either activated or basal transcription. Purified RNA polymerase holoenzyme is able to rescue the transcriptional activation activity of the anti-Srb4 immunodepleted extract. Moreover, we demonstrate that the mediator is needed for basal transcription of a TATA-less promoter.

Activation domain-specific and general transcription stimulation by native histone acetyltransferase complexes.

Recent progress in identifying the catalytic subunits of histone acetyltransferase (HAT) complexes has implicated histone acetylation in the regulation of transcription. Here, we have analyzed the function of two native yeast HAT complexes, SAGA (Spt-Ada-Gcn5 Acetyltransferase) and NuA4 (nucleosome acetyltransferase of H4), in activating transcription from preassembled nucleosomal array templates in vitro. Each complex was tested for the ability to enhance transcription driven by GAL4 derivatives containing either acidic, glutamine-rich, or proline-rich activation domains. On nucleosomal array templates, the SAGA complex selectively stimulates transcription driven by the VP16 acidic activation domain in an acetyl coenzyme A-dependent manner. In contrast, the NuA4 complex facilitates transcription mediated by any of the activation domains tested if allowed to preacetylate the nucleosomal template, indicating a general stimulatory effect of histone H4 acetylation. However, when the extent of acetylation by NuA4 is limited, the complex also preferentially stimulates VP16-driven transcription. SAGA and NuA4 interact directly with the VP16 activation domain but not with a glutamine-rich or proline-rich activation domain. These data suggest that recruitment of the SAGA and NuA4 HAT complexes by the VP16 activation domain contributes to HAT-dependent activation. In addition, extensive H4/H2B acetylation by NuA4 leads to a general activation of transcription, which is independent of activator-NuA4 interactions.

Three classes of mammalian transcription activation domain stimulate transcription in Schizosaccharomyces pombe.

Representatives of three distinct classes of mammalian protein domain activating RNA polymerase II were fused to the yeast GAL4p DNA-binding domain. The resulting fusion proteins were tested in the fission yeast Schizosaccharomyces pombe for their ability to activate transcription of different reporter constructs containing GAL4-binding sites in positions close to or far from the TATA box. The acidic-rich activation domain of VP16 stimulates transcription in S.pombe from proximal and distal positions, suggesting that the mechanism of activation is conserved from man to budding and fission yeasts. Unlike in Saccharomyces cerevisiae, the glutamine-rich activation domains of Sp1, Oct1 and Oct2 activate transcription in S. pombe when tested in a proximal TATA box context. Similarly to mammalian cells, these domains are inactive or weakly active when tested in a distal position. Moreover, the proline-rich activation domains of AP-2 and CTF/NF1 display strong transcriptional activities from a TATA box-proximal position, and weak activities when tested in a remote position. Consequently, proline-rich and glutamine-rich activation domains act differently in S.cerevisiae and mammalian cells, but similarly in S.pombe and mammalian cells.

The Ability of the Inhibitory Domain of the POU Family Transcription Factor Oct-2 to Interfere with Promoter Activation by Different Classes of Activation Domains Is Dependent upon the Nature of the Basal Promoter Elements

The Oct-2 transcription factor contains an inhibitory domain which is able to repress transcription following DNA binding. Here we show that within the neuronally expressed Oct-2.5 form, the inhibitory domain can strongly inhibit activation by transcription factor activation domains which are either composed predominantly of acidic residues or contain the HOB motif, whereas it has a weaker effect or no effect on proline-rich activation domains and on a glutamine-rich domain. In contrast, the isolated inhibitory domain of Oct-2 can efficiently repress all types of activation domains. This effect is observed however, only on TATA box-containing promoters and not on promoters containing an initiator motif. This widespread inhibition of different activation domains and its dependence on the nature of the basal promoter elements indicate that the inhibitory domain is likely to act by contacting a common downstream target of activation domains within the basal transcriptional complex bound at the TATA box rather than quenching specific activation domains by direct interaction. These effects are discussed in terms of the functional role of the inhibitory domain within Oct-2.5 and the mechanism by which it acts.

Functional domains of the heavy metal-responsive transcription regulator MTF-1.

Metallothioneins (MTs) constitute a class of low molecular weight, cysteine-rich, metal binding proteins which are regulated at the level of gene transcription in response to heavy metals and other adverse treatments. We have previously cloned a zinc finger factor (MTF-1) that binds specifically to heavy metal-responsive DNA sequence elements in metallothionein promoters and shown that this factor is essential for basal and heavy metal-induced transcription. Here we report that the C-terminal part of MTF-1 downstream of the DNA binding zinc fingers harbours three different transactivation domains, namely an acidic domain, a proline-rich domain and a domain rich in serine and threonine. When fused to the heterologous DNA binding domain of the yeast factor GAL4 these activation domains function constitutively, i.e. transcription of a GAL4-driven reporter gene is not induced by heavy metals. In search of the region(s) responsible for metal induction, external and internal deletion mutations of mouse and human MTF-1 and chimeric variants thereof were tested with a reporter gene driven by a metal-responsive promoter. The N-terminal part of MTF-1 containing the zinc fingers, which are dependent on zinc for efficient DNA binding, can indeed confer a limited (3- to 4-fold) zinc-responsive transcription when fused to the heterologous activation domain of the viral VP16 protein. Another region containing the acidic and proline-rich activation domains also contributes to metal inducibility, but only in the context of intact MTF-1. This indicates that the activity of MTF-1 results from a complex interplay of different functional domains.

The Oct-2 glutamine-rich and proline-rich activation domains can synergize with each other or duplicates of themselves to activate transcription.

The B-cell POU homeodomain protein Oct-2 contains two transcriptional activation domains, one N terminal and the other C terminal of the central DNA-binding POU domain. The synergistic action of these two activation domains makes Oct-2 a more potent activator of mRNA promoters than the related broadly expressed octamer motif-binding protein Oct-1, which contains an N-terminal but not a C-terminal Oct-2-like activation domain. Both Oct-2 mRNA promoter activation domains were delineated by truncation analysis: the N-terminal Q domain is a 66-amino-acid region rich in glutamines, and the C-terminal P domain is a 42-amino-acid region rich in prolines. The Q and P domains synergized with each other or duplicates of themselves, independently of their N-terminal or C-terminal position relative to the POU domain. The C-terminal P domain, which differentiates Oct-2 from Oct-1, also activated transcription in conjunction with the heterologous GAL4 DNA-binding domain. Oct-2 thus contains three modular functional units, the DNA-binding POU domain and the two P and Q activation domains. An electrophoretic mobility shift assay with a variety of these Oct-2 activators revealed a distinct complex called QA that was dependent on the presence of an active glutamine-rich activation domain and migrated more slowly than the Oct-2-DNA complexes. Formation of the QA complex is consistent with interaction of the glutamine-rich activation domains with a regulatory protein important for the process of transcriptional activation.

The Oct-2 Glutamine-Rich and Proline-Rich Activation Domains Can Synergize with Each Other or Duplicates of Themselves To Activate Transcription

The B-cell POU homeodomain protein Oct-2 contains two transcriptional activation domains, one N terminal and the other C terminal of the central DNA-binding POU domain. The synergistic action of these two activation domains makes Oct-2 a more potent activator of mRNA promoters than the related broadly expressed octamer motif-binding protein Oct-1, which contains an N-terminal but not a C-terminal Oct-2-like activation domain. Both Oct-2 mRNA promoter activation domains were delineated by truncation analysis: the N-terminal Q domain is a 66-amino-acid region rich in glutamines, and the C-terminal P domain is a 42-amino-acid region rich in prolines. The Q and P domains synergized with each other or duplicates of themselves, independently of their N-terminal or C-terminal position relative to the POU domain. The C-terminal P domain, which differentiates Oct-2 from Oct-1, also activated transcription in conjunction with the heterologous GAL4 DNA-binding domain. Oct-2 thus contains three modular functional units, the DNA-binding POU domain and the two P and Q activation domains. An electrophoretic mobility shift assay with a variety of these Oct-2 activators revealed a distinct complex called QA that was dependent on the presence of an active glutamine-rich activation domain and migrated more slowly than the Oct-2-DNA complexes. Formation of the QA complex is consistent with interaction of the glutamine-rich activation domains with a regulatory protein important for the process of transcriptional activation.

Proline-rich activator CTF1 targets the TFIIB assembly step during transcriptional activation.

Activators can stimulate transcription through direct or indirect interactions with general initiation factors. We show here that the proline-rich activation domain of CTF1 (CCAAT-box-binding transcription factor 1) selectively interacts with TFIIB but not with the TATA-binding protein (TBP), whereas previous studies have shown that the acidic activation domain of viral VP16 interacts directly with both TBP and TFIIB. In addition, consistent with studies of acidic activation domains, we demonstrate that the activation domain of CTF1 facilitates the recruitment (or stabilization) of TFIIB within TBP-DNA complexes during preinitiation complex assembly. CTF1-enhanced TFIIB recruitment was observed in both human and yeast systems. The results indicate that the proline-rich activation domain enhances transcription, at least in part, through direct interactions with TFIIB and, with previous observations, suggest models involving either quantitative or qualitative changes in TFIIB-TFIID-promoter interactions that lead to increased utilization of downstream initiation factors.

Transcriptional activation of NFI/CTF1 depends on a sequence motif strongly related to the carboxyterminal domain of RNA polymerase II.

Initiation of RNA polymerase II-directed transcription is mediated by DNA sequence specific activator proteins interacting with components of the basal transcription machinery. NFI/CTF is a family of such binding proteins which have been shown to stimulate transcription via proline-rich activation domains. In order to identify residues crucial for its activator function, a pool of CTF1 mutants was cloned and fused to the bacterial repressor LexA. Transcriptional activation of these constructs was monitored in a Saccharomyces cerevisiae reporter assay. Our studies reveal the existence of a core domain in CTF1 between residues 463 and 508 essential for transcriptional activation functions. It contains the sequence motif SPTSPSYSP, which is strongly related to the heptapeptide repeat YSPTSPS present in the carboxyterminal domain (CTD) of RNA polymerase II. Removal of the entire CTD related motif, as well as substitution of key amino acids therein, abolish CTF1 mediated transcriptional activation.

CTD-like sequences are important for transcriptional activation by the proline-rich activation domain of CTF1.

CTD-like sequences are important for transcriptional activation by the proline-rich activation domain of CTF1.

The upstream activator CTF/NF1 and RNA polymerase II share a common element involved in transcriptional activation.

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II consists of tandem repeats of a heptapeptide with the consensus YSPTSPS. It has been shown that the heptapeptide repeat interacts directly with the general transcription factor TFIID. We report here that the CTD activates transcription when fused to the DNA-binding domain of GAL4. More importantly, we find that the proline-rich transcriptional activation domain of the CCAAT-box-binding factor CTF/NF1 contains a sequence with striking similarity to the heptapeptide repeats of the CTD. We show that this CTD-like motif is essential for the transcriptional activator function of the proline-rich domain of CTF/NF1. Deletion of and point mutations in this CTD-like motif abolish the transcriptional activator function of the proline-rich domain, while natural CTD repeats from RNA polymerase II are fully functional in place of the CTD-like motif. We further show that the proline-rich activation domain of CTF/NF1 interacts directly with the TATA-box-binding protein (TBP), and that a mutation in the CTD-like motif that abolishes transcriptional activation reduces the affinity of the proline-rich domain for TBP. These results demonstrate that a class of proline-rich activator proteins and RNA polymerase II possess a common structural and functional component which can interact with the same target in the general transcription machinery. We discuss the implications of these results for the mechanisms of transcriptional activation in eucaryotes.

Cloning and functional analysis of spliced isoforms of human nuclear factor I-X: interference with transcriptional activation by NFI/CTF in a cell-type specific manner.

Previous studies of the epithelial specificity of the human papillomavirus type 16 (HPV-16) enhancer pointed out an important role of nuclear factor I (NFI). In epithelial cells, NFI proteins are derived from the NFI-C gene and referred to as NFI/CTF. In contrast, fibroblasts, where the enhancer is inactive, express high levels of NFI from the NFI-X gene. To compare NFI-C and NFI-X derived transcription factors, we cloned and functionally investigated two differentially spliced forms of NFI-X from human fibroblasts. NFI-X1 has 95% homology with a transcript previously identified in hamster liver cells. NFI-X2, a spliced variant, misses 41 amino acids of the proline-rich activation domain. NFI-X expression, examined by Northern blots, shows strong cell-type specific variation in comparison with NFI/CTF. While the transcriptional activation domain of NFI-X2, functionally tested as GAL4-fusion protein in epithelial and fibroblast cells, activates transcription from promoter as well as enhancer position similar to NFI/CTF-1, the activation domain of NFI-X1 fails to activate transcription from enhancer position. In Drosophila cells, void of endogenous NFI proteins, full length NFI/CTF-1 and NFI-X2 activate a reporter construct containing only NFI sites as well as the NFI dependent HPV-16 enhancer. In contrast, NFI-X1 fails to activate the HPV-16 enhancer. Furthermore, overexpression of NFI-X1 in epithelial cells down-regulates the HPV-16 enhancer. Our findings suggest that the family of NFI transcription factors should not be viewed as constitutive activators, but rather, that NFI-C and NFI-X have divergent functions after binding in promoter or enhancer position. This property, combined with the differential expression of NFI-X, can achieve cell-type specificity of NFI dependent promoters and enhancers.

Transcriptional activation in yeast by the proline-rich activation domain of human CTF1.

Among eukaryotic transcriptional activators, those with acidic activation domains have been shown to stimulate transcription in organisms ranging from yeast to human. In vivo and in vitro assays have been employed to demonstrate transcriptional enhancement in yeast by the proline-rich activation domain of human CTF1. This result suggests that proline-rich activation domains may, like typical acidic activation domains, functional universally.

Different activation domains stimulate transcription from remote (‘enhancer’) and proximal (‘promoter’) positions.

We reported previously that the lymphocyte-derived octamer transcription factor 2A (Oct-2A or OTF-2A) activated both natural immunoglobulin promoters and synthetic promoters which contain the 'octamer' site, but was unable by itself to stimulate transcription from a remote enhancer position. Here we examine a larger set of transcription factors with respect to their proximal versus remote activation. Since a transcription factor may contain more than one activation domain, we have chosen to study the potential of individual activation domains in the context of fusion proteins that contain the DNA binding domain of GALA. We have identified at least two distinct functional classes of transcriptional activation domains. 'Proximal' activation domains, exemplified by glutamine-rich domains of Oct-1, Oct-2A and Sp1, stimulate transcription only from a position close to the TATA box, usually in response to a remote enhancer. 'General' activation domains, derived from VP16, GAL4, p65 (NF-chi B), TFE3, ITF-1 and ITF-2, can activate transcription from remote as well as proximal positions. These domains contain many acidic amino acids and/or other features such as clusters of serine and threonine. The proline-rich activation domains of AP-2 and CTF/NF1 may represent a third class with considerable promoter activity and low but significant enhancer activity. Furthermore, activation domains of both the acidic and glutamine-rich types seem to have a modular structure, since duplicated subdomains can substitute for the entire domain.

Coactivators for a proline-rich activator purified from the multisubunit human TFIID complex.

The mechanisms of transcriptional activation directed by sequence-specific regulators is central to understanding gene regulation. Here, we report the isolation of coactivators responsible for mediating transcriptional activation by Gal4-Pro, a hybrid regulator containing the proline-rich activation domain of human CTF/NFI. Chromatographic studies indicate that endogenous human TFIID consists of a multisubunit complex containing the TATA-binding protein (TBP), coactivators, and other associated factors. A fraction containing the coactivator activity was separated from the endogenous TBP after disrupting the tightly associated complex with urea. The urea-purified TBP was active for basal level transcription but no longer could support activation by Gal4-Pro. However, when the two separated components were added together, activated levels of transcription were restored in the presence of Gal4-Pro. Immunoaffinity purification of the TFIID complex identifies several polypeptides specifically associated with the endogenous TBP, some or all of which function as coactivators when reconstituted with Gal4-Pro. The isolated coactivators also mediate activation by a chimeric glutamine-rich activator derived from Sp1 but not the Gal4-VP16 activator, suggesting distinct factor requirements for different types of transcriptional regulators.

Analysis of the DNA-binding and activation properties of the human transcription factor AP-2.

The mammalian transcription factor AP-2 is a sequence-specific DNA-binding protein expressed in neural crest lineages and regulated by retinoic acid. Here we report a structure/function analysis of the DNA-binding and transcription activation properties of the AP-2 protein. DNA contact studies indicate that AP-2 binds as a dimer to a palindromic recognition sequence. Furthermore, cross-linking and immunoprecipitation data illustrate that AP-2 exists as a dimer even in the absence of DNA. Examination of cDNA mutants reveals that the sequences responsible for DNA binding are located in the carboxy-terminal half of the protein. In addition, a domain mediating dimerization forms an integral component of this DNA-binding structure. Expression of AP-2 in mammalian cells demonstrates that transcriptional activation requires an additional amino-terminal domain that contains an unusually high concentration of proline residues. This proline-rich activation domain also functions when attached to the heterologous DNA-binding region of the GAL4 protein. This study reveals that although AP-2 shares an underlying modular organization with other transcription factors, the regions of AP-2 involved in transcriptional activation and DNA binding/dimerization have novel sequence characteristics.