Michael Meisterernst

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 CBPs 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.

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.

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.

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.

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.

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.

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.

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.

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.

C-terminal domain of transcription cofactor PC4 reveals dimeric ssDNA binding site.

The crystal structure of human replication and transcription cofactor PC4CTD reveals a dimer with two single-stranded (ss)DNA binding channels running in opposite directions to each other. This arrangement suggests a role in establishment or maintenance of melted DMA at promoters or origins of replication.