Eukaryotic Transcriptional Regulation Research Articles

A compilation of cellular transcription factor interactions with the HIV-1 LTR promoter.

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) represents a model promoter system and the identification and characterisation of cellular proteins that interact with this region has provided a basic understanding about both general eukaryotic and HIV-1 proviral transcriptional regulation. To date a large number of sequence-specific DNA-protein interactions have been described for the HIV-1 LTR. The aim of this report is to provide a comprehensive, updated listing of these HIV-1 LTR interactions. It is intended as a reference point to facilitate on-going studies characterising the identity of cellular proteins interacting with the HIV-1 LTR and the functional role(s) of specific regions of the LTR for HIV-1 replication.

Insulators and Long-Distance Interactions between Regulatory Elements in Higher Eukaryotes

Enhancers can activate a promoter located as far as several hundred kilobases away, which is specific for transcription regulation in higher eukaryotes. Notwithstanding the vast information accumulated on transcription regulation at various levels, the mechanism of long-distance enhancer–promoter interactions is still obscure. Modern views of insulators as elements modulating enhancer–promoter interactions are reviewed.

Regulation of dorso/ventral patterning in the Drosophila embryo by multiple dorsal-interacting proteins.

The Rel family transcription factor, Dorsal, determines cell fate as a function of position along the dorsoventral axis of the Drosophila embryo. This process depends on interactions between Dorsal and a large number of additional proteins present in the early embryo. Cytoplasmic interactions regulate the nuclear uptake of Dorsal, resulting in the establishment of the Dorsal nuclear concentration gradient, which determines the dorsoventral polarity of the embryo. Nuclear protein-protein interactions then enable Dorsal to activate some target genes and to repress others, thereby promoting the division of the embryo into distinct developmental domains. Because of this broad array of regulatory interactions, Dorsal serves as an excellent paradigm for eukaryotic transcriptional regulation.

Genes regulated cooperatively by one or more transcription factors and their identification in whole eukaryotic genomes.

The question addressed here is how cooperative interactions among transcription factors (TFs), a very frequent phenomenon in eukaryotic transcriptional regulation, can be used to identify genes that are regulated by one or more TFs with known DNA binding specificities. Cooperativity may be homotypic, involving binding of only one transcription factor to multiple sites in a gene's regulatory region. It may also be heterotypic, involving binding of more than one TF. Both types of cooperativity have in common that the binding sites for the respective TFs form tightly linked 'clusters', groups of binding sites often more closely associated than expected by chance alone. A statistical technique suitable for the identification of statistically significant homotypic or heterotypic TF binding site clusters in whole eukaryotic genomes is presented. It can be used to identify genes likely to be regulated by the TFs. Application of the technique is illustrated with two transcription factors involved in the cell cycle and mating control of the yeast Saccharomyces cerevisiae, indicating that the results obtained are biologically meaningful. This rapid and inexpensive computational method of generating hypotheses about gene regulation thus generates information that may be used to guide subsequent costly and laborious experimental approaches, and that may aid in the assignment of biological functions to putative open reading frames.

Chicken Histone Deacetylase-2 Controls the Amount of the IgM H-chain at the Steps of Both Transcription of Its Gene and Alternative Processing of Its Pre-mRNA in the DT40 Cell Line

Histone deacetylases (HDACs) are involved in the deacetylation of core histones, which is an important event in transcription regulation in eukaryotes through alterations in the chromatin structure. We cloned cDNAs and genomic DNAs encoding two chicken HDACs (chHDAC-1 and -2), which are preferentially localized in nuclei. Treatment with trichostatin A reduced the HDAC activities in immunoprecipitates obtained with anti-chHDAC-1 and -2 antisera. Using gene targeting techniques, we generated homozygous DT40 mutants, DeltachHDAC-1 and -2, devoid of two alleles of the chHDAC-1 and -2 genes, respectively. The protein patterns on two-dimensional PAGE definitely changed for DeltachHDAC-2, and the amounts of the IgM H- and L-chains increased in it. Of the two IgM H-chain forms, the secreted form mu(s) increased in DeltachHDAC-2, but the membrane-bound form mu(m) decreased. The IgM H-chain gene was transcribed more in DeltachHDAC-2 than in DT40 cells. In the mutant, the alternative processing of IgM H-chain pre-mRNA preferentially occurred, resulting in an increase in the amount of mu(s) mRNA, whereas the stability of the two types of mRNA, mu(s) and mu(m), was unchanged. In DT40 cells, treatment with trichostatin A increased both the amounts of IgM H-chain mRNAs and the switch from mu(m) to mu(s) mRNAs. Based on these results, we propose a model for a role of chHDAC-2 in both the transcription and alternative processing steps, resulting in control of the amount of the mu(s) IgM H-chain in the DT40 cell line.

Combinatorial transcription factors

Combinatorial regulation of eukaryotic transcription is mediated by proteins that associate in a specific manner to form multiprotein DNA-bound complexes. Substantial progress has recently been made towards the understanding of the molecular determinants of the protein-protein and protein-DNA interactions that govern assembly of these complexes. Three-dimensional structures have been determined of the MATα2/MCM1-DNA complex, the p50/p65 Rel homology domain heterodimer bound to DNA, the NFAT/Fos-Jun/DNA quaternary complex, and of the GABPα/β ETS domain-ankyrin repeat heterodimer bound to DNA.

The histone tails of the nucleosome

Reversible acetylation of core histone tails plays an important role in the regulation of eukaryotic transcription, in the formation of repressive chromatin complexes, and in the inactivation of whole chromosomes. The high-resolution X-ray structure of the nucleosome core particle, as well as earlier evidence, suggests that the histone tails are largely responsible for the assembly of nucleosomes into chromatin fibers and implies that the physiological effects of histone acetylation may be achieved by modulation of a dynamic inter-conversion between the fiber and a less condensed nucleofilament structure. In addition, the tails and adjacent regions serve as recognition sites for chromatin assembly and transcription remodeling machinery and the interactions that occur may also be responsive to histone acetylation.

Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes.

Mechanisms controlling transcription and its regulation are fundamental to our understanding of molecular biology and, ultimately, cellular biology. Our knowledge of transcription initiation and integral factors such as RNA polymerase is considerable, and more recently our understanding of the involvement of enhancers and complexes such as holoenzyme and mediator has increased dramatically. However, an understanding of transcriptional repression is also essential for a complete understanding of promoter structure and the regulation of gene expression. Transcriptional repression in eukaryotes is achieved through 'silencers', of which there are two types, namely 'silencer elements' and 'negative regulatory elements' (NREs). Silencer elements are classical, position-independent elements that direct an active repression mechanism, and NREs are position-dependent elements that direct a passive repression mechanism. In addition, 'repressors' are DNA-binding trasncription factors that interact directly with silencers. A review of the recent literature reveals that it is the silencer itself and its context within a given promoter, rather than the interacting repressor, that determines the mechanism of repression. Silencers form an intrinsic part of many eukaryotic promoters and, consequently, knowledge of their interactive role with enchancers and other transcriptional elements is essential for our understanding of gene regulation in eukaryotes.

Role of general and gene-specific cofactors in the regulation of eukaryotic transcription.

Role of general and gene-specific cofactors in the regulation of eukaryotic transcription.

Atomic force microscopic demonstration of DNA looping by GalR and HU.

Regulation of gene transcription in both prokaryotes and eukaryotes involves formation of various DNA-multiprotein complexes of higher order structure through communication between distant regions of DNA. The communication between distant DNA sites occurs by interaction between proteins bound to the sites by looping out the intervening DNA segments. The repression of transcription of two overlapping promoters of the gal operon in Escherichia coli requires Gal repressor (GalR) and the histone-like protein HU. Both in vivo and in vitro data support a proposed HU containing complex responsive to induction in which GalR molecules bound to two distant operator sites interact by looping out DNA. We successfully applied atomic force microscope (AFM) imaging to visualize galDNA complexes with proteins. We report GalR mediated DNA looping in which HU plays an obligatory role by helping GalR tetramerization. Supercoiling of DNA, which is also critical for GalR action, may stabilize the DNA loops by providing an energetically favorable geometry of the DNA.

Visualization of Multicomponent Transcription Factor Complexes on Chromatin and Nonnucleosomal Templatesin Vivo

There is increasing evidence that specific chromatin structures play an important role in the regulation of transcription in eukaryotes. The mouse mammary tumor virus (MMTV) promoter, which is reproducibly assembled into a phased array of six nucleosomes when introduced into cells, represents a particularly well-studied example. The second or B nucleosome of the phased array is disrupted in response to hormone stimulation, allowing the assembly of a preinitiation complex and the activation of transcription.In vitroexperiments demonstrate that the assembly of the proximal promoter into chromatin is sufficient to prevent the binding of transcription factors such as nuclear factor 1. Consequently it is argued that chromatin serves to restrict the access of ubiquitous transcription factors in the absence of hormone stimulation. We have employed anin vivoexonuclease III (ExoIII)/Taqpolymerase footprinting assay to study the hormone-dependent loading of transcription factors on the MMTV promoter. This assay makes use of stable mouse and human cell lines harboring bovine papilloma virus chimeras of the MMTV promoter attached to a reporter gene. To ascertain the significance of protein–chromatin interactions vs protein–DNA interactions, we examined transcription factor binding to chromatin and nonchromatin templates of the MMTV promoter within the same cells. By the use of primers specific for each of the two distinct reporter genes, and restriction enzymes that generate entry sites forExoIII, we can distinguish chromatin and nonnucleosomal templatesin vivo.This system has allowed us to visualize the assembly of multicomponent transcription preinitiation complexes and to ascertain the consequences of defined chromatin structures on the binding of individual transcription factorsin vivo.

Redox Regulation of GA-binding Protein-α DNA Binding Activity

We have investigated the reduction/oxidation (redox) regulation of the heteromeric transcription factor GA-binding protein (GABP). GABP, also known as nuclear respiratory factor 2, regulates the expression of nuclear encoded mitochondrial proteins involved in oxidative phosphorylation, including cytochrome c oxidase subunits IV and Vb, as well as the expression of mitochondrial transcription factor 1. GABP is composed of two subunits, the Ets-related GABP-alpha, which mediates specific DNA binding, and GABP-beta, which forms heterodimers and heterotetramers on DNA sequences containing the PEA3/Ets motif ((C/A)GGA(A/T)(G/A)). We demonstrate here that GABP DNA binding activity and GABP-dependent gene expression in 3T3 cells are inhibited by pro-oxidant conditions. DNA binding of recombinant GABP-alpha was activated by chemical reduction (dithiothreitol) and by thioredoxin; however, GSSG inhibited GABP DNA binding activity. Treatment of GABP-alpha, but not GABP-beta1, with sulfhydryl-alkylating agents also inhibited GABP DNA binding activity. Our results suggest that GABP DNA binding activity is redox-regulated in vivo, possibly by thioredoxin-mediated reduction and by GSSG-mediated oxidation of the GABP-alpha subunit. The regulation of GABP (nuclear respiratory factor 2) DNA binding activity by cellular redox changes provides an important link between mitochondrial and nuclear gene expression and the redox state of the cell.

Control of gene expression by proteolysis

The proteasome and the small protein ubiquitin are key elements in the intracellular pathway of general protein degradation. Recent evidence shows that the proteasome and other less well defined cytoplasmic proteases can participate in specific events which control inducible gene expression. A number of eukaryotic transcriptional regulators, including NF-κB/IκB, p53, c-Jun, Notch, sterol regulated element binding proteins and MAT2α, have recently been shown to be regulated by proteolytic events, a regulation which results in the activation or inactivation of gene expression.

A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p.

Trapoxin is a microbially derived cyclotetrapeptide that inhibits histone deacetylation in vivo and causes mammalian cells to arrest in the cell cycle. A trapoxin affinity matrix was used to isolate two nuclear proteins that copurified with histone deacetylase activity. Both proteins were identified by peptide microsequencing, and a complementary DNA encoding the histone deacetylase catalytic subunit (HD1) was cloned from a human Jurkat T cell library. As the predicted protein is very similar to the yeast transcriptional regulator Rpd3p, these results support a role for histone deacetylase as a key regulator of eukaryotic transcription.

Transcription syndromes and the role of RNA polymerase II general transcription factors in human disease.

Messenger RNA synthesis is a major site for the regulation of gene expression. Eukaryotic messenger RNA synthesis is catalyzed by multisubunit RNA polymerase II (1–3) and proceeds via multiple stages, which are designated preinitiation, initiation, elongation, and termination and which have come to be referred to collectively as the transcription cycle (Fig. 1). The past decade was a watershed for biochemical studies of eukaryotic messenger RNA synthesis. A diverse collection of transcription factors and other nuclear proteins that govern the activity of RNA polymerase II during messenger RNA synthesis was identified and characterized, and unprecedented progress in several key research areas has provided a deeper understanding of the biochemical mechanisms underlying many aspects of eukaryotic transcriptional regulation. First, major breakthroughs in investigations of the structures of eukaryotic protein-coding genes and the role of chromatin in the regulation of their expression were achieved. Chromatin proteins, such as histones and HMG proteins, were found to play crucial roles in gene regulation by packaging genes into inactive or transcriptionally repressed configurations (4–9). Second, many DNA binding transactivators that interact specifically with upstream promoter elements and enhancer sequences located in the promoter-regulatory regions of genes were isolated, classified according to their structures, and found to regulate the expression of specific genes or gene families by controlling the rate of initiation (10) and, as shown more recently, the efficiency of elongation by RNA polymerase II (11–13). Third, chromatin remodeling proteins, such as the multisubunit SWI/SNF (14, 15) and NURF (16, 17) complexes, were discovered and found to play key roles in transcriptional activation by promoting conversion of regions of inactive chromatin into transcriptionally active, open chromatin, thereby allowing DNA binding transactivators and RNA polymerase II access to the promoter-regulatory regions of genes (4, 18–23). Fourth, coactivators, such as the SRB-containing mediator complex (2, 3, 24, 25), CREB binding protein (CBP) 1

Yeast transcriptional regulatory mechanisms.

Transcriptional regulation directly influences many biological phenomena such as cell growth, response to environmental change, development of multicellular organisms, and disease. Transcriptional regulatory mechanisms are fundamentally similar in eukaryotic organisms (93). Components of the basic RNA polymerase II (Pol II) machinery are highly conserved and, in some cases, functionally interchangeable. Transcription factors with similar structures and DNA-binding specificities are found throughout the eukaryotic kingdom, and acidic activation domains stimulate transcription across a wide range of species. Complex promoters with multiple protein binding sites are typical in all eukaryotic organisms, and efficient transcription generally requires the combinatorial and synergistic action of activator proteins that function at long and variable distances from the mRNA initiation site. Molecular mechanisms of eukaryotic transcriptional regulation have been elucidated from the studies that involve a wide variety of genes, promoters, proteins, organisms, and experimental approaches. This review focuses on transcriptional regulatory mechanisms in the baker's yeast Saccharomyces cerevisiae. Studies in yeast have emphasized powerful genetic approaches that are not available in other eukaryotic organisms. As a consequence, yeast is particularly amenable for analyzing transcriptional regulatory mechanisms in vivo under true physiological conditions. Furthermore, classical and molecular yeast genetics has permitted the discovery and functional characterization of transcriptional regulatory proteins that were not identified in biochemical studies. Thus, genetic analysis in yeast has often generated information complementary to that obtained from biochemical studies of transcription in vitro, and it has provided unique insights into mechanisms of eukaryotic transcriptional regulation.

Crystal Structure of the MATa1/MATα2 Homeodomain Heterodimer Bound to DNA

The Saccharomyces cerevisiae MATa1 and MAT alpha 2 homeodomain proteins, which play a role in determining yeast cell type, form a heterodimer that binds DNA and represses transcription in a cell type-specific manner. Whereas the alpha 2 and a1 proteins on their own have only modest affinity for DNA, the a1/alpha 2 heterodimer binds DNA with high specificity and affinity. The three-dimensional crystal structure of the a1/alpha 2 homeodomain heterodimer bound to DNA was determined at a resolution of 2.5 A. The a1 and alpha 2 homeodomains bind in a head-to-tail orientation, with heterodimer contacts mediated by a 16-residue tail located carboxyl-terminal to the alpha 2 homeodomain. This tail becomes ordered in the presence of a1, part of it forming a short amphipathic helix that packs against the a1 homeodomain between helices 1 and 2. A pronounced 60 degree bend is induced in the DNA, which makes possible protein-protein and protein-DNA contacts that could not take place in a straight DNA fragment. Complex formation mediated by flexible protein-recognition peptides attached to stably folded DNA binding domains may prove to be a general feature of the architecture of other classes of eukaryotic transcriptional regulators.

Promotion and Regulation of Ribosomal Transcription in Eukaryotes by RNA Polymerase

This chapter discusses the mechanisms of eukaryotic ribosomal transcription and its regulation. Ribosomal transcription has been studied in an exceptionally wide range of organisms. In eukaryotes, ribosomal gene transcription uses a dedicated set of transcription factors and a specialized ribonucleic acid (RNA) polymerase, the deoxyribonucleic acid (DNA)-dependent RNA polymerase I (RPOI). The resultant precursor-rRNA (pre-rRNA) is neither capped nor polyadenylated and is produced in the nucleolus; a large nuclear structure visible in the light microscope. Moreover, the promotion of ribosomal transcription is generally directed, by a 100- to 150-bp DNA sequence that lies across and, predominantly, upstream of the transcription-initiation site. Promotion requires an activated form of RPOI, the DNA-binding factor upstream transcription factor (UBF), and the TATA binding protein (TBP) I -complex, containing three TATA-box binding protein associated protein (TAF) I s. Upstream binding factor (UBF) binds to both an upstream and an initiation-site promoter element, probably coiling the promoter into a 180-bp loop. This allows the interaction of the TBP,-complex, and subsequent polymerase recruitment. The activity of the promoter is enhanced by a promoter-proximal terminator, by enhancer repeats in the proximal IGS, and by the presence in the IGS of duplicate spacer promoters. Further upstream, other sequences may also modulate transcription.

Lessons in transcriptional regulation learned from studies on immunoglobulin genes.

In this article, we consider basic principles of eukaryotic gene transcriptional regulation which have emerged from studies on immunoglobulin heavy chain and kappa chain genes. Four areas are considered: (1) regulation by distant enhancer elements, (2) multiple regulators in a given element including particular families of regulators which were discovered by studying Ig genes, (3) achieving tissue specificity of transcription by multiple mechanisms and (4) the importance of chromatin structure for transcriptional regulation.

Cloning and characterization of seven human forkhead proteins: binding site specificity and DNA bending.

The forkhead domain is a monomeric DNA binding motif that defines a rapidly growing family of eukaryotic transcriptional regulators. Genetic and biochemical data suggest a central role in embryonic development for genes encoding forkhead proteins. We have used PCR and low stringency hybridization to isolate clones from human cDNA and genomic libraries that represent seven novel forkhead genes, freac-1 to freac-7. The spatial patterns of expression for the seven freac genes range from specific for a single tissue to nearly ubiquitous. The DNA binding specificities of four of the FREAC proteins were determined by selection of binding sites from random sequence oligonucleotides. The binding sites for all four FREAC proteins share a core sequence, RTAAAYA, but differ in the positions flanking the core. Domain swaps between two FREAC proteins identified two subregions within the forkhead domain as responsible for creating differences in DNA binding specificity. Applying a circular permutation assay, we show that binding of FREAC proteins to their cognate sites results in bending of the DNA at an angle of 80-90 degrees.