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M Meisterernst

Publications and source records attributed to M Meisterernst.

At least 37 records · Page 2Linked to original sources
PubMed1997

A conserved tissue-specific structure at a human T-cell receptor beta-chain core promoter.

The T-cell receptor (TCR) beta-chain promoters have been characterized as nonstructured basal promoters that carry a single conserved ubiquitous cyclic AMP-responsive element. Our investigation of the human TCR beta gene uncovers a surprisingly complex and tissue-specific structure at the TCR Vbeta 8.1 promoter. The core of the promoter (positions -42 to +11) is recognized by the lymphoid cell-specific transcription factors Ets-1, LEF1, and AML1 as well as by CREB/ATF-1, as is demonstrated in gel shift and footprinting experiments. With the exception of LEF1, these factors activate transcription in T cells. Binding sites at the core region show little conservation with consensus sites. Nonetheless, CREB, Ets-1, and AML1 bind and activate cooperatively and very efficiently through the nonconsensus binding sites at the core promoter region. Moderate ubiquitous activation is further induced by CREB/ATF and Sp1 factors through proximal upstream elements. The tissue-specific core promoter structure is apparently conserved in other T-cell-specifically expressed genes such as the CD4 gene. Our observations suggest that both the enhancer and the promoter have a complex tissue-specific structure whose functional interplay potentiates T-cell-specific transcription.

Base Sequence↗
PubMed1996

Characterization of the basal inhibitor of class II transcription NC2 from Saccharomyces cerevisiae.

Human NC2 utilizes a unique mechanism of repression of transcription by associating with TBP and inhibition of preinitiation complex formation. Here we have cloned two genes from Saccharomyces cerevisiae and functionally characterized them as yeast NC2. We show that yeast NC2 binds to TBP as a heterodimer and represses RNA polymerase II transcription during assembly of the preinitiation complex. Yeast NC2 is highly homologous to its human counterpart within histone fold domains. C-Terminal regions previously discussed to be important for repression in man are in part not conserved. The human alpha but not the beta subunit efficiently heterodimerizes and represses transcription in combination with the corresponding yeast subunit. Yeast and human NC2 inhibit transcription in the presence of yeast and human TBP. However, repression is optimal within one species. The N-terminus of human TBP supports repression of transcription by human but not by yeast NC2.

Base Sequence↗
PubMed1996

A mechanism for repression of class II gene transcription through specific binding of NC2 to TBP-promoter complexes via heterodimeric histone fold domains.

Negative co-factor 2 (NC2) regulates transcription of the class II genes through binding to TFIID and inhibition of pre-initiation complex formation. We have isolated and cloned NC2, and investigated the molecular mechanism underlying repression of transcription. NC2 consists of two subunits, termed NC2alpha and NC2beta, the latter of which is identical to Dr1. The NC2 subunits dimerize and bind to TATA binding protein (TBP)-promoter complexes via histone fold domains of the H2A-H2B type. Repression of basal transcription requires the histone fold and carboxy-terminal domains of the NC2 subunits. Several mechanisms probably contribute to transcriptional repression. Binding of NC2 inhibits association of TFIIB with TBP-promoter complexes. NC2 binds directly to DNA, and binding of NC2 to TBP-promoter complexes affects the conformation of DNA, which could be one cause for the inhibition of TFIIB. In addition, multimerization of repressor-TBP complexes on DNA might inhibit the assembly of the pre-initiation complex. We suggest that binding of the repressor to TRP-promoter complexes establishes a mechanism that controls the rate of transcription by RNA polymerase II.

Amino Acid Sequence↗
PubMed1996

The human general co-factors.

The human general co-factors were discovered during biochemical fractionation of mammalian nuclear extracts in functional in vitro assays. They appear to act in concert with other co-activators that bind tightly to the TATA-binding protein and RNA polymerase II. Several co-factors have been shown to interact with general transcription factors, leading either to activation or repression of transcription. At least one subgroup of co-factors that enhance the effects of activators on transcription are DNA-binding proteins located in the chromatin. In fact, one co-factor, the repressor NC2, is structurally related to histones. The understanding of the molecular interplay of such components of the initiation complex in the chromatin-including general co-factors, other co-factors, general factors and activators-will be a major challenge in the future.

Animals↗
PubMed1995

Activation of transcription by recombinant upstream stimulatory factor 1 is mediated by a novel positive cofactor.

The transcription factor USF1 belongs to the family of basic helix-loop-helix proteins that are involved in the regulation of various important cellular processes. Here we characterized the factors involved in the activation of transcription by upstream stimulatory factor 1 (USF1) in a reconstituted class II gene transcription system. Activation of transcription by both wild type USF1 and a GAL-USF (amino acids 1-94 of the yeast activator protein GAL4 fused to amino acids 17-196 of USF) fusion protein required the presence of at least one positive cofactor. A novel positive cofactor (PC5) that functions specifically through the activation domain of USF1 was partially purified and biochemicaly distinguished from previously described positive cofactors. The mechanism by which PC5 mediates activation of transcription through USF1 was investigated in order-of-addition experiments. PC5 had to be present during binding of transcription factor (TF) IID to the TATA box to observe transcriptional activation. However, this event alone did not result in transcriptional activation, which also required the presence of the activator and of PC5 after binding of TFIID. Hence, PC5 may enter transcription during binding of TFIID to function in concert with the activator during subsequent steps in transcription.

DNA-Binding Proteins↗
PubMed1995

The coactivator p15 (PC4) initiates transcriptional activation during TFIIA-TFIID-promoter complex formation.

We have analyzed the mechanisms underlying stimulation of transcription by the activator GAL4-AH and the recombinant coactivator p15 (PC4). We show that p15 binds to both double-stranded and single-stranded DNA. Analyses of deletion mutants correlates binding to double-stranded DNA with the ability to mediate activator-dependent transcription. Consistent with this finding, phosphorylation of p15 by casein kinase II inhibits binding to double-stranded DNA and the activity of p15. The functional characterization suggests interactions of p15 with both DNA and components of the TFIID complex. GAL4-AH functions in concert with p15 during formation of TFIIA-TFIID-promoter (DA) complexes, as concluded from order-of-addition experiments. At limiting TFIID concentrations, the number of DA complexes is enhanced. The activator also stimulates transcription moderately after DA complex formation, independently of the concentrations of general transcription factors.

Amino Acid Sequence↗
PubMed1995

Interaction of the COOH-terminal transactivation domain of p65 NF-kappa B with TATA-binding protein, transcription factor IIB, and coactivators.

We show that the transactivating COOH terminus of the p65 subunit of human transcription factor NF-kappa B directly binds the general transcription factors TFIIB and TATA-binding protein (TBP) in vitro. Interaction of p65 with TFIIB required the most COOH-terminal sequence repeat within TFIIB. A functional interaction of TFIIB with p65 was evident from assays in yeast cells. Cotransfection experiments in COS cells revealed that only overexpression of TBP was able to further stimulate p65-dependent transactivation of a reporter gene. The coexpression of neither TBP nor TFIIB was able to relieve squelching, indicating the involvement of additional factors in transactivation by p65. A cell-free assay using highly purified factors revealed a specific transcriptional stimulation through the COOH-terminal activation domain of NF-kappa B by at least one cofactor, PC1, isolated from HeLa cells. These data show that the potent acidic transactivation domains in the COOH terminus of p65 are able to functionally recruit various components of the basic transcription machinery as well as coactivators.

Animals↗
PubMed1994

A novel mediator of class II gene transcription with homology to viral immediate-early transcriptional regulators.

Our investigations of mammalian class II gene transcription resulted in identification, purification, and cloning of the corresponding cDNA of a cellular factor (p15) that mediates the effects of several distinct activators on transcription in vitro. Functional deletion analyses revealed a bipartite structure of p15 comprising an amino-terminal regulatory domain and a carboxy-terminal cryptic DNA-binding domain. We provide evidence that activity of p15 is controlled by protein kinases that target the regulatory domain. Structural and functional similarities, including sequence homology to domains essential for cofactor function, cofactor activity, promiscuity with respect to transcriptional activators, and interactions with components of the basal transcription machinery, relate this novel cellular cofactor to viral immediate-early transcriptional regulators.

Amino Acid Sequence↗
PubMed1994

RNA polymerase II cofactor PC2 facilitates activation of transcription by GAL4-AH in vitro.

We have isolated from a crude Hela cell cofactor fraction (USA) a novel positive cofactor that cooperates with the general transcription machinery to effect efficient stimulation of transcription by GAL4-AH, a derivative of the Saccharomyces cerevisiae regulatory factor GAL4. PC2 was shown to be a 500-kDa protein complex and to be functionally and biochemically distinct from native TFIID and previously identified cofactors. In the presence of native TFIID and other general factors, PC2 was necessary and sufficient for activation by GAL4-AH. Cofactor function was specific for transcriptional activation domains of GAL4-AH. The repressor histone H1 further potentiated but was not required for activation of transcription by GAL4-AH. On the basis of the observation that PC2 exerts entirely positive effects on transcription, we propose a model in which PC2 increases the activity of the preinitiation complex in the presence of an activator, thereby establishing a specific pathway during activation of RNA polymerase II.

Blotting, Western↗
PubMed1994

Repression of basal transcription by HMG2 is counteracted by TFIIH-associated factors in an ATP-dependent process.

A basal repressor of class II gene transcription was identified, purified, and found to be identical to nonhistone chromosomal protein HMG2. HMG2 was shown to inhibit basal transcription under conditions in which transcription templates form soluble complexes with HMG2. Order-of-addition experiments clearly revealed that HMG2 acted after assembly of a TBP-TFIIA-promoter complex and before formation of the fourth phosphodiester bond by RNA polymerase II. Subsequently, an activity that efficiently counteracted repression of transcription by HMG2 in both TBP- and TFIID-containing transcription systems was isolated. Several lines of evidence suggested that antirepression was mediated by a TFIIH-associated factor. The antirepressor first coeluted with TFIIH, was depleted from this fraction by antibodies directed against the TFIIH subunit p62, was dependent on either ATP or dATP, and then was inhibited by the ATP analogs AMP-PNP and ATP gamma S. Relief of HMG2-mediated repression as well as basal promoter function of TFIIH may involve a helicase that coelutes with TFIIH and displays similar nucleotide specificities. Taken together, these data suggest novel consequences of chromatin-associated HMG proteins and they provide direct evidence for a role of TFIIH-associated enzymes in ATP-dependent antirepression of nonhistone chromosomal proteins.

Adenosine Triphosphate↗
PubMed1993

Identification of human DNA topoisomerase I as a cofactor for activator-dependent transcription by RNA polymerase II.

The transcriptional activation of eukaryotic class II genes by sequence-specific regulatory proteins requires cofactors in addition to the general transcription factors. One cofactor (termed PC3) was purified from HeLa cells and identified by sequence analysis and functional assays as human DNA topoisomerase I (EC5.99.1.2). Under identical conditions PC3 mediates both a net activation of transcription by the acidic activator GAL4-AH and repression of basal transcription, thereby leading to a large induction of transcription by the activator. PC3-mediated activation of transcription is dependent on the presence of both the GAL4-AH activation domain and the TATA-binding protein (TBP)-associated-factors (TAFs) in natural transcription factor TFIID, while repression of basal transcription is observed with either TFIID or the derived TBP alone. These results suggest novel functions, apparently through distinct mechanisms, for human DNA topoisomerase I in the regulation of transcription initiation by RNA polymerase II.

Amino Acid Sequence↗
PubMed1993

An alternative pathway for transcription initiation involving TFII-I.

The minimal promoter elements required for initiation by RNA polymerase II include the TATA box and/or an initiator element (Inr) at or near the transcription start site. Studies of the adenovirus major late core promoter (containing both elements) have demonstrated an initiation pathway that involves binding of the transcription factor TFIID (or the derived subunit, the TATA-binding protein TBP (TFIID tau)) to the TATA element, which is facilitated by transcription factor TFIIA, followed by sequential interactions of other general factors. Here we describe a novel pathway that requires an intact Inr and the Inr-binding factor TFII-I (ref. 3). Sequential addition of the general factors generated TFII-I-dependent preinitiation complexes different from those formed with TFIIA. Furthermore, TBP bound cooperatively (with only TFII-I) to an Inr-containing TATA-less promoter, suggesting a means for activation of TATA-less promoters, which nonetheless require TFIID (refs 9-11). These observations provide support for functionally distinct pathways which could be subject to differential regulation by specific activators or repressors.

Adenoviridae↗
PubMed1992

Transcriptional regulation of the HIV-1 promoter by NF-kappa B in vitro.

NF-kappa B, purified from HeLa cell cytosol, and a recombinant p50 subunit of NF-kappa B alone (expressed in and purified from bacteria) both stimulated transcription from the HIV-1 promoter in vitro (at least up to 15-fold). A deletion analysis of the p50 subunit revealed that transcriptional activation was mediated by the conserved c-rel-related domain. I kappa B-beta (or a related protein), which binds to the p65 but not the p50 subunit of NF-kappa B, inhibited stimulation by natural NF-kappa B but not by recombinant p50. Experiments employing a purified transcription system revealed that efficient induction of transcription by both natural NF-kappa B or recombinant p50 required a cofactor fraction in addition to the general initiation factors. Combined with DNA-binding experiments, these studies suggest a role of p50 homodimers in transcriptional activation of certain promoters, with a possible preference for those carrying symmetric NF-kappa B recognition sites, and a potential role of I kappa B-beta in direct transcriptional regulation within the nucleus.

Base Sequence↗
PubMed1991

Cooperative interaction of an initiator-binding transcription initiation factor and the helix-loop-helix activator USF.

Transcription initiation by mammalian RNA polymerase II is effected by multiple common factors interacting through minimal promoter elements and regulated by gene-specific factors interacting with distal control elements. Minimal promoter elements that can function independently or together, depending on the specific promoter, include the upstream TATA box and a pyrimidine-rich initiator (Inr) overlapping the transcription start site. The binding of TFIID to the TATA element promotes the assembly of other factors into a preinitiation complex but factors which function at the Inr have not been defined. We show here that a novel factor (TFII-I) binds specifically to Inr elements, supports basal transcription from the adenovirus major late promoter and is immunologically related to the helix-loop-helix activator USF. We further show that TFII-I also binds to the upstream high-affinity USF site (E box), that USF also binds to the Inr, and that TFII-I and USF interact cooperatively at both Inr and E box sites. Thus, TFII-I represents a novel type of transcription initiation factor whose interactions at multiple promoter elements may aid novel communication mechanisms between upstream regulatory factors and the general transcriptional machinery.

Animals↗
PubMed1991

Family of proteins that interact with TFIID and regulate promoter activity.

A family of proteins was shown to bind cooperatively with TFIID to core promoters, as previously demonstrated for the general initiation factor TFIIA. These factors form distinct complexes with TFIID, fail to bind DNA in the absence of TFIID, differ chromatographically from TFIIA, and compete with TFIIA for binding to TFIID. Our results suggest the formation of heterogeneous preinitiation complexes at the step involving TFIIA interactions. This establishes a molecular switch that regulates basal level transcription in vitro and has consequences for transcriptional activation by gene-specific activators.

DNA-Binding Proteins↗
PubMed1991

Activation of class II gene transcription by regulatory factors is potentiated by a novel activity.

A novel activity (USA) stimulated activator-dependent transcription in a reconstituted system in conjunction with natural TFIID, resulting in 10- to 50-fold levels of induction by regulatory factors. USA mediated a modest induction by USF in conjunction with either recombinant human TFIID, intact yeast TFIID, or the evolutionarily conserved C-terminal portion of yeast TFIID. Upon further purification, USA was resolved into two components that had opposite effects on core promoter activity and that in combination potentiated activator function. Gel mobility shift experiments indicated physical interactions between the inhibitory activity and TFIID, suggesting that the additional components (cofactors) associate with the preinitiation complex both to reduce promoter activity in the absence and to increase promoter activity in the presence of transcriptional activators.

Adenoviruses, Human↗
PubMed1990

Recombinant yeast TFIID, a general transcription factor, mediates activation by the gene-specific factor USF in a chromatin assembly assay.

The TATA box-binding transcription factor TFIID from Saccharomyces cerevisiae was tested for its ability to mediate regulatory factor functions both in a cell-free system reconstituted with other general initiation factors (purified from HeLa cells) and in a combined nucleosome assembly-transcription system. In the latter assay recombinant yeast TFIID, expressed in and purified from bacteria, was sufficient to prevent nucleosome assembly-mediated repression and to mediate transcriptional enhancement of the adenovirus major late promoter by the gene-specific activator USF. In contrast, recombinant yeast TFIID was unable to mediate activation by USF in the system reconstituted only with purified general factors. Under the same conditions a partially purified natural yeast TFIID was able to mediate activation by both USF and Sp1 (assayed with the human immunodeficiency virus promoter), but to a lesser extent than observed with a partially purified natural human TFIID. The implications of these findings are discussed with respect to the structure of the yeast and human TATA factors and the possible involvement either of specific TFIID modifications or of coactivators.

Animals↗