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M Meisterernst

Publications and source records attributed to M Meisterernst.

At least 19 recordsLinked to original sources
PubMed2001

Crystal structure of negative cofactor 2 recognizing the TBP-DNA transcription complex.

The X-ray structure of a ternary complex of Negative Cofactor 2 (NC2), the TATA box binding protein (TBP), and DNA has been determined at 2.6 A resolution. The N termini of NC2 alpha and beta resemble histones H2A and H2B, respectively, and form a heterodimer that binds to the bent DNA double helix on the underside of the preformed TBP-DNA complex via electrostatic interactions. NC2beta contributes to inhibition of TATA-dependent transcription through interactions of its C-terminal alpha helix with a conserved hydrophobic feature on the upper surface of TBP, which in turn positions the penultimate alpha helix of NC2beta to block recognition of the TBP-DNA complex by transcription factor IIB. Further regulatory implications of the NC2 heterodimer structure are discussed.

Amino Acid Sequence↗
PubMed2001

Isolation and characterization of a novel gene from the DiGeorge chromosomal region that encodes for a mediator subunit.

Hemizygous deletions on chromosome 22q11.2 result in developmental disorders referred to as DiGeorge syndrome (DGS)/velocardiofacial syndrome (VCFS). We report the isolation of a novel gene, PCQAP (PC2 glutamine/Q-rich-associated protein), that maps to the DiGeorge typically deleted region and encodes a protein identified as a subunit of the large multiprotein complex PC2. PC2 belongs to the family of the human Mediator complexes, which exhibit coactivator function in RNA polymerase II transcription. Furthermore, we cloned the homologous mouse Pcqap cDNA. There is 83% amino acid identity between the human and the mouse predicted protein sequences, with 96% similarity at the amino- and carboxy-terminal ends. To assess the potential involvement of PCQAP in DGS/VCFS, its developmental expression pattern was analyzed. In situ hybridization of mouse embryos at different developmental stages revealed that Pcqap is ubiquitously expressed. However, higher expression was detected in the frontonasal region, pharyngeal arches, and limb buds. Moreover, analysis of subjects carrying a typical 22q11 deletion revealed that the human PCQAP gene was deleted in all patients. Many of the structures affected in DGS/VCFS evolve from Pcqap-expressing cells. Together with the observed haploinsufficiency of PCQAP in DGS/VCFS patients, this finding is consistent with a possible role for this novel Mediator subunit in the development of some of the structures affected in DGS/VCFS.

Amino Acid Sequence↗
PubMed2001

The chromatin structure of the dual c-myc promoter P1/P2 is regulated by separate elements.

The proto-oncogene c-myc is transcribed from a dual promoter P1/P2, with transcription initiation sites 160 base pairs apart. Here we have studied the transcriptional activation of both promoters on chromatin templates. c-myc chromatin was reconstituted on stably transfected, episomal, Epstein-Barr virus-derived vectors in a B cell line. Episomal P1 and P2 promoters showed only basal activity but were strongly inducible by histone deacetylase inhibitors. The effect of promoter mutations on c-myc activity, chromatin structure, and E2F binding was studied. The ME1a1 binding site between P1 and P2 was required for the maintenance of an open chromatin configuration of the dual c-myc promoters. Mutation of this site strongly reduced the sensitivity of the core promoter region of P1/P2 to micrococcal nuclease and prevented binding of polymerase II (pol II) at the P2 promoter. In contrast, mutation of the P2 TATA box also abolished binding of pol II at the P2 promoter but did not affect the chromatin structure of the P1/P2 core promoter region. The E2F binding site adjacent to ME1a1 is required for repression of the P2 promoter but not the P1 promoter, likely by recruitment of histone deacetylase activity. Chromatin precipitation experiments with E2F-specific antibodies revealed binding of E2F-1, E2F-2, and E2F-4 to the E2F site of the c-myc promoter in vivo if the E2F site was intact. Taken together, the analyses support a model with a functional hierarchy for regulatory elements in the c-myc promoter region; binding of proteins to the ME1a1 site provides a nucleosome-free region of chromatin near the P2 start site, binding of E2F results in transcriptional repression without affecting polymerase recruitment, and the TATA box is required for polymerase recruitment.

Base Sequence↗
PubMed2001

Novel critical role of a human Mediator complex for basal RNA polymerase II transcription.

Human Mediator complexes have been described as important bridging factors that enhance the effect of activators in purified systems and in chromatin. Here we report a novel basal function of a human Mediator complex. A monoclonal antibody was generated that depleted the majority of Mediator components from crude cell extracts. The removal of human Mediator abolished transcription by RNA polymerase II. This was observed on all genes tested, on TATA-containing and TATA-less promoters, both in the presence and absence of activators. To identify the relevant complex a combined biochemical and immunopurification protocol was applied. Two variants termed Mediator and basal Mediator were functionally and structurally distinguished. Basal Mediator function relies on additional constraints, which is reflected in the observation that it is essential in crude but not in purified systems. We conclude that basal Mediator is a novel general transcription factor of RNA polymerase II.

Antibodies, Monoclonal↗
PubMed2000

Functional interaction between the HIV transactivator Tat and the transcriptional coactivator PC4 in T cells.

The human immunodeficiency virus (HIV) transactivator Tat is a potent activator of transcription from the HIV long terminal repeat and is essential for efficient viral gene expression and replication. Tat has been shown to interact with components of the basal transcription machinery and transcriptional activators. Here we identify the cellular coactivator PC4 as a Tat-interacting protein using the yeast two-hybrid system and confirmed this interaction both in vitro and in vivo by coimmunoprecipitation. We found that this interaction has a functional outcome in that PC4 overexpression enhanced activation of the HIV long terminal repeat in transient transfection studies in a Tat-dependent manner. The domains of PC4 and Tat required for the interaction were mapped. In vitro binding studies showed that the basic transactivation-responsive binding domain of Tat is required for the interaction with PC4. The minimum region of PC4 required for Tat binding was amino acids 22-91, whereas mutation of the lysine-rich domain between amino acids 22 and 43 prevented interaction with Tat. Tat-PC4 interactions may be controlled by phosphorylation, because phosphorylation of PC4 by casein kinase II inhibited interactions with Tat both in vivo and in vitro. We propose that PC4 may be involved in linking Tat to the basal transcription machinery.

Amino Acid Sequence↗
PubMed2000

A single point mutation in TFIIA suppresses NC2 requirement in vivo.

Negative cofactor 2 (NC2) is a dimeric histone-fold complex that represses RNA polymerase II transcription through binding to TATA-box-binding protein (TBP) and inhibition of the general transcription factors TFIIA and TFIIB. Here we study molecular mechanisms of repression by human NC2 in vivo in yeast. Yeast NC2 genes are essential and can be exchanged with human NC2. The physiologically relevant regions of NC2 have been determined and shown to match the histone-fold dimerization motif. A suppressor screen based upon limiting concentrations of NC2beta yielded a cold-sensitive mutant in the yeast TFIIA subunit Toa1. The single point mutation in Toa1 alleviates the requirement for both subunits of NC2. Biochemical characterization indicated that mutant (mt)-Toa1 dimerizes well with Toa2; it supports specific recognition of the TATA box by TBP but forms less stable TBP-TFIIA-DNA complexes. Wild-type but not the mt-Toa1 can relieve NC2 effects in purified transcription systems. These data provide evidence for a dimeric NC2 complex that is in an equilibrium with TFIIA after the initial binding of TBP to promoter TATA boxes.

Base Sequence↗
PubMed2000

The NC2 repressor is dispensable in yeast mutated for the Sin4p component of the holoenzyme and plays roles similar to Mot1p in vivo.

NC2 (Dr1/DRAP1) and Mot1p are global repressors of transcription that have been isolated in both Saccharomyces cerevisiae and humans. NC2 is a dimeric histone-fold complex that represses RNA polymerase II transcription through binding to TBP and inhibition of TFIIA and TFIIB. Mot1p is an ATPase that removes DNA-bound TBP upon ATP hydrolysis. In this work, we studied the core promoter specificity of NC2 in vivo using a strain that carries mutated NC2beta activity. We show that NC2, like Mot1p, is required for transcription of the HIS3 and HIS4 TATA-less core promoters. Furthermore, whereas neither Mot1p nor NC2 appear to function as repressors of the HIS3 gene in cells growing exponentially in glucose, we find that both are required for repression of the HIS3 TATA promoter when cells go through the diauxic shift. Thus, the activity of these factors is similarly regulated depending upon the physiological conditions, and it appears that core promoters activated or repressed by them in vivo might be distinguishable by whether or not they contain a canonical TATA sequence. Finally, although NC2 is an essential factor for yeast viability, we isolated a mutation in a non-essential component of the holoenzyme, Sin4p, that bypasses the requirement for NC2.

Adenosine Triphosphatases↗
PubMed2000

Mechanisms of transcriptional activation of cAMP-responsive element-binding protein CREB.

The CREB-CREM transcription factors are the main gene regulatory effectors of the cAMP signaling pathway. The investigations of this family of transcription factors had a profound impact on the understanding of signaling-induced gene transcription. Here we discuss some key aspects of the underlying biology, review transcriptional activation by CREB proteins through transcription cofactors and present novel insights into the context- and position-specific function of CREB on complex genes.

Activating Transcription Factor 1↗
PubMed1999

Conserved cAMP responsive element and core promoter complex are critical for specificity of the distal T-cell receptor beta chain enhancer for its native promoter.

The Vbeta 8.1 promoter is regulated by T-cell-specific and ubiquitous transcription factors, which bind immediately upstream of and inside the core promoter region. The various Vbeta promoters contain two conserved elements, a cAMP responsive element (CRE) located upstream of the core promoter and a basal initiator flanked by two regulatory motifs. Here we have studied the interplay between the distal enhancer and its native promoter. We show that the remote enhancer acts specifically through its native promoter. Specific enhancer-promoter interplay is mediated through the conserved regions of the Vbeta promoters. Importantly, the conserved CRE serves as a functional recognition element for the enhancer whereas it barely contributes to promoter activity. The other conserved regions surrounding the initiation site are critical for activators that bind at and function through the core promoter region and thereby regulate both promoter and enhancer activity. The enhancer is highly sensitive to E1A-12S, which represses both general and specific enhancer activities. Enhancer activity and promoter-enhancer specificity is, at least in part, mediated by the coactivators CBP/p300.

Adenovirus E1A Proteins↗
PubMed1998

Interaction of PC4 with melted DNA inhibits transcription.

PC4 is a nuclear DNA-binding protein that stimulates activator-dependent class II gene transcription in vitro. Recent biochemical and X-ray analyses have revealed a unique structure within the C-terminal domain of PC4 that binds tightly to unpaired double-stranded (ds)DNA. The cellular function of this evolutionarily conserved dimeric DNA-binding fold is unknown. Here we demonstrate that PC4 represses transcription through this motif. Interaction with melted promoters is not required for activator-dependent transcription in vitro. The inhibitory activity is attenuated on bona fide promoters by (i) transcription factor TFIIH and (ii) phosphorylation of PC4. PC4 remains a potent inhibitor of transcription in regions containing unpaired ds DNA, in single-stranded DNA that can fold into two antiparallel strands, and on DNA ends. Our observations are consistent with a novel inhibitory function of PC4.

Amino Acid Sequence↗
PubMed1998

The acetyltransferase activity of CBP stimulates transcription.

The CBP co-activator protein possesses an intrinsic acetyltransferase (AT) activity capable of acetylating nucleosomal histones, as well as other proteins such as the transcription factors TFIIE and TFIIF. In addition, CBP associates with two other TSs, P/CAF and SRC1. We set out to establish whether the intrinsic AT activity of CBP contributes to transcriptional activation. We show that a region of CBP, encompassing the previously defined histone AT (HAT) domain, can stimulate transcription when tethered to a promoter. The stimulatory effect of this activation domain shows some promoter preference and is dependent on AT activity. Analysis of 14 point mutations reveals a direct correlation between CBP's ability to acetylate histones in vitro and to activate transcription in vivo. We also find that the HAT domains of CBP and P/CAF share sequence similarity. Four conserved motifs are identified, three of which are analogous to motifs A, B and D, found in other N-acetyltransferases. The fourth motif, termed E, is unique to CBP and P/CAF. Mutagenesis shows that all four motifs in CBP contribute to its HAT activity in vitro and its ability to activate transcription in vivo. These results demonstrate that the AT activity of CBP is directly involved in stimulating gene transcription. The identification of specific HAT domain motifs, conserved between CBP and P/CAF, should facilitate the identification of other members of this AT family.

Acetyltransferases↗
PubMed1998

High-affinity DNA binding by the C-terminal domain of the transcriptional coactivator PC4 requires simultaneous interaction with two opposing unpaired strands and results in helix destabilization.

The general transcriptional cofactor PC4 enhances transcription from various promoters and functions with a wide range of transcriptional activators. Earlier studies have suggested that this enhancement originates mostly from stabilization of the TATA-box/TFIID/TFIIA complex by simultaneous interaction of PC4 with transactivation domains of upstream-binding factors and the basal factor TFIIA. However, the C-terminal half of the protein also has been shown to exhibit substantial ssDNA binding properties, to which as yet no clear function has been assigned. We have investigated the interaction of this domain with various DNA structures and report that high-affinity binding, characterized by an equilibrium dissociation constant in the nanomolar range, requires either a heteroduplex containing a minimum of about eight mismatches, or alternatively a single-stranded DNA molecule consisting of 16 to 20 nucleotides. Furthermore, both juxtaposed single strands of a heteroduplex are protected by the C-terminal domain of PC4 in DNase I footprinting experiments, whereas the double-stranded regions do not appear to be contacted. We conclude from these observations that the role of PC4 ssDNA binding is likely to involve simultaneous interaction with opposing strands in internally melted duplexes, or the induction of a pronounced distortion in the local structure of ssDNA that results in a similar juxtaposed arrangement of single strands. In addition, we have observed that both the PC4 C-terminal domain and the intact PC4 destabilize dsDNA and we discuss the possible involvement of PC4 in promoter opening and other strand displacement events.

Binding Sites↗
PubMed1998

Involvement of negative cofactor NC2 in active repression by zinc finger-homeodomain transcription factor AREB6.

The transcription factor AREB6 contains a homeodomain flanked by two clusters of Krüppel type C2H2 zinc fingers. AREB6 binds to the E-box consensus sequence, CACCTGT, through either the N- or the C-terminal zinc finger cluster. To gain insights into the molecular mechanism by which AREB6 activates and represses gene expression, we analyzed the domain structure of AREB6 in the context of a heterologous DNA-binding domain by transient-transfection assays. The C-terminal region spanning amino acids 1011 to 1124 was identified as a conventional acidic activation domain. The region containing amino acids 754 to 901, which was identified as a repression domain, consists of 40% hydrophobic amino acids displaying no sequence similarities to other known repression domains. This region repressed transcription in vitro in a HeLa nuclear extract but not in reconstituted transcription systems consisting of transcription factor IID (TFIID), TFIIB, TFIIE, TFIIH/F, and RNA polymerase II. The addition of recombinant negative cofactor NC2 (NC2alpha/DRAP1 and NC2beta/Dr1) to the reconstituted transcription system restored the activity of the AREB6 repression domain. We further demonstrated interactions between the AREB6 repression domain and NC2alpha in yeast two-hybrid assay. Our findings suggest a mechanism of transcriptional repression that is mediated by the general cofactor NC2.

Animals↗
PubMed1997

Poly(ADP-ribose) polymerase enhances activator-dependent transcription in vitro.

Mammalian cells contain activities that amplify the effects of activators on class II gene transcription in vitro. The molecular identity of several of these cofactor activities is still unknown. Here we identify poly(ADP-ribose) polymerase (PARP) as one functional component of the positive cofactor 1 activity. PARP enhances transcription by acting during preinitiation complex formation, but at a step after binding of transcription factor IID. This transcriptional activation requires the amino-terminal DNA-binding domain, but not the carboxyl-terminal catalytic region. In purified systems, coactivator function requires a large molar excess of PARP over the number of templates, as reported for other DNA-binding cofactors such as topoisomerase I. PARP effects on supercoiled templates are DNA concentration-dependent and do not depend on damaged DNA. The PARP coactivator function is suppressed by NAD+, probably as a result of auto-ADP-ribosylation. These observations provide another example of the potentiation of trancription by certain DNA-binding cofactors and may point to interactions of PARP with RNA polymerase II-associated factors in special situations.

Animals↗
PubMed1997

Requirement of cofactors for RXR/RAR-mediated transcriptional activation in vitro.

Using crude in vitro systems, we have previously shown that RXR/RAR heterodimers are able to activate transcription from the RAR beta 2 promoter in a retinoid-dependent manner. Here we demonstrate that cofactors distinct from general transcription factors or receptors are required to mediate retinoic acid-dependent transcription in vitro.

Cell Extracts↗
PubMed1997

Specific repression of Tax trans-activation by TAR RNA-binding protein TRBP.

The human T-cell leukemia virus type 1 (HTLV-1)-encoded Tax protein activates transcription from the long terminal repetition via association with host cellular factors. In this study, we searched for cellular proteins that interact with Tax and modulate its activity by using the yeast two-hybrid system. One of the strongest interactors was found to be identical with TRBP, which was previously shown to bind to the RNA encoded by the Tat response element of human immunodeficiency virus type 1. Interactions are demonstrated with Escherichia coli-expressed proteins in vitro and in mammalian cells, using one- and two-hybrid systems, and with antibodies that coprecipitate Tax and TRBP at physiological TRBP concentrations. Moreover, TRBP, when directed into the cytoplasm, is capable of preventing transport of Tax into the nucleus. A 60-amino-acid polypeptide suffices for binding to Tax. TRBP inhibits activation of transcription by both Tax and GAL4-Tax fusion proteins. Inhibition is specific for Tax and is not seen with the other activators tested. Our data are consistent with the interpretation that TRBP inhibits the interplay of Tax with the transcription machinery or accessory factors.

Binding Sites↗
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↗