Transcription Complex Assembly Research Articles

Modulation of RNA Polymerase Assembly Dynamics in Transcriptional Regulation

The interaction of transcription factors with target genes is highly dynamic. Whether the dynamic nature of these interactions is merely an intrinsic property of transcription factors or serves a regulatory role is unknown. Here we have used single-cell fluorescence imaging combined with computational modeling and chromatin immunoprecipitation to analyze transcription complex dynamics in gene regulation during the cell cycle in living cells. We demonstrate a link between the dynamics of RNA polymerase I (RNA Pol I) assembly and transcriptional output. We show that transcriptional upregulation is accompanied by prolonged retention of RNA Pol I components at the promoter, resulting in longer promoter dwell time, and an increase in the steady-state population of assembling polymerase. As a consequence, polymerase assembly efficiency and, ultimately, the rate of entry into processive elongation are elevated. Our results show that regulation of rDNA transcription in vivo occurs via modulation of the efficiency of transcription complex subunit capture and assembly.

Doublesex and the Regulation of Sexual Dimorphism in Drosophila melanogaster: STRUCTURE, FUNCTION, AND MUTAGENESIS OF A FEMALE-SPECIFIC DOMAIN

The DSX (Doublesex) transcription factor regulates somatic sexual differentiation in Drosophila. Female and male isoforms (DSX F and DSX M) are formed due to sex-specific RNA splicing. DNA recognition, mediated by a shared N-terminal zinc module (the DM domain), is enhanced by a C-terminal dimerization element. Sex-specific extension of this element in DSX F and DSX M leads to assembly of distinct transcriptional preinitiation complexes. Here, we describe the structure of the extended C-terminal dimerization domain of DSX F as determined by multidimensional NMR spectroscopy. The core dimerization element is well ordered, giving rise to a dense network of interresidue nuclear Overhauser enhancements. The structure contains dimer-related UBA folds similar to those defined by x-ray crystallographic studies of a truncated domain. Whereas the proximal portion of the female tail extends helix 3 of the UBA fold, the distal tail is disordered. Ala substitutions in the proximal tail disrupt the sex-specific binding of IX (Intersex), an obligatory partner protein and putative transcriptional coactivator; IX-DSX F interaction is, by contrast, not disrupted by truncation of the distal tail. Mutagenesis of the UBA-like dimer of DSX F highlights the importance of steric and electrostatic complementarity across the interface. Two temperature-sensitive mutations at this interface have been characterized in yeast model systems. One weakens a network of solvated salt bridges, whereas the other perturbs the underlying nonpolar interface. These mutations confer graded gene-regulatory activity in yeast within a physiological temperature range and so may provide novel probes for genetic analysis of a sex-specific transcriptional program in Drosophila development.

Different Requirements of the SWI/SNF Complex for Robust Nucleosome Displacement at Promoters of Heat Shock Factor and Msn2- and Msn4-Regulated Heat Shock Genes

The stress response in yeast cells is regulated by at least two classes of transcription activators-HSF and Msn2/4, which differentially affect promoter chromatin remodeling. We demonstrate that the deletion of SNF2, an ATPase activity-containing subunit of the chromatin remodeling SWI/SNF complex, eliminates histone displacement, RNA polymerase II recruitment, and heat shock factor (HSF) binding at the HSP12 promoter while delaying these processes at the HSP82 and SSA4 promoters. Out of the three promoters, the double deletion of MSN2 and MSN4 eliminates both chromatin remodeling and HSF binding only at the HSP12 promoter, suggesting that Msn2/4 activators are primary determinants of chromatin disassembly at the HSP12 promoter. Unexpectedly, during heat shock the level of Msn2/4 at the HSP12 promoter declines. This is likely a result of promoter-targeted Msn2/4 degradation associated with transcription complex assembly. While histone displacement kinetic profiles bear clear promoter specificity, the kinetic profiles of recovery from heat shock for all analyzed genes display an equal or even higher nucleosome return rate, which is to some extent delayed by the deletion of SNF2.

PIAS proteins as regulators of small ubiquitin-related modifier (SUMO) modifications and transcription

Transcriptional activity of signal-dependent transcription factors, including nuclear receptors, relies on interacting co-regulator proteins, many of which possess protein-modifying activity. SUMOs (small ubiquitin-related modifiers) and their conjugation pathway components act as co-regulator proteins for numerous transcription factors that also are often targets for SUMO modification. PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] proteins promote SUMOylation in a manner that resembles the action of RING-type ubiquitin E3 ligases. PIAS proteins were initially named for their ability to interact with STAT proteins and inhibit their activity, but their interactions and functions are not restricted to the STATs. Moreover, PIAS proteins do not operate merely as SUMO E3s, since their co-regulator effects are often independent of their RING finger but dependent on their SIM (SUMO-interacting motif) or SAP (scaffold attachment factor-A/B/acinus/PIAS) domain capable of interacting with DNA. The modulator activity imparted by the PIAS/SUMO system involves altered subnuclear targeting and/or assembly of transcription complexes. PIAS proteins may act as platforms that facilitate both removal and recruitment of other regulatory proteins in the transcription complexes.

Recruitment of Rad50 and MCM Complexes on the Human Beta Globin Locus Control Region (LCR): A Novel Role for LCR Hypersensitive Site 4 (HS4).

The human beta globin locus control region (LCR) aids in the establishment of an open chromatin configuration thereby facilitating the expression of linked downstream globin genes in a temporal and spatial manner along the developmental ontogeny. The LCR also functions as a classical enhancer of the beta like globin genes. The four DNAase-I hypersensitive regions (HS 1–4) on the LCR are believed to act as nucleation centers for the assembly of multi-protein transcriptional complexes that interact with the distant gene promoters. Present work is focused on characterizing the protein complex assemblies on the HS4 region and delineating their function in the context of goblin gene regulation. Our finding shows an assembly of a 2–5 MDa complex on the HS4 locus that is both erythroid restricted and sequence specific. Electrophoretic mobility shift assays (EMSA) were performed using K562 and HeLa nuclear extracts and ∼35mer double stranded oligonucleotides with 7 bp overlaps tiling the entire core HS4 sequence. We detected prominent K562 specific bands formed with an AT rich 39 bp HS4 sequence. Sequence specificity of DNA-protein complex formation on this AT rich sequence was determined by competitive EMSA assays. Gel supershift assays with a wide range of antibodies and subsequent partial purification of this multiprotein complex followed by the LC-MS/MS analysis reveal the presence of a mini chromosome maintenance (MCM) complex that is associated with the DNA replication, DNA repair associated Rad50-Mre-11-NBS-1 complex, histone acetyl transferase p300 and DNA binding transcription factors ILF2 (NF45) and ILF3 (NF90). The association of these proteins was determined by co-immunoprecipitation experiments. Chromatin immunoprecipitation (ChIP) experiments demonstrate the in vivo recruitment of these proteins on the HS4 region of the LCR. We are currently working on further characterization and functional studies of this complex and its significance on the role of the LCR in DNA replication and repair.

ANCCA, an estrogen-regulated AAA+ ATPase coactivator for ERα, is required for coregulator occupancy and chromatin modification

AAA+ proteins play crucial roles in diverse biological processes via their ATPase-driven remodeling of macromolecular complexes. Here we report our identification of an evolutionarily conserved AAA+ protein, ANCCA/pro2000, endowed with a bromodomain that is strongly induced by estrogen in human breast cancer cells and is a direct target of protooncogene ACTR/AIB1/SRC-3. We found that ANCCA associates directly with estrogen-bound estrogen receptor (ER) alpha and ACTR. It is selectively recruited, upon estrogen stimulation, to a subset of ERalpha target genes including cyclin D1, c-myc, and E2F1 and is required for their estrogen-induced expression as well as breast cancer cell proliferation. Further studies indicate that ANCCA binds and hydrolyzes ATP and is critical for recruitment of coregulator CBP and histone hyperacetylation at the ER target chromatin. Moreover, mutations at the ATP binding motifs rendered ANCCA defective as a coactivator in mediating estrogen induction of gene expression. Together, our findings reveal an unexpected layer of regulatory mechanism in hormone signaling mediated by ANCCA and suggest that hormone-induced assembly of transcriptional coregulator complexes at chromatin is a process facilitated by AAA+ ATPase proteins.

Chromatin is wonderful stuff

Chromatin molecules have properties that set them aside from all other biomacromolecules in the cell. (i) Chromosomes, which are single chromatin molecules, are the largest macromolecules in eukaryotic cells. (ii) Chromatin molecules carry the cell's genetic and epigenetic information and all control elements that regulate and, importantly, orchestrate expression of tenths of thousands of genes. (iii) Chromatin fibers are extremely highly folded inside the cell nucleus. Moreover, chromatin folding varies considerably from cell to cell, even in otherwise identical cells. Despite this, chromatin can be accurately duplicated and faithfully separated over the two daughter cells during cell division. (iv) The highly folded chromatin fiber acts as a scaffold that spatially organizes processes inside the nucleus, such as transcription and replication. This chromatin-based compartmentalization of the nucleus is a key element in the regulation of genome function. (v) Chromatin-associated processes often are single molecule processes in which proteins that freely diffuse inside the nucleus have to find and assemble on unique sequence elements, e.g. a specific promoter, of the chromatin fiber. (vi) The structure of chromatin shows remarkable plasticity. Depending on regulatory signals its chemical structure changes continuously (e.g. due to histone modifications), its protein composition alters (e.g. incorporation of variant histones and the assembly and disassembly of transcription complexes) and folding inside the nucleus may change dramatically (e.g. depending on epigenetic changes and changes in transcriptional activity). These properties all contribute to and probably are essential for proper functioning of the eukaryotic genome. Research aiming to understand chromatin properties and unravel underlying physical principles and molecular mechanisms is hampered by the fact that we have no tools to directly visualize and analyze the chromatin fiber inside the cell. Also, chromatin structure in part depends on weak interactions that are lost upon opening the cell, making biochemical approaches difficult. To unravel chromatin-related enigmas we have to rely on combining a broad range of strategies and techniques. This special issue addresses a number of important aspects of chromatin research that are not yet encountered often in the main stream scientific literature. In the first paper Van Holde and Zlatanova [1] discuss our present understanding of what probably is a basic folding state of chromatin, the 30 nm fiber. One way to unravel principles of chromatin folding is the development of predictive computational models, which define precise questions that can be addressed experimentally. Langowski and Heermann [2] present a sample of this by modeling the nucleosomal fiber and analyzing its properties, such as flexibility as a function of linker histone H1 binding. Hancock [3] discusses an often overlooked aspect of biological organization in general and chromatin structure in particular: macromolecular crowding. It is made highly plausible that crowding phenomena are a key element in defining chromatin structure in vivo. The paper of Fakan and Van Driel [4] discusses an intriguing aspect of spatial organization of our genome. Transcription and replication occur exclusively at the surface of compact subchromosomal chromatin domains, which poses major constrains to the way the chromatin fiber is folded inside the interphase nucleus. Dorman et al. [5] E.R. Dorman, A.M. Bushey and V.G. Corces, The role of insulator elements in large-scale chromatin structure in interphase, Sem Cell Dev Biol 18 (2007), pp. 682-690. Article | PDF (1131 K) | View Record in Scopus | Cited By in Scopus (17)[5] summarize our understanding of the role of insulator elements in the genome in the spatial organization of the chromatin fiber. Insulator sequences may constitute a major class of chromatin-chromatin interactions. Sexton et al. [6] describe another important type of interchromatin interactions: transcription factories. In this type of structures transcriptionally active genes from different part of the chromatin fiber and from different chromosomes come together in the interphase nucleus, imposing further constrains on the large-scale folding pattern. Chuang and Belmont [7] address the issue of directed movement of parts of the chromatin fiber in relation to compartmentalization of the interphase nucleus. Finally, Goetze et al. [8] discuss various aspects of the large-scale organization of the chromatin fiber in mammalian cells. Together, these papers give a good overview of the different ways structure-function relationships of the chromatin fiber are addressed and summarize the main questions that we have to deal with in the forthcoming years.

Multienzyme assembly of a p53 transcription complex

Proline isomerization of the p53 transactivation domain has a central role in coordinating kinase signaling pathways that assemble the core p53 transcription complex.

The New Biomimetic Chemistry: Artificial Transcription Factors

While many research programs have focused on the challenge of developing small molecules that can inhibit protein-protein interactions, some researchers have taken the problem one step further by attempting to develop small molecules that mimic the essential features of an entire protein. An area of particular interest has been in the field of artificial transcription factors (ATFs), where the essential function of some transcription factors is to recruit and promote the assembly of a larger transcription complex, leading to the expression of a gene of interest. The goal of synthesizing small-molecule ATFs holds promise as a means to independently control the expression of genes such as those that are misregulated in cancer and disease.

Inhibition of transcription at radiation-induced nuclear foci of phosphorylated histone H2AX in mammalian cells

Double-strand DNA breaks (DSBs) induced by ionizing radiation can be visualized in human cells using antibodies against Ser-139 phosphorylated histone H2AX (gamma-H2AX). Large gamma-H2AX foci are seen in the nucleus fixed 1 hour after irradiation and their number corresponds to the number of DSBs, allowing analysis of these genome lesions after low doses. We estimated whether transcription is affected in chromatin domains containing gamma-H2AX by following in vivo incorporation of 5-bromouridine triphosphate (BrUTP) loaded by cell scratching (run-on assay). We found that BrUTP incorporation is strongly suppressed at gamma-H2AX foci, suggesting that H2AX phosphorylation inhibits transcription. This is not caused by preferential association of gamma-H2AX foci with constitutive or facultative heterochromatin, which was visualized in irradiated cells using antibodies against histone H3 trimethylated at lysine-9 (H3-K9m3) or histone H3 trimethylated at lysine-27 (H3-K27m3). Apparently, formation of gamma-H2AX induces changes of chromatin that inhibit assembly of transcription complexes without heterochromatin formation. Inhibition of transcription by phosphorylation of histone H2AX can decrease chromatin movement at DSBs and frequency of misjoining of DNA ends.

Zinc fingers 1 and 7 of yeast TFIIIA are essential for assembly of a functional transcription complex on the 5 S RNA gene

The binding of transcription factor (TF) IIIA to the internal control region of the 5 S RNA gene is the first step in the assembly of a DNA–TFIIIA–TFIIIC– TFIIIB transcription complex, which promotes accurate transcription by RNA polymerase III. With the use of mutations that are predicted to disrupt the folding of a zinc finger, we have examined the roles of zinc fingers 1 through 7 of yeast TFIIIA in the establishment of a functional transcription complex both in vitro and in vivo. Our data indicate that, in addition to their role in DNA binding, the first and seventh zinc fingers contribute other essential roles in the assembly of an active transcription complex. Alanine-scanning mutagenesis identified residues within zinc finger 1 that are not required for DNA binding but are required for incorporation of TFIIIC into the TFIIIA–DNA complex. Although disruption of zinc finger 2 or 3 had a deleterious effect on the activity of TFIIIA both in vitro and in vivo, we found that increasing the level of their in vivo expression allowed these mutant proteins to support cell viability. Disruption of zinc fingers 4, 5 or 6 had minimal effect on the DNA binding and TF activities of TFIIIA.

Repression of RUNX1 Activity by EVI1: A New Role of EVI1 in Leukemogenesis

Recurring chromosomal translocations observed in human leukemia often result in the expression of fusion proteins that are DNA-binding transcription factors. These altered proteins acquire new dimerization properties that result in the assembly of inappropriate multimeric transcription complexes that deregulate hematopoietic programs and induce leukemogenesis. Recently, we reported that the fusion protein AML1/MDS1/EVI1 (AME), a product of a t(3;21)(q26;q22) associated with chronic myelogenous leukemia and acute myelogenous leukemia, displays a complex pattern of self-interaction. Here, we show that the 8th zinc finger motif of MDS1/EVI1 is an oligomerization domain involved not only in interaction of AME with itself but also in interactions with the parental proteins, RUNX1 and MDS1/EVI1, from which AME is generated. Because the 8th zinc finger motif is also present in the oncoprotein EVI1, we have evaluated the effects of the interaction between RUNX1 and EVI1 in vitro and in vivo. We found that in vitro, this interaction alters the ability of RUNX1 to bind to DNA and to regulate a reporter gene, whereas in vivo, the expression of the isolated 8th zinc finger motif of EVI1 is sufficient to block the granulocyte colony-stimulating factor-induced differentiation of 32Dcl3 cells, leading to cell death. As EVI1 is not detected in normal bone marrow cells, these data suggest that its inappropriate expression could contribute to hematopoietic transformation in part by a new mechanism that involves EVI1 association with key hematopoietic regulators, leading to their functional impairment.

Site Specific Acetylation of p53 Directs Selective Transcription Complex Assembly on p53 Target Gene Promoters

Site Specific Acetylation of p53 Directs Selective Transcription Complex Assembly on p53 Target Gene Promoters

Site-specific Acetylation of p53 Directs Selective Transcription Complex Assembly

Histone deacetylase (HDAC) inhibitors are being investigated as possible adjuvant therapies for a number of diseases, including cancer. In addition to stabilization of acetylated histones, HDAC inhibitors stabilize the acetylation of a number of transcription factors, including p53. This study investigates the action of two HDAC inhibitors, CG-1521 and trichostatin A, which stabilize Ac-Lys-373 p53 and Ac-Lys-382 p53, respectively, in LNCaP prostate cancer cells. Real-time PCR demonstrates that CG-1521 induces p21 transcription whereas trichostatin A does not alter the steady state level of p21 mRNA. Co-immunoprecipitation demonstrates that the selective acetylation of p53 directs the recruitment of mutually exclusive coactivator complexes on the p53 response elements in the p21 promoter. Furthermore, the co-activator complexes initiate the recruitment of the components of the basal transcription apparatus to the basal promoter with markedly different outcomes because only Ac-Lys-373 p53 promotes the assembly of the basal transcriptional apparatus on the p21 promoter. These data highlight the profound effects of post-translational modification, including acetylation, on the function of p53. The data also suggest a novel and critically important role for protein acetylation/deacetylation in the assembly of active transcription processes that may be as important as classical phosphorylation/dephosphorylation.

Cell-permeable hypoxia-inducible factor-1 (HIF-1) antagonists function as tumor radiosensitizers

Hypoxia is a common phenomenon in human solid tumors and has been considered as an important, independent negative prognostic factor for response to treatment and survival of tumor patients. Hypoxia-inducible factor-1 (HIF-1) is the central transcription factor which is activated by hypoxia and modulates the expression of many genes involved in cell metabolism, proliferation, apoptosis, angiogenesis. Recently, it has been reported that HIF-1 contributes to tumor radioresistance by upregulating survivin expression under hypoxic conditions. Moreover, in hypoxic tumor cells, HIF-1 dependent signal transduction pathway is activated and could be further enhanced by radiation, thereby providing survival signals to adjacent vascular endothelial cells by upregulation of VEGF and bFGF and resulting in tumor radioresistance through vascular radioprotection. Recent research revealed that the stability of HIF-1alpha, one of the two subunits of HIF-1, determines the whole HIF-1 activity and the C-terminal transactivation domain of HIF-1alpha could reduce HIF-1 activity when overexpressed in tumor cells by disruption of the assembly of HIF-1 transcription complex. Therefore, we postulate that fusion with protein transduction domains would overcome the inability of C-terminal transactivation domain of HIF-1alpha to cross cellular membrane. Thus the recombinant fusion proteins could serve as cell-permeable HIF-1 antagonists, function as both inhibitors of tumor angiogenesis and tumor radiosensitizers, and would be widely used in clinical settings to improve tumor response to radiotherapy.

A TATA binding protein regulatory network that governs transcription complex assembly

A portion of the assembly process involving the regulation of the TATA binding protein (TBP) throughout the yeast genome is modeled and experimentally tested.

Reverse Transcription Complex: The Key Player of the Early Phase of HIV Replication

The early events of retroviral replication following virus–cell fusion and preceding integration of the viral genome into cellular chromatin occur within a context of a large multimolecular complex termed reverse transcription or preintegration complex. While some of the functions of these complexes (e.g., reverse transcription) are well described, most other events involved in assembly and processing of reverse transcription complexes, such as viral uncoating and nuclear transport, are poorly understood, and available data are very controversial. This article reviews the current literature on structure and function of reverse transcription complexes, focusing on the least known and most controversial steps.

A Novel C to A Substitution in the CCAAT Box of beta Globin Gene.

The promoter of the human beta-globin gene contains three positive cis-acting elements required for maximal transcription: the CACCC box located between −86 and −90, the CCAAT box located between −72 and − 76 and the TATA box located between −28 and −31 relative to the start site of transcription. Naturally occurring mutations within the TATA and the CACCC box regions have been recorded in patients with beta+ thalassemia. Mutations within the TATA box disrupt assembly of the basal transcription complex, while mutations at the CACCC box prevent binding of an erythroid-specific transcription factor EKLF. Surprisingly, no mutations have so far been identified in the highly conserved element CCAAT box and the transcription factors responsible for the regulatory activity of the CCAAT site in vivo have been less intensively studied. We report a novel mutation −76 C>A (HBB c. −126) detected by sequencing analysis of beta globin gene in a Italian beta+ thalassemic patient. The transversion C>A hits the first nucleotide in the CCAAT box of the beta globin gene. The carrier, a male 44 years old, shows a mild hypochromic and microcytic anaemia with reduced mean corpuscular volume and mean corpuscular haemoglobin (MCV 75 fl, MCH 25 pg) and Hb A2 level slightly increased (3.9%). Recently, studies in vitro in gel-shift and reporter assays, investigating the transcriptional activity of human beta globin CCAAT box, have identified five factors: NF-Y (CP1) a ubiquitous CCAAT box binding complex, GATA-1 an erythroid-specific transcription factor, C/EBPbeta, C/EBPgamma and C/EBPdelta members of CCAAT/enhancer-binding protein family involved in hemapoietic regulation. This represents the first report of a natural mutation of the human beta-globin CCAAT box and confirms its functional significance for in vivo transcription.

The nuclear receptor co-repressor RIP140 controls the expression of metabolic gene networks

NRs (nuclear receptors) regulate the expression of specific gene networks in target cells by recruiting cofactor complexes involved in chromatin remodelling and in the assembly of transcription complexes. The importance of activating gene expression, in metabolic tissues, is well established, but the contribution of transcriptional inhibition is less well defined. In this review, we highlight a crucial role for RIP140 (receptor-interacting protein 140), a transcriptional co-repressor for NR, in the regulation of metabolic gene expression. Many genes involved in lipid and carbohydrate metabolism are repressed by RIP140 in adipose and muscle. The repressive function of RIP140 results from its ability to bridge NRs to repressive enzyme complexes that modify DNA and histones. In the absence of RIP140, expression from many metabolic genes is increased so that mice exhibit a lean phenotype and resistance to high-fat-diet-induced obesity and display increased glucose tolerance and insulin sensitivity. We propose that a functional interplay between transcriptional activators and the co-repressor RIP140 is an essential process in metabolic regulation.

Signal Transduction Pathways of Inflammatory Gene Expressions and Therapeutic Implications

Intercellular adhesion molecule-1 (ICAM-1), an inducible cell adhesion glycoprotein of the immunoglobulin supergene family and cyclooxygenase-2 (COX-2), an inducible prostaglandin G/H synthase, are overexpressed by proinflammatory mediators in a wide variety of cell types. These stimuli increase ICAM-1 or COX-2 expression primarily through activation of ICAM-1 or COX-2 gene transcription. The architecture of the ICAM-1 or COX-2 promoter is complex, containing a large number of binding site for inducible transcription factors, the most important of which is NF-kB. NF-kB acts in concert with other transcription factors or transcriptional coactivators which facilitate the assembly of distinct stereospecific transcription complexes on the ICAM-1 or COX-2 promoter. These transcription complexes presumably mediate the induction of ICAM-1 or COX-2 expression in different cell types and in response to different stimuli. In this review, I summarize the current understanding of ICAM-1 and COX-2 gene regulation with a particular emphasis on the transcription factors or coactivators, and signal transduction pathways critical for their expression. A PKC-dependent c-Src pathway activating NF-kB or GAS to enhance ICAM-1 or COX-2 gene expression is discussed. Furthermore, natural products and novel agents targeting on the transcription factor with potential anti-inflammation and anti-tumor activity are also discussed.