Assembly Of General Transcription Factors Research Articles

Transcriptional response to stress is pre-wired by promoter and enhancer architecture

Programs of gene expression are executed by a battery of transcription factors that coordinate divergent transcription from a pair of tightly linked core initiation regions of promoters and enhancers. Here, to investigate how divergent transcription is reprogrammed upon stress, we measured nascent RNA synthesis at nucleotide-resolution, and profiled histone H4 acetylation in human cells. Our results globally show that the release of promoter-proximal paused RNA polymerase into elongation functions as a critical switch at which a gene’s response to stress is determined. Highly transcribed and highly inducible genes display strong transcriptional directionality and selective assembly of general transcription factors on the core sense promoter. Heat-induced transcription at enhancers, instead, correlates with prior binding of cell-type, sequence-specific transcription factors. Activated Heat Shock Factor 1 (HSF1) binds to transcription-primed promoters and enhancers, and CTCF-occupied, non-transcribed chromatin. These results reveal chromatin architectural features that orient transcription at divergent regulatory elements and prime transcriptional responses genome-wide.

Understanding the Mechanisms of Human RNA Polymerase II Transcription using Single Molecule Fluorescence Colocalization

Transcription is the first step in cellular gene expression and a key regulatory point for determining how a cell responds to stimuli. In mammalian cells, transcription is initiated by the formation of a pre-initiation complex (PIC), an assembly of general transcription factors (GTFs) and RNA polymerase II (Pol II), on promoter DNA. Our goal is to use single molecule approaches to address mechanisms of PIC formation and activity that have previously been unattainable using traditional biochemical approaches. We have developed a novel single molecule total internal reflection fluorescence (TIRF) microscopy assay that allows us to visualize human Pol II transcriptional activity on individual DNA templates. With this single molecule setup, we have performed experiments to address the pathways by which active pre-initiation complexes can form. Ultimately we found that Pol II and three GTFs can form a quaternary complex in the absence of promoter DNA, indicating that a stable network of interactions exists between these proteins independent of promoter DNA.We are also developing a single molecule system to monitor dynamic protein-protein interactions that facilitate activation of transcription. It has long been known that transcriptional activators interact with subunits of transcription factor IID (TFIID) to potentiate high levels of transcription, however, the biophysical parameters defining these interactions are almost entirely uncharacterized. We have developed a novel method to label transcriptional activators and monitor their interactions with transcription factor TFIID in real-time using single molecule fluorescence colocalization. We observe that the activation domains from different activators exhibit different kinetic parameters for interactions with TFIID. We are positioned to address previously unanswered questions regarding how important protein-protein interactions between TFIID and transcriptional activators impact gene expression.

Structural visualization of key steps in human transcription initiation.

Eukaryotic transcription initiation requires the assembly of general transcription factors into a pre-initiation complex that ensures the accurate loading of RNA polymerase II at the transcription start site. The molecular mechanism and function of this assembly have remained elusive due to lack of structural information. We have used an in vitro reconstituted system to study the stepwise assembly of human TBP, TFIIA, TFIIB, Pol II, TFIIF, TFIIE, and TFIIH onto promoter DNA using cryo-electron microscopy. Our structural analyses provide pseudo-atomic models at various stages of transcription initiation that illuminate critical molecular interactions, including how TFIIF engages Pol II and promoter DNA to stabilize both the closed PIC and the open-promoter complex and regulate start site selection. Comparison of open versus closed pre-initiation complexes, combined with the localization of the TFIIH helicases XPD and XPB, supports a DNA translocation model of XPB and explains its essential role in promoter opening.

Sgf29p Facilitates the Recruitment of TATA Box Binding Protein but Does Not Alter SAGA’s Global Structural Integrity in Vivo

Although Sgf29p has been biochemically implicated as a component of SAGA (Spt-Ada-Gcn5 acetyltransferase), its precise mechanism of action in transcription is not clearly understood in vivo. Here, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation (ChIP) assay in conjunction with transcriptional and mutational analyses, we show that Sgf29p along with other SAGA components is recruited to the upstream activating sequence (UAS) of a SAGA-regulated gene, GAL1, in an activation domain-dependent manner. However, Sgf29p does not alter the recruitment of Spt20p that maintains the overall structural and functional integrity of SAGA. The recruitment of other SAGA components such as TAF10p, TAF12p, and Ubp8p to the GAL1 UAS is also not altered in the absence of Sgf29p. Interestingly, we find that the recruitment of TBP (TATA box binding protein that nucleates the assembly of general transcription factors to form the preinitiation complex for transcriptional initiation) to the core promoter of GAL1 is weakened in Δsgf29. Likewise, Sgf29p also enhances the recruitment of TBP to other SAGA-regulated promoters. Such weakening of recruitment of TBP to these promoters subsequently decreases the level of transcription. Taken together, these results support the idea that SAGA-associated Sgf29p facilitates the recruitment of TBP (and hence transcription) without altering the global structural integrity of SAGA in vivo.

TBP homologues in embryo transcription: who does what?

One of the crucial events in eukaryotic gene regulation is the assembly of general transcription factors and RNA polymerase II (Pol II) into a pre‐initiation complex (PIC) at promoter regions. The textbook description of mRNA synthesis initiation requires the recruitment and binding of the TATA‐binding protein (TBP)—a component of the TFIID complex—to the TATA box (Butler & Kadonaga, 2002). Several studies are now changing this idea by showing that TBP‐independent Pol II transcription can occur (Paulson et al , 2002; Wieczorek et al , 1998), and that other types of TBP‐related factors (TRFs) can mediate Pol II transcription of development regulator genes in vertebrate embryos in the absence of TBP (Martianov et al , 2002; Muller et al , 2001; Veenstra et al , 2000). In addition to TBP, two other TRFs have been characterized in vertebrates and were suggested to function in Pol II transcription initiation: TBP‐like factor (TLF, also called TRF2, TLP or TBPL1) and TBP2 (also called TRF3 or TBPL2; Bartfai et al , 2004; Dantonel et al , 1999; Jallow et al , 2004; Persengiev et al , 2003, and references therein). Given the diversity of TBP‐related proteins (Fig 1), several important questions arise. Is there any functional specialization for TBP and TBP‐related factors in vivo ? Do the requirements of these factors for transcriptional activation vary according to the groups of genes involved in specific cellular processes? One direct way to address these questions is to investigate the role of these factors during early development, as distinct groups of ‘developmental’ genes can then be analysed. Furthermore, the embryo switches from a genomic ‘transcription off’ to a ‘transcription on’ state, which in Xenopus and zebrafish occurs during the midblastula transition (MBT). Figure 1. Schematic representation and domain comparison of TATA‐binding protein‐related factors. TBP from zebrafish ( Danio rerio ; dr) and …

Analysis of in vivo targets of transcriptional activators by fluorescence resonance energy transfer

Gene expression in eukaryotes is largely controlled at the level of transcriptional initiation by gene-specific activators. Transcriptional activators stimulate the assembly of general transcription factors on the promoter to form a preinitiation complex (PIC). Such a stimulated assembly of PIC is believed to result from a direct interaction between the activator and one or more components of the transcription machinery, termed the “target”. Based primarily upon in vitro protein–protein interaction experiments, a variety of factors have been proposed to be the direct targets of activators. However, whether any of these are bona fide in vivo targets required for stimulation of PIC assembly and hence transcriptional activation remains mostly unknown, primarily because of lack of appropriate experimental methods. Using a confocal microscopy-based FRET (fluorescence resonance energy transfer) assay, we have recently identified the target of a prototypic activator, Gal4p, in living yeast cells. Here, we describe the FRET assay in general for analysis of the targets of transcriptional activators in living yeast cells. Such an assay can also be used as a general method to monitor protein–protein interactions in vivo.

Core promoter-selective RNA polymerase II transcription

The initiation of mRNA synthesis in eukaryotic cells is a complex and highly regulated process that requires the assembly of general transcription factors and RNAP II (RNA polymerase II; also abbreviated as Pol II) into a pre-initiation complex at the core promoter. The core promoter is defined as the minimal DNA region that is sufficient to direct low levels of activator-independent (basal) transcription by RNAP II in vitro. The core promoter typically extends approx. 40 bp up- and down-stream of the start site of transcription and can contain several distinct core promoter sequence elements. Core promoters in higher eukaryotes are highly diverse in structure, and each core promoter sequence element is only found in a subset of genes. So far, only TATA box and INR (initiator) element have been shown to be capable of directing accurate RNAP II transcription initiation independent of other core promoter elements. Computational analysis of metazoan genomes suggests that the prevalence of the TATA box has been overestimated in the past and that the majority of human genes are TATA-less. While TATA-mediated transcription initiation has been studied in great detail and is very well understood, very little is known about the factors and mechanisms involved in the function of the INR and other core promoter elements. Here we summarize our current understanding of the factors and mechanisms involved in core promoter-selective transcription and discuss possible pathways through which diversity in core promoter architecture might contribute to combinatorial gene regulation in metazoan cells.

Systematic analysis of essential yeast TAFs in genome-wide transcription and preinitiation complex assembly.

The general transcription factor TFIID is composed of the TATA box binding protein (TBP) and a set of conserved TBP-associated factors (TAFs). Here we report the completion of genome-wide expression profiling analyses of yeast strains bearing temperature-sensitive mutations in each of the 13 essential TAFs. The percentage of the yeast genome dependent on each TAF ranges from 3% (TAF2) to 59-61% (TAF9). Approximately 84% of yeast genes are dependent upon one or more TAFs and 16% of yeast genes are TAF independent. In addition, this complete analysis defines three distinct classes of yeast promoters whose transcriptional requirements for TAFs differ substantially. Using this collection of temperature-sensitive mutants, we show that in all cases the transcriptional dependence for a TAF can be explained by a requirement for TBP recruitment and assembly of the preinitiation complex (PIC). Unexpectedly, these assembly experiments reveal that TAF11 and TAF13 appear to provide the critical functional contacts with TBP during PIC assembly. Collectively, our results confirm and extend the proposal that individual TAFs have selective transcriptional roles and distinct functions.

Review of the in Vivo Functions of the p160 Steroid Receptor Coactivator Family

The p160 steroid receptor coactivator (SRC) gene family contains three homologous members, which serve as transcriptional coactivators for nuclear receptors and certain other transcription factors. These coactivators interact with ligand-bound nuclear receptors to recruit histone acetyltransferases and methyltransferases to specific enhancer/promotor regions, which facilitates chromatin remodeling, assembly of general transcription factors, and transcription of target genes. This minireview summarizes our current knowledge about the molecular structures, molecular mechanisms, temporal and spatial expression patterns, and biological functions of the SRC family. In particular, this article highlights the roles of SRC-1 (NCoA-1), SRC-2 (GRIP1, TIF2, or NCoA-2) and SRC-3 (p/CIP, RAC3, ACTR, AIB1, or TRAM-1) in development, organ function, endocrine regulation, and nuclear receptor function, which are defined by characterization of the genetically manipulated animal models. Furthermore, this article also reviews our current understanding of the role of SRC-3 in breast cancer and discusses possible mechanisms for functional specificity and redundancy among SRC family members.

TAF(II)250: a transcription toolbox.

Activation of RNA-polymerase-II-dependent transcription involves conversion of signals provided by gene-specific activator proteins into the synthesis of messenger RNA. This conversion requires dynamic structural changes in chromatin and assembly of general transcription factors (GTFs) and RNA polymerase II at core promoter sequence elements surrounding the transcription start site of genes. One hallmark of transcriptional activation is the interaction of DNA-bound activators with coactivators such as the TATA-box binding protein (TBP)-associated factors (TAF(II)s) within the GTF TFIID. TAF(II)250 possesses a variety of activities that are likely to contribute to the initial steps of RNA polymerase II transcription. TAF(II)250 is a scaffold for assembly of other TAF(II)s and TBP into TFIID, TAF(II)250 binds activators to recruit TFIID to particular promoters, TAF(II)250 regulates binding of TBP to DNA, TAF(II)250 binds core promoter initiator elements, TAF(II)250 binds acetylated lysine residues in core histones, and TAF(II)250 possesses protein kinase, ubiquitin-activating/conjugating and acetylase activities that modify histones and GTFs. We speculate that these activities achieve two goals--(1) they aid in positioning and stabilizing TFIID at particular promoters, and (2) they alter chromatin structure at the promoter to allow assembly of GTFs--and we propose a model for how TAF(II)250 converts activation signals into active transcription.

A role for activator-mediated TFIIB recruitment in diverse aspects of transcriptional regulation

Transcription by RNA polymerase II in eukaryotic cells requires the ordered assembly of general transcription factors on the promoter to form a preinitiation complex. Transcriptional activator proteins (activators) stimulate transcription by increasing the rate and/or extent of preinitiation complex assembly. We have shown previously that acidic activators increase the stable association of TFIIB on the promoter, a process we refer to as 'recruitment'. In this study, we provide evidence that diverse activators facilitate TFIIB assembly by a related mechanism. We then investigate the activator-mediated assembly of TFIIB with regard to two aspects of transcription: the distance-dependence of activator function, and reinitiation. We have previously described amino-acid-substitution mutants of TFIIB that are able to support an activator-independent basal level of transcription but do not respond to acidic activators. We now show that these mutants also do not respond to other classes of activators. We demonstrate that this defect is due to a failure of the activators to recruit the mutant TFIIB to the promoter. Activators often lose activity as their distance from the initiation site is increased. We show that this impaired transcriptional activity correlates with a decrease in TFIIB recruitment. Finally, we find that following the initiation of transcription, TFIIB dissociates from the promoter, requiring the activator-mediated reassembly of TFIIB in the preinitiation complex for each new round of transcription. We have provided evidence that diverse activators recruit TFIIB to the promoter by a related mechanism. This central step in transcriptional activation is sensitive to promoter architecture, and is required for each new round of transcription.

Eukaryotic activators function during multiple steps of preinitiation complex assembly.

Eukaryotic activator proteins (activators) stimulate transcription by increasing assembly of the preinitiation complex. We have developed methods to quantify the stable assembly of general transcription factors into transcriptional complexes in response to activators. We show that activators function during at least two stages of preinitiation complex assembly: first, to recruit the general transcription factor TFIIB, and then at a second step, after TFIIB entry. It is at this second step that the TATA-box binding protein associated factors act. This step also seems to be critical for activators to stimulate transcription synergistically.

ICP4, the major transcriptional regulatory protein of herpes simplex virus type 1, forms a tripartite complex with TATA-binding protein and TFIIB.

The ICP4 protein of herpes simplex virus can either increase or decrease the rate of transcription mediated by RNA polymerase II, depending on the target promoter. The interplay of DNA-protein and protein-protein contacts determining ICP4 function has yet to be characterized, and consequently the molecular mechanism by which the protein acts remains unclear. ICP4 can transactivate minimal promoters containing only TATA homologies, and therefore it is reasonable to hypothesize that ICP4 works by influencing the TATA-dependent assembly of general transcription factors via specific protein-protein interactions. This study directly addresses this hypothesis by determining whether ICP4 affects the assembly of general transcription factors on templates bearing a TATA box and an ICP4-binding site. Using gel retardation and footprinting assays, we found that ICP4 forms a tripartite complex with TFIIB and either the TATA-binding protein (TBP) or TFIID. The formation of this complex was not the result of simple tripartite occupancy of the DNA but the consequence of protein-protein interactions. In the presence of all three proteins, the affinity of ICP4 and TBP for their respective binding sites was substantially increased. Using mutant derivatives of ICP4 and defective versions of promoters, we also demonstrated that the ability of ICP4 to regulate gene expression correlated with its ability to form a tripartite complex with TFIIB and TBP in vitro.

Requirement for acidic amino acid residues immediately N-terminal to the conserved domain of Saccharomyces cerevisiae TFIID.

TFIID binds to TATA boxes and initiates the assembly of general transcription factors and pol II on promoters. TFIID proteins from various species consist of a highly conserved carboxy terminal domain and very divergent amino terminal domains. We investigated the function of the non-conserved amino terminal domain (residues 1-60) of Saccharomyces cerevisiae TFIID (YIID, 240 residues) by testing the ability of a series of YIID amino terminal deletion mutants to complement a YIID deficient yeast strain. Mutants with deletions up to amino acid 48 restored the YIID deficient yeast strain to an apparently wild type phenotype. However, deletion up to position 57 or 60 produced yeast strains which formed extremely small colonies. Moreover, overexpression of YIID delta 2-57 or YIID delta 3-60 protein in the presence of wild type YIID resulted in a dominant-negative inhibition of growth. No difference between the basal transcriptional activity of wild type YIID and these amino terminal deletion mutants was observed in vitro. However, transcriptional activation in vivo of promoter-lacZ fusions showed that the YIID delta 2-57 deletion affects the ability of certain promoters (CUP1 and an HSP UAS-CYC1 promoter hybrid promoter) to respond to upstream factor stimulation. At least one inducible promoter, PHO5, was not affected by this deletion. The defect produced by YIID delta 2-57 was due to the deletion of several acidic residues present between residues 48 and 57. The results show that the conserved carboxy terminal domain of YIID is sufficient for cell viability. However, an acidic region just amino terminal to the conserved domain is required for normal growth and transcription control in most yeast strains.

Mechanism of action of an acidic transcriptional activator in vitro

Transcription of a eukaryotic structural gene by RNA polymerase II requires the ordered assembly of general transcription factors on the promoter to form a preinitiation complex. Here we analyze affinity-purified complexes at various stages of assembly to determine the mechanism of action of an acidic transcriptional activator. We show that the activator can function in the absence of ATP and stimulates transcription by increasing the number of functional preinitiation complexes. The activator effects this increase by recruiting the general transcription factor TFIIB to the promoter. Using protein affinity chromatography we demonstrate a specific interaction between an acidic activating region and TFIIB. Based on these combined results, we propose that TFIIB is a direct target of an acidic activator.