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Levesque MG, Picketts DJ. It Takes a Village of Chromatin Remodelers to Regulate rDNA Expression. Int J Mol Sci 2025; 26:1772. [PMID: 40004235 PMCID: PMC11855044 DOI: 10.3390/ijms26041772] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2024] [Revised: 02/09/2025] [Accepted: 02/16/2025] [Indexed: 02/27/2025] Open
Abstract
Ribosome biogenesis is one of the most fundamental and energetically demanding cellular processes. In humans, the ribosomal DNA (rDNA) repeats span a large region of DNA and comprise 200 to 600 copies of a ~43 kb unit spread over five different chromosomes. Control over ribosome biogenesis is closely tied to the regulation of the chromatin environment of this large genomic region. The proportion of rDNA loci which are active or silent is altered depending on the proliferative or metabolic state of the cell. Repeat silencing is driven by epigenetic changes culminating in a repressive heterochromatin environment. One group of proteins facilitating these epigenetic changes in response to growth or metabolic demands are ATP-dependent chromatin remodeling protein complexes that use ATP hydrolysis to reposition nucleosomes. Indeed, some chromatin remodelers are known to have indispensable roles in regulating the chromatin environment of rDNA. In this review, we highlight these proteins and their complexes and describe their mechanistic roles at rDNA. We also introduce the developmental disorders arising from the dysfunction of these proteins and discuss how the consequent dysregulation of rDNA loci may be reflected in the phenotypes observed.
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Affiliation(s)
- Mathieu G. Levesque
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada;
- Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - David J. Picketts
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada;
- Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
- Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
- Department of Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
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2
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Bi X. Hmo1: A versatile member of the high mobility group box family of chromosomal architecture proteins. World J Biol Chem 2024; 15:97938. [PMID: 39156122 PMCID: PMC11325855 DOI: 10.4331/wjbc.v15.i1.97938] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/13/2024] [Revised: 07/21/2024] [Accepted: 08/01/2024] [Indexed: 08/08/2024] Open
Abstract
Eukaryotic chromatin consisting of nucleosomes connected by linker DNA is organized into higher order structures, which is facilitated by linker histone H1. Formation of chromatin compacts and protects the genome, but also hinders DNA transactions. Cells have evolved mechanisms to modify/remodel chromatin resulting in chromatin states suitable for genome functions. The high mobility group box (HMGB) proteins are non-histone chromatin architectural factors characterized by one or more HMGB motifs that bind DNA in a sequence nonspecific fashion. They play a major role in chromatin dynamics. The Saccharomyces cerevisiae (yeast hereafter) HMGB protein Hmo1 contains two HMGB motifs. However, unlike a canonical HMGB protein that has an acidic C-terminus, Hmo1 ends with a lysine rich, basic, C-terminus, resembling linker histone H1. Hmo1 exhibits characteristics of both HMGB proteins and linker histones in its multiple functions. For instance, Hmo1 promotes transcription by RNA polymerases I and II like canonical HMGB proteins but makes chromatin more compact/stable like linker histones. Recent studies have demonstrated that Hmo1 destabilizes/disrupts nucleosome similarly as other HMGB proteins in vitro and acts to maintain a common topological architecture of genes in yeast genome. This minireview reviews the functions of Hmo1 and the underlying mechanisms, highlighting recent discoveries.
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Affiliation(s)
- Xin Bi
- Department of Biology, University of Rochester, Rochester, NY 14627, United States
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3
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Daiß JL, Griesenbeck J, Tschochner H, Engel C. Synthesis of the ribosomal RNA precursor in human cells: mechanisms, factors and regulation. Biol Chem 2023; 404:1003-1023. [PMID: 37454246 DOI: 10.1515/hsz-2023-0214] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2023] [Accepted: 07/04/2023] [Indexed: 07/18/2023]
Abstract
The ribosomal RNA precursor (pre-rRNA) comprises three of the four ribosomal RNAs and is synthesized by RNA polymerase (Pol) I. Here, we describe the mechanisms of Pol I transcription in human cells with a focus on recent insights gained from structure-function analyses. The comparison of Pol I-specific structural and functional features with those of other Pols and with the excessively studied yeast system distinguishes organism-specific from general traits. We explain the organization of the genomic rDNA loci in human cells, describe the Pol I transcription cycle regarding structural changes in the enzyme and the roles of human Pol I subunits, and depict human rDNA transcription factors and their function on a mechanistic level. We disentangle information gained by direct investigation from what had apparently been deduced from studies of the yeast enzymes. Finally, we provide information about how Pol I mutations may contribute to developmental diseases, and why Pol I is a target for new cancer treatment strategies, since increased rRNA synthesis was correlated with rapidly expanding cell populations.
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Affiliation(s)
- Julia L Daiß
- Regensburg Center for Biochemistry, University of Regensburg, D-93053 Regensburg, Germany
| | - Joachim Griesenbeck
- Regensburg Center for Biochemistry, University of Regensburg, D-93053 Regensburg, Germany
| | - Herbert Tschochner
- Regensburg Center for Biochemistry, University of Regensburg, D-93053 Regensburg, Germany
| | - Christoph Engel
- Regensburg Center for Biochemistry, University of Regensburg, D-93053 Regensburg, Germany
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Hori RT, Moshahid Khan M, Xiao J, Hargrove PW, Moss T, LeDoux MS. Behavioral and molecular effects of Ubtf knockout and knockdown in mice. Brain Res 2022; 1793:148053. [PMID: 35973608 PMCID: PMC10908547 DOI: 10.1016/j.brainres.2022.148053] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2022] [Revised: 07/10/2022] [Accepted: 08/10/2022] [Indexed: 11/17/2022]
Abstract
The UBTF E210K neuroregression syndrome is caused by de novo dominant mutations in UBTF (NM_014233.3:c.628G > A, p.Glu210Lys). In humans, onset is typically at 2.5 to 3 years and characterized by slow progression of global motor, cognitive and behavioral dysfunction. Other potentially pathogenic UBTF variants have been reported in humans with severe neurological disease and it remains undetermined if the UBTF E210K mutation operates via gain- and/or loss-of-function. Here we examine the behavioral, cognitive, motor, and molecular effects of Ubtf knockout and knockdown in mice as a means of gauging the role of loss-of-function in humans. Ubtf+/- mice show progression of behavioral (dominance tube), cognitive (cross maze), and mild motor abnormalities from 3 to 18 months. At 18 months, Ubtf+/- mice had more slips on a raised 9-mm round beam task, shorter latencies to fall on the accelerated rotarod, reduced open field vertical and jump counts, and significant deficits in spatial learning and memory. Via crosses to Nestin-Cre (NesCre) mice we found that homozygous Ubtf deletion limited to the central nervous system was embryonic lethal. Tamoxifen-induced homozygous knockdown of Ubtf in adult mice with the Cre-ERT2 system was associated with precipitous deterioration in neurological functioning. At the molecular level, 18-month-old Ubtf+/- mice showed mild increases in cerebellar 53BP1 immunoreactivity. These findings show that UBTF is essential for embryogenesis and survival in adults, and the deleterious effects of UBTF haploinsufficiency progress with age. Loss-of-function mechanisms may contribute, in part, to the human UBTF E210K neuroregression syndrome.
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Affiliation(s)
- Roderick T Hori
- Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, TN 38163, USA
| | - Mohammad Moshahid Khan
- Department of Neurology, University of Tennessee Health Science Center, Memphis, TN 38163, USA; Department of Physical Therapy, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
| | - Jianfeng Xiao
- Department of Neurology, University of Tennessee Health Science Center, Memphis, TN 38163, USA
| | - Phillip W Hargrove
- Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, TN 38163, USA
| | - Tom Moss
- Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Québec, Canada
| | - Mark S LeDoux
- Department of Psychology, University of Memphis, Memphis, TN 38152, USA; Veracity Neuroscience, Memphis, TN, 38157, USA.
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5
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Tremblay MG, Sibai DS, Valère M, Mars JC, Lessard F, Hori RT, Khan MM, Stefanovsky VY, LeDoux MS, Moss T. Ribosomal DNA promoter recognition is determined in vivo by cooperation between UBTF1 and SL1 and is compromised in the UBTF-E210K neuroregression syndrome. PLoS Genet 2022; 18:e1009644. [PMID: 35139074 PMCID: PMC8863233 DOI: 10.1371/journal.pgen.1009644] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2021] [Revised: 02/22/2022] [Accepted: 01/12/2022] [Indexed: 11/18/2022] Open
Abstract
Transcription of the ~200 mouse and human ribosomal RNA genes (rDNA) by RNA Polymerase I (RPI/PolR1) accounts for 80% of total cellular RNA, around 35% of all nuclear RNA synthesis, and determines the cytoplasmic ribosome complement. It is therefore a major factor controlling cell growth and its misfunction has been implicated in hypertrophic and developmental disorders. Activation of each rDNA repeat requires nucleosome replacement by the architectural multi-HMGbox factor UBTF to create a 15.7 kbp nucleosome free region (NFR). Formation of this NFR is also essential for recruitment of the TBP-TAFI factor SL1 and for preinitiation complex (PIC) formation at the gene and enhancer-associated promoters of the rDNA. However, these promoters show little sequence commonality and neither UBTF nor SL1 display significant DNA sequence binding specificity, making what drives PIC formation a mystery. Here we show that cooperation between SL1 and the longer UBTF1 splice variant generates the specificity required for rDNA promoter recognition in cell. We find that conditional deletion of the TAF1B subunit of SL1 causes a striking depletion of UBTF at both rDNA promoters but not elsewhere across the rDNA. We also find that while both UBTF1 and -2 variants bind throughout the rDNA NFR, only UBTF1 is present with SL1 at the promoters. The data strongly suggest an induced-fit model of RPI promoter recognition in which UBTF1 plays an architectural role. Interestingly, a recurrent UBTF-E210K mutation and the cause of a pediatric neurodegeneration syndrome provides indirect support for this model. E210K knock-in cells show enhanced levels of the UBTF1 splice variant and a concomitant increase in active rDNA copies. In contrast, they also display reduced rDNA transcription and promoter recruitment of SL1. We suggest the underlying cause of the UBTF-E210K syndrome is therefore a reduction in cooperative UBTF1-SL1 promoter recruitment that may be partially compensated by enhanced rDNA activation.
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Affiliation(s)
- Michel G. Tremblay
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Québec, Canada
| | - Dany S. Sibai
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Québec, Canada
- Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Québec, Canada
| | - Melissa Valère
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Québec, Canada
- Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Québec, Canada
| | - Jean-Clément Mars
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Québec, Canada
- Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Québec, Canada
| | - Frédéric Lessard
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Québec, Canada
| | | | - Mohammad Moshahid Khan
- Departments of Neurology and Anatomy & Neurobiology, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America
| | - Victor Y. Stefanovsky
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Québec, Canada
| | - Mark S. LeDoux
- Department of Psychology, University of Memphis, Memphis TN and Veracity Neuroscience LLC, Memphis, Tennessee, United States of America
| | - Tom Moss
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Québec, Canada
- Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Québec, Canada
- * E-mail:
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6
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Justilien V, Lewis KC, Meneses KM, Jamieson L, Murray NR, Fields AP. Protein kinase Cι promotes UBF1-ECT2 binding on ribosomal DNA to drive rRNA synthesis and transformed growth of non-small-cell lung cancer cells. J Biol Chem 2020; 295:8214-8226. [PMID: 32350115 DOI: 10.1074/jbc.ra120.013175] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2020] [Revised: 04/23/2020] [Indexed: 01/31/2023] Open
Abstract
Epithelial cell-transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor for Rho GTPases that is overexpressed in many cancers and involved in signal transduction pathways that promote cancer cell proliferation, invasion, and tumorigenesis. Recently, we demonstrated that a significant pool of ECT2 localizes to the nucleolus of non-small-cell lung cancer (NSCLC) cells, where it binds the transcription factor upstream binding factor 1 (UBF1) on the promoter regions of ribosomal DNA (rDNA) and activates rDNA transcription, transformed cell growth, and tumor formation. Here, we investigated the mechanism by which ECT2 engages UBF1 on rDNA promoters. Results from ECT2 mutagenesis indicated that the tandem BRCT domain of ECT2 mediates binding to UBF1. Biochemical and MS-based analyses revealed that protein kinase Cι (PKCι) directly phosphorylates UBF1 at Ser-412, thereby generating a phosphopeptide-binding epitope that binds the ECT2 BRCT domain. Lentiviral shRNA knockdown and reconstitution experiments revealed that both a functional ECT2 BRCT domain and the UBF1 Ser-412 phosphorylation site are required for UBF1-mediated ECT2 recruitment to rDNA, elevated rRNA synthesis, and transformed growth. Our findings provide critical molecular insight into ECT2-mediated regulation of rDNA transcription in cancer cells and offer a rationale for therapeutic targeting of UBF1- and ECT2-stimulated rDNA transcription for the management of NSCLC.
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Affiliation(s)
- Verline Justilien
- Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida, USA
| | - Kayla C Lewis
- Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida, USA
| | - Kayleah M Meneses
- Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida, USA
| | - Lee Jamieson
- Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida, USA
| | - Nicole R Murray
- Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida, USA
| | - Alan P Fields
- Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida, USA
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7
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Hannig K, Babl V, Hergert K, Maier A, Pilsl M, Schächner C, Stöckl U, Milkereit P, Tschochner H, Seufert W, Griesenbeck J. The C-terminal region of Net1 is an activator of RNA polymerase I transcription with conserved features from yeast to human. PLoS Genet 2019; 15:e1008006. [PMID: 30802237 PMCID: PMC6415870 DOI: 10.1371/journal.pgen.1008006] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2018] [Revised: 03/13/2019] [Accepted: 02/05/2019] [Indexed: 01/25/2023] Open
Abstract
RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA) in all eukaryotes, accounting for the major part of transcriptional activity in proliferating cells. Although basal Pol I transcription factors have been characterized in diverse organisms, the molecular basis of the robust rRNA production in vivo remains largely unknown. In S. cerevisiae, the multifunctional Net1 protein was reported to stimulate Pol I transcription. We found that the Pol I-stimulating function can be attributed to the very C-terminal region (CTR) of Net1. The CTR was required for normal cell growth and Pol I recruitment to rRNA genes in vivo and sufficient to promote Pol I transcription in vitro. Similarity with the acidic tail region of mammalian Pol I transcription factor UBF, which could partly functionally substitute for the CTR, suggests conserved roles for CTR-like domains in Pol I transcription from yeast to human. The production of ribosomes, cellular factories of protein synthesis, is an essential process driving proliferation and cell growth. Ribosome biogenesis is controlled at the level of synthesis of its components, ribosomal proteins and ribosomal RNA. In eukaryotes, RNA polymerase I is dedicated to transcribe the ribosomal RNA. RNA polymerase I has been identified as a potential target for cell proliferation inhibition. Here we describe the C-terminal region of Net1 as an activator of RNA polymerase I transcription in baker’s yeast. In the absence of this activator RNA polymerase I transcription is downregulated and cell proliferation is strongly impaired. Strikingly, this activator might be conserved in human cells, which points to a general mechanism. Our discovery will help to gain a better understanding of the molecular basis of ribosomal RNA synthesis and may have implications in developing strategies to control cellular growth.
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Affiliation(s)
- Katharina Hannig
- Institut für Biochemie, Genetik und Mikrobiologie, Universität Regensburg, Regensburg, Germany
| | - Virginia Babl
- Institut für Biochemie, Genetik und Mikrobiologie, Universität Regensburg, Regensburg, Germany
| | - Kristin Hergert
- Institut für Biochemie, Genetik und Mikrobiologie, Universität Regensburg, Regensburg, Germany
| | - Andreas Maier
- Institut für Biochemie, Genetik und Mikrobiologie, Universität Regensburg, Regensburg, Germany
| | - Michael Pilsl
- Institut für Biochemie, Genetik und Mikrobiologie, Universität Regensburg, Regensburg, Germany
| | - Christopher Schächner
- Institut für Biochemie, Genetik und Mikrobiologie, Universität Regensburg, Regensburg, Germany
| | - Ulrike Stöckl
- Institut für Biochemie, Genetik und Mikrobiologie, Universität Regensburg, Regensburg, Germany
| | - Philipp Milkereit
- Institut für Biochemie, Genetik und Mikrobiologie, Universität Regensburg, Regensburg, Germany
- * E-mail: (PM); (HT); (WS); (JG)
| | - Herbert Tschochner
- Institut für Biochemie, Genetik und Mikrobiologie, Universität Regensburg, Regensburg, Germany
- * E-mail: (PM); (HT); (WS); (JG)
| | - Wolfgang Seufert
- Institut für Biochemie, Genetik und Mikrobiologie, Universität Regensburg, Regensburg, Germany
- * E-mail: (PM); (HT); (WS); (JG)
| | - Joachim Griesenbeck
- Institut für Biochemie, Genetik und Mikrobiologie, Universität Regensburg, Regensburg, Germany
- * E-mail: (PM); (HT); (WS); (JG)
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8
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Sorrentino A, Rienzo M, Ciccodicola A, Casamassimi A, Abbondanza C. Human PRDM2: Structure, function and pathophysiology. BIOCHIMICA ET BIOPHYSICA ACTA. GENE REGULATORY MECHANISMS 2018; 1861:S1874-9399(18)30071-3. [PMID: 29883756 DOI: 10.1016/j.bbagrm.2018.06.002] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/15/2018] [Revised: 06/04/2018] [Accepted: 06/04/2018] [Indexed: 12/22/2022]
Abstract
PRDM2/RIZ is a member of a superfamily of histone/protein methyltransferases (PRDMs), which are characterized by the conserved N-terminal PR domain, with methyltransferase activity and zinc finger arrays at the C-terminus. Similar to other family members, two main protein types, known as RIZ1 and RIZ2, are produced from the PRDM2 locus differing by the presence or absence of the PR domain. The imbalance in their respective amounts may be an important cause of malignancy, with the PR-positive isoform commonly lost or downregulated and the PR-negative isoform always being present at higher levels in cancer cells. Interestingly, the RIZ1 isoform also represents an important target of estradiol action downstream of the interaction with hormone receptor. Furthermore, the imbalance between the two products could also be a molecular basis for other human diseases. Thus, understanding the molecular mechanisms underlying PRDM2 function could be useful in the pathophysiological context, with a potential to exploit this information in clinical practice.
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Affiliation(s)
- A Sorrentino
- Department of Precision Medicine, University of Campania "Luigi Vanvitelli", Naples, Italy; Department of Science and Technology, University of Naples "Parthenope", Naples, Italy
| | - M Rienzo
- Department of Environmental, Biological, and Pharmaceutical Sciences and Technologies, University of Campania "Luigi Vanvitelli", Caserta, Italy
| | - A Ciccodicola
- Department of Science and Technology, University of Naples "Parthenope", Naples, Italy; Institute of Genetics and Biophysics "Adriano Buzzati Traverso", CNR, Naples, Italy
| | - A Casamassimi
- Department of Precision Medicine, University of Campania "Luigi Vanvitelli", Naples, Italy
| | - C Abbondanza
- Department of Precision Medicine, University of Campania "Luigi Vanvitelli", Naples, Italy.
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9
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Smith ML, Cui W, Jackobel AJ, Walker-Kopp N, Knutson BA. Reconstitution of RNA Polymerase I Upstream Activating Factor and the Roles of Histones H3 and H4 in Complex Assembly. J Mol Biol 2018; 430:641-654. [PMID: 29357286 PMCID: PMC9746128 DOI: 10.1016/j.jmb.2018.01.003] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2017] [Revised: 01/04/2018] [Accepted: 01/04/2018] [Indexed: 12/16/2022]
Abstract
RNA polymerase I (Pol I) transcription in Saccharomyces cerevisiae requires four separate factors that recruit Pol I to the promoter to form a pre-initiation complex. Upstream Activating Factor (UAF) is one of two multi-subunit complexes that regulate pre-initiation complex formation by binding to the ribosomal DNA promoter and by stimulating recruitment of downstream Pol I factors. UAF is composed of Rrn9, Rrn5, Rrn10, Uaf30, and histones H3 and H4. We developed a recombinant Escherichia coli-based system to coexpress and purify transcriptionally active UAF complex and to investigate the importance of each subunit in complex formation. We found that no single subunit is required for UAF assembly, including histones H3 and H4. We also demonstrate that histone H3 is able to interact with each UAF-specific subunit, and show that there are at least two copies of histone H3 and one copy of H4 present in the complex. Together, our results provide a new model suggesting that UAF contains a hybrid H3-H4 tetramer-like subcomplex.
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Affiliation(s)
- Marissa L. Smith
- Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, United States
| | - Weidong Cui
- Department of Chemistry, Washington University in St. Louis, One Brookings Drive, St. Louis, MO 63130, United States
| | - Ashleigh J. Jackobel
- Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, United States
| | - Nancy Walker-Kopp
- Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, United States
| | - Bruce A. Knutson
- Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, United States
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10
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Toro C, Hori RT, Malicdan MCV, Tifft CJ, Goldstein A, Gahl WA, Adams DR, Fauni HB, Wolfe LA, Xiao J, Khan MM, Tian J, Hope KA, Reiter LT, Tremblay MG, Moss T, Franks AL, Balak C, C4RCD Research Group, LeDoux MS. A recurrent de novo missense mutation in UBTF causes developmental neuroregression. Hum Mol Genet 2018; 27:691-705. [PMID: 29300972 PMCID: PMC5886272 DOI: 10.1093/hmg/ddx435] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2017] [Revised: 11/29/2017] [Accepted: 12/19/2017] [Indexed: 12/17/2022] Open
Abstract
UBTF (upstream binding transcription factor) exists as two isoforms; UBTF1 regulates rRNA transcription by RNA polymerase 1, whereas UBTF2 regulates mRNA transcription by RNA polymerase 2. Herein, we describe 4 patients with very similar patterns of neuroregression due to recurrent de novo mutations in UBTF (GRCh37/hg19, NC_000017.10: g.42290219C > T, NM_014233.3: c.628G > A) resulting in the same amino acid change in both UBTF1 and UBTF2 (p.Glu210Lys [p.E210K]). Disease onset in our cohort was at 2.5 to 3 years and characterized by slow progression of global motor, cognitive and behavioral dysfunction. Notable early features included hypotonia with a floppy gait, high-pitched dysarthria and hyperactivity. Later features included aphasia, dystonia, and spasticity. Speech and ambulatory ability were lost by the early teens. Magnetic resonance imaging showed progressive generalized cerebral atrophy (supratentorial > infratentorial) with involvement of both gray and white matter. Patient fibroblasts showed normal levels of UBTF transcripts, increased expression of pre-rRNA and 18S rRNA, nucleolar abnormalities, markedly increased numbers of DNA breaks, defective cell-cycle progression, and apoptosis. Expression of mutant human UBTF1 in Drosophila neurons was lethal. Although no loss-of-function variants are reported in the Exome Aggregation Consortium (ExAC) database and Ubtf-/- is early embryonic lethal in mice, Ubtf+/- mice displayed only mild motor and behavioral dysfunction in adulthood. Our data underscore the importance of including UBTF E210K in the differential diagnosis of neuroregression and suggest that mainly gain-of-function mechanisms contribute to the pathogenesis of the UBTF E210K neuroregression syndrome.
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Affiliation(s)
- Camilo Toro
- Undiagnosed Diseases Program and Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA
| | - Roderick T Hori
- Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN, USA
| | - May Christine V Malicdan
- Undiagnosed Diseases Program and Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA
| | - Cynthia J Tifft
- Undiagnosed Diseases Program and Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA
| | - Amy Goldstein
- Division of Child Neurology, Department of Pediatrics, Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA
| | - William A Gahl
- Undiagnosed Diseases Program and Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA
| | - David R Adams
- Undiagnosed Diseases Program and Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA
| | - Harper B Fauni
- Undiagnosed Diseases Program and Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA
| | - Lynne A Wolfe
- Undiagnosed Diseases Program and Office of the Clinical Director, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA
| | - Jianfeng Xiao
- Departments of Neurology and Anatomy & Neurobiology, University of Tennessee Health Science Center, Memphis, TN, USA
| | - Mohammad M Khan
- Departments of Neurology and Anatomy & Neurobiology, University of Tennessee Health Science Center, Memphis, TN, USA
| | - Jun Tian
- Departments of Neurology and Anatomy & Neurobiology, University of Tennessee Health Science Center, Memphis, TN, USA
| | - Kevin A Hope
- Integrated Program in Biological Sciences, University of Tennessee Health Science Center, Memphis, TN, USA
| | - Lawrence T Reiter
- Departments of Neurology and Anatomy & Neurobiology, University of Tennessee Health Science Center, Memphis, TN, USA
- Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN, USA
| | - Michel G Tremblay
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Canada
- Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, QC, Canada
| | - Tom Moss
- Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Canada
- Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, QC, Canada
| | - Alexis L Franks
- Division of Child Neurology, Department of Pediatrics, Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA
| | - Chris Balak
- Center for Rare Childhood Disorders (C4RCD), Translational Genomics Research Institute (TGen), Phoenix, AZ, USA
| | - C4RCD Research Group
- Center for Rare Childhood Disorders (C4RCD), Translational Genomics Research Institute (TGen), Phoenix, AZ, USA
| | - Mark S LeDoux
- Departments of Neurology and Anatomy & Neurobiology, University of Tennessee Health Science Center, Memphis, TN, USA
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11
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Internal Associations of the Acidic Region of Upstream Binding Factor Control Its Nucleolar Localization. Mol Cell Biol 2017; 37:MCB.00218-17. [PMID: 28874518 DOI: 10.1128/mcb.00218-17] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2017] [Accepted: 08/25/2017] [Indexed: 01/24/2023] Open
Abstract
Upstream binding factor (UBF) is a member of the high-mobility group (HMG) box protein family, characterized by multiple HMG boxes and a C-terminal acidic region (AR). UBF is an essential transcription factor for rRNA genes and mediates the formation of transcriptionally active chromatin in the nucleolus. However, it remains unknown how UBF is specifically localized to the nucleolus. Here, we examined the molecular mechanisms that localize UBF to the nucleolus. We found that the first HMG box (HMG box 1), the linker region (LR), and the AR cooperatively regulate the nucleolar localization of UBF1. We demonstrated that the AR intramolecularly associates with and attenuates the DNA binding activity of HMG boxes and confers the structured DNA preference to HMG box 1. In contrast, the LR was found to serve as a nuclear localization signal and compete with HMG boxes to bind the AR, permitting nucleolar localization of UBF1. The LR sequence binds DNA and assists the stable chromatin binding of UBF. We also showed that the phosphorylation status of the AR does not clearly affect the localization of UBF1. Our results strongly suggest that associations of the AR with HMG boxes and the LR regulate UBF nucleolar localization.
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12
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Wu M, Wei W, Chen J, Cong R, Shi T, Bouvet P, Li J, Wong J, Du JX. Acidic domains differentially read histone H3 lysine 4 methylation status and are widely present in chromatin-associated proteins. SCIENCE CHINA. LIFE SCIENCES 2017; 60:138-151. [PMID: 28194553 DOI: 10.1007/s11427-016-0413-3] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/26/2016] [Accepted: 01/09/2017] [Indexed: 02/03/2023]
Abstract
Histone methylation is believed to provide binding sites for specific reader proteins, which translate histone code into biological function. Here we show that a family of acidic domain-containing proteins including nucleophosmin (NPM1), pp32, SET/TAF1β, nucleolin (NCL) and upstream binding factor (UBF) are novel H3K4me2-binding proteins. These proteins exhibit a unique pattern of interaction with methylated H3K4, as their binding is stimulated by H3K4me2 and inhibited by H3K4me1 and H3K4me3. These proteins contain one or more acidic domains consisting mainly of aspartic and/or glutamic residues that are necessary for preferential binding of H3K4me2. Furthermore, we demonstrate that the acidic domain with sufficient length alone is capable of binding H3K4me2 in vitro and in vivo. NPM1, NCL and UBF require their acidic domains for association with and transcriptional activation of rDNA genes. Interestingly, by defining acidic domain as a sequence with at least 20 acidic residues in 50 continuous amino acids, we identified 655 acidic domain-containing protein coding genes in the human genome and Gene Ontology (GO) analysis showed that many of the acidic domain proteins have chromatin-related functions. Our data suggest that acidic domain is a novel histone binding motif that can differentially read the status of H3K4 methylation and is broadly present in chromatin-associated proteins.
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Affiliation(s)
- Meng Wu
- Shanghai Key Laboratory of Regulatory Biology, the Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, 200241, China
| | - Wei Wei
- Shanghai Key Laboratory of Regulatory Biology, the Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, 200241, China
| | - Jiwei Chen
- Shanghai Key Laboratory of Regulatory Biology, the Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, 200241, China
| | - Rong Cong
- Université de Lyon, Ecole Normale Supérieure de Lyon, CNRS USR 3010, Laboratoire Joliot-Curie, Lyon, 69364, France
| | - Tieliu Shi
- Shanghai Key Laboratory of Regulatory Biology, the Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, 200241, China
| | - Philippe Bouvet
- Université de Lyon, Ecole Normale Supérieure de Lyon, CNRS USR 3010, Laboratoire Joliot-Curie, Lyon, 69364, France
- Université de Lyon, Centre de Recherche en Cancérologie de Lyon, Cancer Cell Plasticity Department, UMR INSERM 1052 CNRS 5286, Centre Léon Bérard, Lyon, France
| | - Jiwen Li
- Shanghai Key Laboratory of Regulatory Biology, the Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, 200241, China
| | - Jiemin Wong
- Shanghai Key Laboratory of Regulatory Biology, the Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, 200241, China.
- Collaborative Innovation Center for Cancer Medicine, Sun Yat-Sen University Cancer Center, Guangzhou, 510060, China.
| | - James X Du
- Shanghai Key Laboratory of Regulatory Biology, the Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, 200241, China.
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13
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Kasahara K, Higashino A, Unzai S, Yoshikawa H, Kokubo T. Oligomerization of Hmo1 mediated by box A is essential for DNA binding in vitro and in vivo. Genes Cells 2016; 21:1333-1352. [PMID: 27860073 DOI: 10.1111/gtc.12449] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2016] [Accepted: 10/05/2016] [Indexed: 11/30/2022]
Abstract
Hmo1, a member of HMGB family proteins in Saccharomyces cerevisiae, binds to and regulates the transcription of genes encoding ribosomal RNA and ribosomal proteins. The functional motifs of Hmo1 include two HMG-like motifs, box A and box B, and a C-terminal tail. To elucidate the molecular roles of the HMG-like boxes in DNA binding in vivo, we analyzed the DNA-binding activity of various Hmo1 mutants using ChIP or reporter assays that enabled us to conveniently detect Hmo1 binding to the promoter of RPS5, a major target gene of Hmo1. Our mutational analyses showed that box B is a bona fide DNA-binding motif and that it also plays other important roles in cell growth. However, box A, especially its first α-helix, contributes to DNA binding of Hmo1 by inducing self-assembly of Hmo1. Intriguingly, box A mediated formation of oligomers of more than two proteins on DNA in vivo. Furthermore, duplication of the box B partially alleviates the requirement for box A. These findings suggest that the principal role of box A is to assemble multiple box B in the appropriate orientation, thereby stabilizing the binding of Hmo1 to DNA and nucleating specific chromosomal architecture on its target genes.
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Affiliation(s)
- Koji Kasahara
- Isotope Center, Tokyo University of Agriculture, Setagaya, Tokyo, Japan
| | - Ayako Higashino
- Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan
| | - Satoru Unzai
- Department of Frontier Bioscience, Hosei University, Koganei, Tokyo, Japan
| | | | - Tetsuro Kokubo
- Graduate School of Medical Life Science, Yokohama City University, Yokohama, Kanagawa, Japan
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14
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Huang S, Xu X, Wang G, Lu G, Xie W, Tao W, Zhang H, Jiang Q, Zhang C. DNA replication initiator Cdc6 also regulates ribosomal DNA transcription initiation. J Cell Sci 2016; 129:1429-40. [PMID: 26872786 DOI: 10.1242/jcs.178723] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2015] [Accepted: 02/06/2016] [Indexed: 01/28/2023] Open
Abstract
RNA-polymerase-I-dependent ribosomal DNA (rDNA) transcription is fundamental to rRNA processing, ribosome assembly and protein synthesis. However, how this process is initiated during the cell cycle is not fully understood. By performing a proteomic analysis of transcription factors that bind RNA polymerase I during rDNA transcription initiation, we identified that the DNA replication initiator Cdc6 interacts with RNA polymerase I and its co-factors, and promotes rDNA transcription in G1 phase in an ATPase-activity-dependent manner. We further showed that Cdc6 is targeted to the nucleolus during late mitosis and G1 phase in a manner that is dependent on B23 (also known as nucleophosmin, NPM1), and preferentially binds to the rDNA promoter through its ATP-binding domain. Overexpression of Cdc6 increases rDNA transcription, whereas knockdown of Cdc6 results in a decreased association of both RNA polymerase I and the RNA polymerase I transcription factor RRN3 with rDNA, and a reduction of rDNA transcription. Furthermore, depletion of Cdc6 impairs the interaction between RRN3 and RNA polymerase I. Taken together, our data demonstrate that Cdc6 also serves as a regulator of rDNA transcription initiation, and indicate a mechanism by which initiation of rDNA transcription and DNA replication can be coordinated in cells.
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Affiliation(s)
- Shijiao Huang
- The MOE Key Laboratory of Cell Proliferation and Differentiation and the State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China
| | - Xiaowei Xu
- The MOE Key Laboratory of Cell Proliferation and Differentiation and the State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China
| | - Guopeng Wang
- The MOE Key Laboratory of Cell Proliferation and Differentiation and the State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China
| | - Guoliang Lu
- The MOE Key Laboratory of Cell Proliferation and Differentiation and the State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China
| | - Wenbing Xie
- The MOE Key Laboratory of Cell Proliferation and Differentiation and the State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China
| | - Wei Tao
- The MOE Key Laboratory of Cell Proliferation and Differentiation and the State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China
| | - Hongyin Zhang
- Cancer Research Center, Peking University Hospital, Peking University, Beijing 100871, China
| | - Qing Jiang
- The MOE Key Laboratory of Cell Proliferation and Differentiation and the State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China
| | - Chuanmao Zhang
- The MOE Key Laboratory of Cell Proliferation and Differentiation and the State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China
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15
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Thapar R. Structure-specific nucleic acid recognition by L-motifs and their diverse roles in expression and regulation of the genome. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2015; 1849:677-87. [PMID: 25748361 DOI: 10.1016/j.bbagrm.2015.02.006] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/18/2014] [Revised: 02/05/2015] [Accepted: 02/24/2015] [Indexed: 01/08/2023]
Abstract
The high-mobility group (HMG) domain containing proteins regulate transcription, DNA replication and recombination. They adopt L-shaped folds and are structure-specific DNA binding motifs. Here, I define the L-motif super-family that consists of DNA-binding HMG-box proteins and the L-motif of the histone mRNA binding domain of stem-loop binding protein (SLBP). The SLBP L-motif and HMG-box domains adopt similar L-shaped folds with three α-helices and two or three small hydrophobic cores that stabilize the overall fold, but have very different and distinct modes of nucleic acid recognition. A comparison of the structure, dynamics, protein-protein and nucleic acid interactions, and regulation by PTMs of the SLBP and the HMG-box L-motifs reveals the versatile and diverse modes by which L-motifs utilize their surfaces for structure-specific recognition of nucleic acids to regulate gene expression.
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Affiliation(s)
- Roopa Thapar
- BioSciences at Rice-Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892, USA.
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16
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Albert B, Colleran C, Léger-Silvestre I, Berger AB, Dez C, Normand C, Perez-Fernandez J, McStay B, Gadal O. Structure-function analysis of Hmo1 unveils an ancestral organization of HMG-Box factors involved in ribosomal DNA transcription from yeast to human. Nucleic Acids Res 2013; 41:10135-49. [PMID: 24021628 PMCID: PMC3905846 DOI: 10.1093/nar/gkt770] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Ribosome biogenesis is a major metabolic effort for growing cells. In Saccharomyces cerevisiae, Hmo1, an abundant high-mobility group box protein (HMGB) binds to the coding region of the RNA polymerase I transcribed ribosomal RNAs genes and the promoters of ∼70% of ribosomal protein genes. In this study, we have demonstrated the functional conservation of eukaryotic HMGB proteins involved in ribosomal DNA (rDNA) transcription. We have shown that when expressed in budding yeast, human UBF1 and a newly identified Sp-Hmo1 (Schizosaccharomyces pombe) localize to the nucleolus and suppress growth defect of the RNA polymerase I mutant rpa49-Δ. Owing to the multiple functions of both proteins, Hmo1 and UBF1 are not fully interchangeable. By deletion and domains swapping in Hmo1, we identified essential domains that stimulate rDNA transcription but are not fully required for stimulation of ribosomal protein genes expression. Hmo1 is organized in four functional domains: a dimerization module, a canonical HMGB motif followed by a conserved domain and a C-terminal nucleolar localization signal. We propose that Hmo1 has acquired species-specific functions and shares with UBF1 and Sp-Hmo1 an ancestral function to stimulate rDNA transcription.
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Affiliation(s)
- Benjamin Albert
- LBME du CNRS, Université de Toulouse, 118 route de Narbonne, F-31000 Toulouse, France, Laboratoire de Biologie Moléculaire Eucaryote, Université de Toulouse, 118 route de Narbonne, F-31000 Toulouse, France and Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, University Road, Galway, Ireland
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17
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Goodfellow SJ, Zomerdijk JCBM. Basic mechanisms in RNA polymerase I transcription of the ribosomal RNA genes. Subcell Biochem 2013; 61:211-36. [PMID: 23150253 PMCID: PMC3855190 DOI: 10.1007/978-94-007-4525-4_10] [Citation(s) in RCA: 133] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
RNA Polymerase (Pol) I produces ribosomal (r)RNA, an essential component of the cellular protein synthetic machinery that drives cell growth, underlying many fundamental cellular processes. Extensive research into the mechanisms governing transcription by Pol I has revealed an intricate set of control mechanisms impinging upon rRNA production. Pol I-specific transcription factors guide Pol I to the rDNA promoter and contribute to multiple rounds of transcription initiation, promoter escape, elongation and termination. In addition, many accessory factors are now known to assist at each stage of this transcription cycle, some of which allow the integration of transcriptional activity with metabolic demands. The organisation and accessibility of rDNA chromatin also impinge upon Pol I output, and complex mechanisms ensure the appropriate maintenance of the epigenetic state of the nucleolar genome and its effective transcription by Pol I. The following review presents our current understanding of the components of the Pol I transcription machinery, their functions and regulation by associated factors, and the mechanisms operating to ensure the proper transcription of rDNA chromatin. The importance of such stringent control is demonstrated by the fact that deregulated Pol I transcription is a feature of cancer and other disorders characterised by abnormal translational capacity.
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Affiliation(s)
- Sarah J. Goodfellow
- Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee , Dundee DD1 5EH , UK
| | - Joost C. B. M. Zomerdijk
- Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee , Dundee DD1 5EH , UK
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18
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Abstract
In this report, we employed a lentiviral RNA interference screen to discover nucleolar DEAD/DEAH-box helicases involved in RNA polymerase I (Pol I)-mediated transcriptional activity. Our screen identified DHX33 as an important modulator of 47S rRNA transcription. We show that DHX33 is a cell cycle-regulated nucleolar protein that associates with ribosomal DNA (rDNA) loci, where it interacts with the RNA Pol I transcription factor upstream binding factor (UBF). DHX33 knockdown decreased the association of Pol I with rDNA and caused a dramatic decrease in levels of rRNA synthesis. Wild-type DHX33 overexpression, but not a DNA binding-defective mutant, enhanced 47S rRNA synthesis by promoting the association of RNA polymerase I with rDNA loci. In addition, an NTPase-defective DHX33 mutant (K94R) acted as a dominant negative mutant, inhibiting endogenous rRNA synthesis. Moreover, DHX33 deficiency in primary human fibroblasts triggered a nucleolar p53 stress response, resulting in an attenuation of proliferation. Thus, we show the mechanistic importance of DHX33 in rRNA transcription and proliferation.
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19
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Kong R, Zhang L, Hu L, Peng Q, Han W, Du X, Ke Y. hALP, a novel transcriptional U three protein (t-UTP), activates RNA polymerase I transcription by binding and acetylating the upstream binding factor (UBF). J Biol Chem 2010; 286:7139-48. [PMID: 21177859 PMCID: PMC3044971 DOI: 10.1074/jbc.m110.173393] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Transcription of ribosome RNA precursor (pre-rRNA) and pre-rRNA processing are coordinated by a subset of U three proteins (UTPs) known as transcriptional UTPs (t-UTPs), which participate in pre-rRNA transcription in addition to participation in 18 S rRNA processing. However, the mechanism by which t-UTPs function in pre-rRNA transcription remains undetermined. In the present study, we identified hALP, a histone acetyl-transferase as a novel t-UTP. We first showed that hALP is nucleolar, and is associated with U3 snoRNA and required for 18 S rRNA processing. Moreover, depletion of hALP resulted in a decreased level of 47 S pre-rRNA. Ectopic expression of hALP activated the rDNA promoter luciferase reporter and knockdown of hALP inhibited the reporter. In addition, hALP bound rDNA. Taken together these data identify hALP as a novel t-UTP. Immunoprecipitation and GST pulldown experiments showed that hALP binds the upstream binding factor (UBF) in vivo and in vitro. It is of importance that hALP acetylated UBF depending on HAT in vivo, and hALP but not hALP (ΔHAT) facilitated the nuclear translocation of the RNA polymerase I (Pol I)-associated factor 53 (PAF53) from the cytoplasm and promoted the association of UBF with PAF53. Thus, we provide a mechanism in which a novel t-UTP activates Pol I transcription by binding and acetylating UBF.
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Affiliation(s)
- Ruirui Kong
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University School of Oncology, Beijing Cancer Hospital & Institute, Beijing 100142, China
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20
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Peng Q, Wu J, Zhang Y, Liu Y, Kong R, Hu L, Du X, Ke Y. 1A6/DRIM, a novel t-UTP, activates RNA polymerase I transcription and promotes cell proliferation. PLoS One 2010; 5:e14244. [PMID: 21151873 PMCID: PMC2998426 DOI: 10.1371/journal.pone.0014244] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2010] [Accepted: 11/18/2010] [Indexed: 11/18/2022] Open
Abstract
BACKGROUND Ribosome biogenesis is required for protein synthesis and cell proliferation. Ribosome subunits are assembled in the nucleolus following transcription of a 47S ribosome RNA precursor by RNA polymerase I and rRNA processing to produce mature 18S, 28S and 5.8S rRNAs. The 18S rRNA is incorporated into the ribosomal small subunit, whereas the 28S and 5.8S rRNAs are incorporated into the ribosomal large subunit. Pol I transcription and rRNA processing are coordinated processes and this coordination has been demonstrated to be mediated by a subset of U3 proteins known as t-UTPs. Up to date, five t-UTPs have been identified in humans but the mechanism(s) that function in the t-UTP(s) activation of Pol I remain unknown. In this study we have identified 1A6/DRIM, which was identified as UTP20 in our previous study, as a t-UTP. In the present study, we investigated the function and mechanism of 1A6/DRIM in Pol I transcription. METHODOLOGY/PRINCIPAL FINDINGS Knockdown of 1A6/DRIM by siRNA resulted in a decreased 47S pre-rRNA level as determined by Northern blotting. Ectopic expression of 1A6/DRIM activated and knockdown of 1A6/DRIM inhibited the human rDNA promoter as evaluated with luciferase reporter. Chromatin immunoprecipitation (ChIP) experiments showed that 1A6/DRIM bound UBF and the rDNA promoter. Re-ChIP assay showed that 1A6/DRIM interacts with UBF at the rDNA promoter. Immunoprecipitation confirmed the interaction between 1A6/DRIM and the nucleolar acetyl-transferase hALP. It is of note that knockdown of 1A6/DRIM dramatically inhibited UBF acetylation. A finding of significance was that 1A6/DRIM depletion, as a kind of nucleolar stress, caused an increase in p53 level and inhibited cell proliferation by arresting cells at G1. CONCLUSIONS We identify 1A6/DRIM as a novel t-UTP. Our results suggest that 1A6/DRIM activates Pol I transcription most likely by associating with both hALP and UBF and thereby affecting the acetylation of UBF.
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MESH Headings
- Cell Line, Tumor
- Cell Proliferation
- DNA, Ribosomal/genetics
- Genes, p53
- Glucuronosyltransferase/genetics
- Humans
- Models, Genetic
- Promoter Regions, Genetic
- RNA Interference
- RNA Polymerase I/genetics
- RNA, Ribosomal, 18S/genetics
- RNA, Ribosomal, 28S/genetics
- RNA, Ribosomal, 5.8S/genetics
- Transcription, Genetic
- Tumor Suppressor Protein p53/metabolism
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Affiliation(s)
- Qunhui Peng
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Genetics Laboratory, Peking University School of Oncology, Beijing Cancer Hospital & Institute, Beijing, China
- Cancer Research Center, Peking University Health Science Center, Beijing, China
| | - Jianguo Wu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Genetics Laboratory, Peking University School of Oncology, Beijing Cancer Hospital & Institute, Beijing, China
- Cancer Research Center, Peking University Health Science Center, Beijing, China
| | - Ying Zhang
- Department of Cell Biology, Peking University Health Science Center, Beijing, China
- Cancer Research Center, Peking University Health Science Center, Beijing, China
| | - Yun Liu
- Department of Cell Biology, Peking University Health Science Center, Beijing, China
- Cancer Research Center, Peking University Health Science Center, Beijing, China
| | - Ruirui Kong
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Genetics Laboratory, Peking University School of Oncology, Beijing Cancer Hospital & Institute, Beijing, China
- Cancer Research Center, Peking University Health Science Center, Beijing, China
| | - Lelin Hu
- Department of Cell Biology, Peking University Health Science Center, Beijing, China
- Cancer Research Center, Peking University Health Science Center, Beijing, China
| | - Xiaojuan Du
- Department of Cell Biology, Peking University Health Science Center, Beijing, China
- Cancer Research Center, Peking University Health Science Center, Beijing, China
| | - Yang Ke
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Genetics Laboratory, Peking University School of Oncology, Beijing Cancer Hospital & Institute, Beijing, China
- Cancer Research Center, Peking University Health Science Center, Beijing, China
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Jiménez-Vidal M, Srivastava J, Putney LK, Barber DL. Nuclear-localized calcineurin homologous protein CHP1 interacts with upstream binding factor and inhibits ribosomal RNA synthesis. J Biol Chem 2010; 285:36260-6. [PMID: 20720019 DOI: 10.1074/jbc.m110.165555] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Calcineurin homologous protein 1 (CHP1) is a widely expressed, 22-kDa myristoylated EF-hand Ca(2+)-binding protein that shares a high degree of similarity with the regulatory B subunit of calcineurin (65%) and with calmodulin (59%). CHP1 localizes to the plasma membrane, the Golgi apparatus, and the nucleus and functions to regulate trafficking of early secretory vesicles, activation of T cells, and expression and transport of the Na-H exchanger NHE1. Although CHP1 contains nuclear export signals, whether its nuclear and cytoplasmic localization is regulated and has distinct functions remain unknown. We show that CHP1 is predominantly in the nucleus in quiescent fibroblasts, is translocated to cytoplasmic compartments with growth medium, and that translocation is inhibited by mutations in the nuclear export motifs. In a screen for proteins co-precipitating with CHP1 in quiescent cells we identified the upstream binding factor UBF, a DNA-binding protein and component of the RNA polymerase I complex regulating RNA synthesis. The CHP1-UBF interaction is restricted to the nucleus and inhibited by Ca(2+). Nuclear retention of CHP1 attenuates the abundance of UBF in the nucleolus and inhibits RNA synthesis when quiescent cells are transferred to growth medium. These data show UBF as a newly identified CHP1-binding protein and regulation of RNA synthesis as a newly identified function for nuclear-localized CHP1, which is distinct from CHP1 functions in the cytosol.
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Affiliation(s)
- Maite Jiménez-Vidal
- Department of Cell and Tissue Biology, University of California, San Francisco, California 94143, USA
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22
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Sanij E, Poortinga G, Sharkey K, Hung S, Holloway TP, Quin J, Robb E, Wong LH, Thomas WG, Stefanovsky V, Moss T, Rothblum L, Hannan KM, McArthur GA, Pearson RB, Hannan RD. UBF levels determine the number of active ribosomal RNA genes in mammals. ACTA ACUST UNITED AC 2008; 183:1259-74. [PMID: 19103806 PMCID: PMC2606969 DOI: 10.1083/jcb.200805146] [Citation(s) in RCA: 157] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
In mammals, the mechanisms regulating the number of active copies of the approximately 200 ribosomal RNA (rRNA) genes transcribed by RNA polymerase I are unclear. We demonstrate that depletion of the transcription factor upstream binding factor (UBF) leads to the stable and reversible methylation-independent silencing of rRNA genes by promoting histone H1-induced assembly of transcriptionally inactive chromatin. Chromatin remodeling is abrogated by the mutation of an extracellular signal-regulated kinase site within the high mobility group box 1 domain of UBF1, which is required for its ability to bend and loop DNA in vitro. Surprisingly, rRNA gene silencing does not reduce net rRNA synthesis as transcription from remaining active genes is increased. We also show that the active rRNA gene pool is not static but decreases during differentiation, correlating with diminished UBF expression. Thus, UBF1 levels regulate active rRNA gene chromatin during growth and differentiation.
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Affiliation(s)
- Elaine Sanij
- Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia
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23
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Prieto JL, McStay B. Pseudo-NORs: a novel model for studying nucleoli. BIOCHIMICA ET BIOPHYSICA ACTA 2008; 1783:2116-23. [PMID: 18687368 DOI: 10.1016/j.bbamcr.2008.07.004] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/23/2008] [Revised: 07/08/2008] [Accepted: 07/08/2008] [Indexed: 11/21/2022]
Abstract
Nucleolar organiser regions (NORs) are comprised of tandem arrays of ribosomal gene (rDNA) repeats that are transcribed by RNA polymerase I (Pol I), ultimately resulting in formation of a nucleolus. Upstream binding factor (UBF), a DNA binding protein and component of the Pol I transcription machinery, binds extensively across the rDNA repeat in vivo. Pseudo-NORs are tandem arrays of a heterologous DNA sequence with high affinity for UBF introduced into human chromosomes. In this review we describe how analysis of pseudo-NORs has provided important insights into nucleolar formation. Pseudo-NORs mimic endogenous NORs in a number of important respects. On metaphase chromosomes both appear as secondary constrictions comprised of undercondensed chromatin. The transcriptional silence of pseudo-NORs provides a platform for studying the transcription independent recruitment of factors required for nucleolar formation by this specialised chromatin structure. During interphase, pseudo-NORs appear as distinct and novel sub-nuclear bodies. Analysis of these bodies and comparison to their endogenous counterpart has provided insights into nucleolar formation and structure.
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Affiliation(s)
- José-Luis Prieto
- Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY Scotland, UK
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24
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Abstract
In eukaryotes, the genes encoding ribosomal RNAs (rDNA) exist in two distinct epigenetic states that can be distinguished by a specific chromatin structure that is maintained throughout the cell cycle and is inherited from one cell to another. The fact that even in proliferating cells with a high demand of protein synthesis a fraction of rDNA is silenced provides a unique possibility to decipher the mechanism underlying epigenetic regulation of rDNA. This chapter summarizes our knowledge of the molecular mechanisms that establish and propagate the epigenetic state of rRNA genes, unraveling a complex interplay of DNA methyltransferases and histone-modifying enzymes that act in concert with chromatin remodeling complexes and RNA-guided mechanisms to define the transcriptional state of rDNA. We also review the critical role of the RNA polymerase I transcription factor UBF in the formation of active nucleolar organizer regions (NORs) and maintenance of the euchromatic state of rRNA genes.
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Affiliation(s)
- Brian McStay
- Biomedical Research Center, Ninewells Hospital, University of Dundee, Dundee DD1 9SY, United Kingdom.
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25
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Gorski JJ, Pathak S, Panov K, Kasciukovic T, Panova T, Russell J, Zomerdijk JCBM. A novel TBP-associated factor of SL1 functions in RNA polymerase I transcription. EMBO J 2007; 26:1560-8. [PMID: 17318177 PMCID: PMC1829371 DOI: 10.1038/sj.emboj.7601601] [Citation(s) in RCA: 75] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2006] [Accepted: 01/22/2007] [Indexed: 11/09/2022] Open
Abstract
In mammalian RNA polymerase I transcription, SL1, an assembly of TBP and associated factors (TAFs), is essential for preinitiation complex formation at ribosomal RNA gene promoters in vitro. We provide evidence for a novel component of SL1, TAF(I)41 (MGC5306), which functions in Pol I transcription. TAF(I)41 resides at the rDNA promoter in the nucleolus and co-purifies and co-immunoprecipitates with SL1. TAF(I)41 immunodepletion from nuclear extracts dramatically reduces Pol I transcription; addition of SL1 restores the ability of these extracts to support Pol I transcription. In cells, siRNA-mediated decreased expression of TAF(I)41 leads to loss of SL1 from the rDNA promoter in vivo, with concomitant loss of Pol I from the rDNA and reduced synthesis of the pre-rRNA. Extracts from these cells support reduced levels of Pol I transcription; addition of SL1 to the extracts raises the level of Pol I transcription. These data suggest that TAF(I)41 is integral to transcriptionally active SL1 and imply a role for SL1, including the TAF(I)41 subunit, in Pol I recruitment and, therefore, preinitiation complex formation in vivo.
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Affiliation(s)
- Julia J Gorski
- Division of Gene Regulation and Expression, College of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, UK
| | - Shalini Pathak
- Division of Gene Regulation and Expression, College of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, UK
| | - Kostya Panov
- Division of Gene Regulation and Expression, College of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, UK
| | - Taciana Kasciukovic
- Division of Gene Regulation and Expression, College of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, UK
| | - Tanya Panova
- Division of Gene Regulation and Expression, College of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, UK
| | - Jackie Russell
- Division of Gene Regulation and Expression, College of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, UK
| | - Joost C B M Zomerdijk
- Division of Gene Regulation and Expression, College of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, UK
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Panova TB, Panov KI, Russell J, Zomerdijk JCBM. Casein kinase 2 associates with initiation-competent RNA polymerase I and has multiple roles in ribosomal DNA transcription. Mol Cell Biol 2006; 26:5957-68. [PMID: 16880508 PMCID: PMC1592790 DOI: 10.1128/mcb.00673-06] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Mammalian RNA polymerase I (Pol I) complexes contain a number of associated factors, some with undefined regulatory roles in transcription. We demonstrate that casein kinase 2 (CK2) in human cells is associated specifically only with the initiation-competent Pol Ibeta isoform and not with Pol Ialpha. Chromatin immunoprecipitation analysis places CK2 at the ribosomal DNA (rDNA) promoter in vivo. Pol Ibeta-associated CK2 can phosphorylate topoisomerase IIalpha in Pol Ibeta, activator upstream binding factor (UBF), and selectivity factor 1 (SL1) subunit TAFI110. A potent and selective CK2 inhibitor, 3,8-dibromo-7-hydroxy-4-methylchromen-2-one, limits in vitro transcription to a single round, suggesting a role for CK2 in reinitiation. Phosphorylation of UBF by CK2 increases SL1-dependent stabilization of UBF at the rDNA promoter, providing a molecular mechanism for the stimulatory effect of CK2 on UBF activation of transcription. These positive effects of CK2 in Pol I transcription contrast to that wrought by CK2 phosphorylation of TAFI110, which prevents SL1 binding to rDNA, thereby abrogating the ability of SL1 to nucleate preinitiation complex (PIC) formation. Thus, CK2 has the potential to regulate Pol I transcription at multiple levels, in PIC formation, activation, and reinitiation of transcription.
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Affiliation(s)
- Tatiana B Panova
- Division of Gene Regulation and Expression, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom
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27
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Lin CY, Navarro S, Reddy S, Comai L. CK2-mediated stimulation of Pol I transcription by stabilization of UBF-SL1 interaction. Nucleic Acids Res 2006; 34:4752-66. [PMID: 16971462 PMCID: PMC1635259 DOI: 10.1093/nar/gkl581] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
High levels of rRNA synthesis by RNA polymerase I are important for cell growth and proliferation. In vitro studies have indicated that the formation of a stable complex between the HMG box factor [Upstream binding factor (UBF)] and SL1 at the rRNA gene promoter is necessary to direct multiple rounds of Pol I transcription initiation. The recruitment of SL1 to the promoter occurs through protein interactions with UBF and is regulated by phosphorylation of UBF. Here we show that the protein kinase CK2 co-immunoprecipitates with the Pol I complex and is associated with the rRNA gene promoter. Inhibition of CK2 kinase activity reduces Pol I transcription in cultured cells and in vitro. Significantly, CK2 regulates the interaction between UBF and SL1 by counteracting the inhibitory effect of HMG boxes five and six through the phosphorylation of specific serines located at the C-terminus of UBF. Transcription reactions with immobilized templates indicate that phosphorylation of CK2 phosphoacceptor sites in the C-terminal domain of UBF is important for promoting multiple rounds of Pol I transcription. These data demonstrate that CK2 is recruited to the rRNA gene promoter and directly regulates Pol I transcription re-initiation by stabilizing the association between UBF and SL1.
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Affiliation(s)
| | | | - Sita Reddy
- Department of Biochemistry and Molecular Biology, Institute for Genetic Medicine, Keck School of Medicine, University of Southern California2250 Alcazar Street, Los Angeles, CA, 90033, USA
| | - Lucio Comai
- To whom correspondence should be addressed. Tel: +1 323 442 3950; Fax: +1 323 441 2764;
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28
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Panov KI, Friedrich JK, Russell J, Zomerdijk JCBM. UBF activates RNA polymerase I transcription by stimulating promoter escape. EMBO J 2006; 25:3310-22. [PMID: 16858408 PMCID: PMC1523182 DOI: 10.1038/sj.emboj.7601221] [Citation(s) in RCA: 79] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2006] [Accepted: 06/09/2006] [Indexed: 01/10/2023] Open
Abstract
Ribosomal RNA gene transcription by RNA polymerase I (Pol I) is the driving force behind ribosome biogenesis, vital to cell growth and proliferation. The key activator of Pol I transcription, UBF, has been proposed to act by facilitating recruitment of Pol I and essential basal factor SL1 to rDNA promoters. However, we found no evidence that UBF could stimulate recruitment or stabilization of the pre-initiation complex (PIC) in reconstituted transcription assays. In this, UBF is fundamentally different from archetypal activators of transcription. Our data imply that UBF exerts its stimulatory effect on RNA synthesis, after PIC formation, promoter opening and first phosphodiester bond formation and before elongation. We provide evidence to suggest that UBF activates transcription in the transition between initiation and elongation, at promoter escape by Pol I. This novel role for UBF in promoter escape would allow control of rRNA synthesis at active rDNA repeats, independent of and complementary to the promoter-specific targeting of SL1 and Pol I during PIC assembly. We posit that stimulation of promoter escape could be a general mechanism of activator function.
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Affiliation(s)
- Kostya I Panov
- Division of Gene Regulation and Expression, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, UK
| | - J Karsten Friedrich
- Division of Gene Regulation and Expression, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, UK
| | - Jackie Russell
- Division of Gene Regulation and Expression, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, UK
| | - Joost C B M Zomerdijk
- Division of Gene Regulation and Expression, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, UK
- Division of Gene Regulation and Expression, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, UK. Tel.: +44 1382 384242; Fax: +44 1382 388072; E-mail:
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29
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Stefanovsky V, Langlois F, Gagnon-Kugler T, Rothblum LI, Moss T. Growth factor signaling regulates elongation of RNA polymerase I transcription in mammals via UBF phosphorylation and r-chromatin remodeling. Mol Cell 2006; 21:629-39. [PMID: 16507361 DOI: 10.1016/j.molcel.2006.01.023] [Citation(s) in RCA: 163] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2005] [Revised: 12/14/2005] [Accepted: 01/17/2006] [Indexed: 01/21/2023]
Abstract
Synthesis of the 45S rRNA by RNA polymerase I limits cell growth. Knowledge of the mechanism of its regulation is therefore key to understanding growth control. rRNA transcription is believed to be regulated solely at initiation/promoter release. However, we found that stimulation of endogenous 45S rRNA synthesis by epidermal growth factor (EGF) and serum failed to induce an increase in RNA polymerase I engagement on the rRNA genes, despite robust enhancement of 45S rRNA synthesis. Further, endogenous transcription elongation rates were measured and found to be directly proportional to 45S rRNA synthesis. Thus, elongation is a rate-limiting step for rRNA synthesis in vivo. ERK phosphorylation of the HMG boxes of UBF, an RNA polymerase I factor essential for transcription enhancement, was shown to directly regulate elongation by inducing the remodeling of ribosomal gene chromatin. The data suggest a mechanism for coordinating the cotranscriptional assembly of preribosomal particles.
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Affiliation(s)
- Victor Stefanovsky
- Cancer Research Centre and Department of Medical Biology, Laval University, Hôtel-Dieu de Québec, 11 côte du Palais, G1R 2J6 Québec, Canada
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30
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Abstract
The rRNAs constitute the catalytic and structural components of the ribosome, the protein synthesis machinery of cells. The level of rRNA synthesis, mediated by Pol I (RNA polymerase I), therefore has a major impact on the life and destiny of a cell. In order to elucidate how cells achieve the stringent control of Pol I transcription, matching the supply of rRNA to demand under different cellular growth conditions, it is essential to understand the components and mechanics of the Pol I transcription machinery. In this review, we discuss: (i) the molecular composition and functions of the Pol I enzyme complex and the two main Pol I transcription factors, SL1 (selectivity factor 1) and UBF (upstream binding factor); (ii) the interplay between these factors during pre-initiation complex formation at the rDNA promoter in mammalian cells; and (iii) the cellular control of the Pol I transcription machinery.
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Affiliation(s)
- Jackie Russell
- Division of Gene Regulation and Expression, School of Life Sciences, University of Dundee, Wellcome Trust Biocentre, Dundee DD1 5EH, U.K
| | - Joost C.B.M. Zomerdijk
- Division of Gene Regulation and Expression, School of Life Sciences, University of Dundee, Wellcome Trust Biocentre, Dundee DD1 5EH, U.K
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31
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Friedrich JK, Panov KI, Cabart P, Russell J, Zomerdijk JCBM. TBP-TAF complex SL1 directs RNA polymerase I pre-initiation complex formation and stabilizes upstream binding factor at the rDNA promoter. J Biol Chem 2005; 280:29551-8. [PMID: 15970593 PMCID: PMC3858828 DOI: 10.1074/jbc.m501595200] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Knowledge of the role of components of the RNA polymerase I transcription machinery is paramount to understanding regulation of rDNA expression. We describe key findings for the roles of essential transcription factor SL1 and activator upstream binding factor (UBF). We demonstrate that human SL1 can direct accurate Pol I transcription in the absence of UBF and can interact with the rDNA promoter independently and stably, consistent with studies of rodent SL1 but contrary to previous reports of human SL1. UBF itself does not bind stably to rDNA but rapidly associates and dissociates. We show that SL1 significantly reduces the rate of dissociation of UBF from the rDNA promoter. Our findings challenge the idea that UBF activates transcription through recruitment of SL1 at the rDNA promoter and suggest that the rate of pre-initiation complex (PIC) formation is primarily determined by the rate of association of SL1, rather than UBF, with the promoter. Therefore, we propose that SL1 directs PIC formation, functioning in core promoter binding, RNA polymerase I recruitment, and UBF stabilization and that SL1-promoter complex formation is a necessary prerequisite to the assembly of functional and stable PICs that include the UBF activator in mammalian cells.
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Affiliation(s)
| | - Kostya I. Panov
- Division of Gene Regulation and Expression, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom
| | | | - Jackie Russell
- Division of Gene Regulation and Expression, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom
| | - Joost C. B. M. Zomerdijk
- Division of Gene Regulation and Expression, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom
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32
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Zhang X, Zhang J, Li X, Xu J, Huang H, Chen Q, Wu J, Shi Y. Compact molten globule-like state of hUBF HMG Box1 at extremely low pH. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2005; 1748:66-73. [PMID: 15752694 DOI: 10.1016/j.bbapap.2004.12.002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/21/2004] [Revised: 11/28/2004] [Accepted: 12/15/2004] [Indexed: 11/18/2022]
Abstract
Using far and near-UV CD, ANS fluorescence and 2D NMR spectroscopy, an acid-induced partly folded state (A state) at extremely low pH for hUBF HMG Box1 was identified and characterized. As compared to the native state (N), the A state has similar secondary structure, less compact pack with larger amounts of exposed hydrophobic surface, and narrower chemical shift dispersion in (1)H-(15)N HSQC spectrum, which implies that it is a molten globule (MG)-like species. On the other hand, substantial tertiary contacts and cooperative thermal denaturing transition indicate that the A state is closer-relative to the classic MG-to the native folded state. In addition, when the solution pH is adjusted to neutrality, the protein in the A state refolds to the native state easily. All these data suggest that the A state of hUBF HMG Box1 could represent a potential folding intermediate on protein folding pathway.
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Affiliation(s)
- Xuecheng Zhang
- School of Life Science, University of Science and Technology of China, People's Republic of China
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33
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Chen D, Dundr M, Wang C, Leung A, Lamond A, Misteli T, Huang S. Condensed mitotic chromatin is accessible to transcription factors and chromatin structural proteins. ACTA ACUST UNITED AC 2004; 168:41-54. [PMID: 15623580 PMCID: PMC2171683 DOI: 10.1083/jcb.200407182] [Citation(s) in RCA: 158] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/03/2022]
Abstract
During mitosis, chromosomes are highly condensed and transcription is silenced globally. One explanation for transcriptional repression is the reduced accessibility of transcription factors. To directly test this hypothesis and to investigate the dynamics of mitotic chromatin, we evaluate the exchange kinetics of several RNA polymerase I transcription factors and nucleosome components on mitotic chromatin in living cells. We demonstrate that these factors rapidly exchange on and off ribosomal DNA clusters and that the kinetics of exchange varies at different phases of mitosis. In addition, the nucleosome component H1c-GFP also shows phase-specific exchange rates with mitotic chromatin. Furthermore, core histone components exchange at detectable levels that are elevated during anaphase and telophase, temporally correlating with H3-K9 acetylation and recruitment of RNA polymerase II before the onset of bulk RNA synthesis at mitotic exit. Our findings indicate that mitotic chromosomes in general and ribosomal genes in particular, although highly condensed, are accessible to transcription factors and chromatin proteins. The phase-specific exchanges of nucleosome components during late mitotic phases are consistent with an emerging model of replication independent core histone replacement.
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Affiliation(s)
- Danyang Chen
- Department of Cell and Molecular Biology, The Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
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34
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Abstract
In vitro production (IVP) of porcine embryos including in vitro maturation (IVM) of oocytes followed by in vitro fertilization (IVF) and in vitro culture (IVC) of the resultant embryos may result in live offspring, but it is still associated with great inefficiencies probably due to incomplete cytoplasmic maturation of the oocytes in vitro. Therefore, fundamental knowledge on the regulation of transcription during the oocyte growth phase when the messengers and protein synthetic machinery necessary for oocyte developmental competence are formed, is of great importance. In mammals, synthesis of RNA, up to 60-70% of which is ribosomal (rRNA), increases during oocyte growth and reaches a peak at the beginning of follicular antrum formation. In oocytes at the end of the growth phase, acquisition of full meiotic competence coincides with a markedly decreased rRNA transcriptional activity in the gametes. Our recent studies on the porcine oocyte growth phase have revealed a deeper molecular and biological insight into the complex regulation of rRNA transcription at different stages of follicular development. The data indicate that the so-called pocket protein, p130, is involved in the down-regulation of rRNA transcription at the end of the oocyte growth phase through an inhibition of the action of upstream binding factor (UBF). The latter protein is necessary for the function of RNA polymerase I (RNA Pol I), which is the actual enzyme driving rRNA gene transcription. Moreover, rRNA transcription also appears to be down-regulated by a decrease in the expression of mRNA encoding PAF53, an RNA Pol I-associated factor also required for the polymerase to exert its action. At the ultrastructural level, these molecular changes are paralleled by marginalization of the fibrillar centres of the oocyte nucleolus followed by compaction of the nucleolus into an inactive sphere of fibrils.
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Affiliation(s)
- B Bjerregaard
- Department of Animal and Veterinary Basic Sciences, The Royal Veterinary and Agricultural University, Groennegaardsvej 7, DK-1870 Frederiksberg C, Denmark
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35
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Affiliation(s)
- Lucio Comai
- Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
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36
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Halkidou K, Logan IR, Cook S, Neal DE, Robson CN. Putative involvement of the histone acetyltransferase Tip60 in ribosomal gene transcription. Nucleic Acids Res 2004; 32:1654-65. [PMID: 15016909 PMCID: PMC390321 DOI: 10.1093/nar/gkh296] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Tip60 is a histone acetyltransferase (HAT) implicated in a wide range of cellular functions, including mRNA synthesis and DNA repair. In the present report we propose a model based on which Tip60 is actively involved in ribosomal gene transcription through acetylation of UBF, a ribosomal specific transcription factor, as well as through its direct recruitment to the human ribosomal gene promoter, as shown by chromatin immunoprecipitation experiments. Electron microscopy studies revealed that Tip60 resides in sites of active rDNA transcription within the nucleolus, while it co-localizes with UBF as shown by confocal microscopy. In addition, in vivo transcription assays demonstrated that the nucleolar fraction of Tip60 localizes to sites of newly synthesized rRNA. Finally, functional assays established that Tip60 complexes with, and targets UBF for acetylation. The present study underlines the importance of acetylation in rDNA transcription and directly implicates Tip60 in the process of ribosomal gene transcription.
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Affiliation(s)
- Kalipso Halkidou
- Prostate Research Group, School of Surgical and Reproductive Sciences, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK
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37
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Bjerregaard B, Wrenzycki C, Philimonenko VV, Hozak P, Laurincik J, Niemann H, Motlik J, Maddox-Hyttel P. Regulation of Ribosomal RNA Synthesis During the Final Phases of Porcine Oocyte Growth. Biol Reprod 2003; 70:925-35. [PMID: 14627545 DOI: 10.1095/biolreprod.103.020941] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022] Open
Abstract
In porcine oocytes, acquisition of meiotic competence coincides with a decrease of general transcriptional activity at the end of the oocyte growth phase and, specifically, of ribosomal RNA (rRNA) synthesis in the nucleolus. The present study investigated the regulation of rRNA synthesis during porcine oocyte growth. Localization and expression of components involved in regulation of the rRNA synthesis (the RNA polymerase I-associated factor PAF53, upstream binding factor [UBF], and the pocket proteins p130 and pRb) were assessed by immunocytochemistry and semiquantitative reverse transcription-polymerase chain reaction and correlated with ultrastructural analysis and autoradiography following [3H]uridine incubation in growing and fully grown porcine oocytes. In addition, meiotic resumption, ultrastructure, and expression of p130, UBF, and PAF53 were analyzed in growing and fully grown porcine oocytes cultured with 100 microM butyrolactone I (BL-I), a potent inhibitor of cyclin-dependent kinases, to gain insight concerning the regulation of rRNA transcription during meiotic arrest. Immunocytochemical analysis demonstrated that p130 became colocalized with UBF and PAF53 and that the intensity of the PAF53 labeling decreased toward the end of the oocyte growth phase. These data suggest that the decrease in rRNA synthesis is regulated through inhibition of UBF by p130 as well as by decreased availability of PAF53. Moreover, expression of mRNA encoding PAF53 was decreased at the end of the oocyte growth phase. At the morphological level, these events coincided with inactivation of the nucleolus, as visualized by the transformation of the fibrillogranular nucleolus to an electron-dense fibrillar sphere with remnants of the fibrillar centers at the surface. Meiotic inhibition with 100 microM BL-I had a detrimental effect on the ability of porcine oocytes to resume meiosis and on nucleolus morphology, resulting in a lack of RNA synthetic capability as the fibrillar components, where rRNA transcription and initial processing occur, condensed or even disintegrated.
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Affiliation(s)
- Bolette Bjerregaard
- Department of Anatomy and Physiology, Royal Veterinary and Agricultural University, 1870 Frederiksberg, Denmark
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38
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Grueneberg DA, Pablo L, Hu KQ, August P, Weng Z, Papkoff J. A functional screen in human cells identifies UBF2 as an RNA polymerase II transcription factor that enhances the beta-catenin signaling pathway. Mol Cell Biol 2003; 23:3936-50. [PMID: 12748295 PMCID: PMC155208 DOI: 10.1128/mcb.23.11.3936-3950.2003] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2002] [Revised: 10/21/2002] [Accepted: 02/28/2003] [Indexed: 11/20/2022] Open
Abstract
beta-Catenin signaling plays an important role in the development of many organisms and has a key part in driving the malignant transformation of epithelial cells comprising a variety of cancers. beta-Catenin can activate gene expression through its association with transcription factors of the lymphoid enhancer factor 1 (LEF-1)/T-cell factor (TCF) family. We designed a screen in human cells to identify novel genes that activate a beta-catenin-LEF/TCF-responsive promoter and isolated the high-mobility group box transcription factor, UBF2. UBF1 and UBF2 are splice variants of a common precursor RNA. Although UBF1 has been shown to activate RNA polymerase I-regulated genes, the function of UBF2 has remained obscure. Here, we show for the first time that both UBF1 and UBF2 activate RNA polymerase II-regulated promoters. UBF2 associates with LEF-1, as shown by coimmunoprecipitation experiments, and potentiates transcriptional activation stimulated by LEF-1/beta-catenin from a synthetic promoter with multimerized LEF/TCF binding sites and a natural cyclin D1 promoter with consensus LEF/TCF binding sites. Downregulation of endogenous UBF expression using an RNA interference approach reduces transcriptional activation of a beta-catenin-LEF/TCF-responsive promoter by means of overexpressed beta-catenin, further implicating UBF as a transcriptional enhancer of the beta-catenin pathway.
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39
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Lin CY, Tuan J, Scalia P, Bui T, Comai L. The cell cycle regulatory factor TAF1 stimulates ribosomal DNA transcription by binding to the activator UBF. Curr Biol 2002; 12:2142-6. [PMID: 12498690 DOI: 10.1016/s0960-9822(02)01389-1] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Control of ribosome biogenesis is a potential mechanism for the regulation of cell size during growth, and a key step in regulating ribosome production is ribosomal RNA synthesis by RNA polymerase I (Pol I). In humans, Pol I transcription requires the upstream binding factor UBF and the selectivity factor SL1 to assemble coordinately on the promoter. UBF is an HMG box-containing factor that binds to the rDNA promoter and activates Pol I transcription through its acidic carboxy-terminal tail. Using UBF (284-670) as bait in a yeast two-hybrid screen, we have identified an interaction between UBF and TAF1, a factor involved in the transcription of cell cycle and growth regulatory genes. Coimmunoprecipitation and protein-protein interaction assays confirmed that TAF1 binds to UBF. Confocal microscopy showed that TAF1 colocalizes with UBF in Hela cells, and cell fractionation experiments provided further evidence that a portion of TAF1 is localized in the nucleolus, the organelle devoted to ribosomal DNA transcription. Cotransfection and in vitro transcription assays showed that TAF1 stimulates Pol I transcription in a dosage-dependent manner. Thus, TAF1 may be involved in the coordinate expression of Pol I- and Pol II-transcribed genes required for protein biosynthesis and cell cycle progression.
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Affiliation(s)
- Chih-Yin Lin
- Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA
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Yuan X, Zhao J, Zentgraf H, Hoffmann-Rohrer U, Grummt I. Multiple interactions between RNA polymerase I, TIF-IA and TAF(I) subunits regulate preinitiation complex assembly at the ribosomal gene promoter. EMBO Rep 2002; 3:1082-7. [PMID: 12393749 PMCID: PMC1307603 DOI: 10.1093/embo-reports/kvf212] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
In mammals, growth-dependent regulation of rRNA synthesis is brought about by the transcription initiation factor TIF-IA. TIF-IA is associated with a fraction of the TBP-containing factor TIF-IB/SL1 and the initiation-competent form of RNA polymerase I (Pol I). We investigated the mechanisms that down-regulate cellular pre-rRNA synthesis and demonstrate that nutrient starvation, density arrest and protein synthesis inhibitors inactivate TIF-IA and impair the association of TIF-IA with Pol I. Moreover, we used a panel of TIF-IA deletion mutants to map the domains that mediate the interaction of TIF-IA with Pol I and TIF-IB/SL1. We found that amino acids 512-609 interact with two subunits of Pol I, RPA43 and PAF67, whereas a short, conserved motif (LARAK, amino acids 411-415) is required for the association of TIF-IA with TAF(I)95 and TAF(I)68. The results uncover an interphase for essential protein-protein interactions that facilitate Pol I preinitiation complex formation.
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Affiliation(s)
- Xuejun Yuan
- Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
| | - Jian Zhao
- Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
| | - Hanswalter Zentgraf
- Applied Tumor Virology, German Cancer Research Center, D-69120 Heidelberg, Germany
| | - Urs Hoffmann-Rohrer
- Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
| | - Ingrid Grummt
- Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
- Tel: +49 6221 423423; Fax: +49 6221 423404;
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41
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Yang W, Zeng W, Zhou D, Shi Y. Cloning, expression, secondary structure characterization of HMG box 1 of hUBF from E. coli and its binding to DNA. BIOCHIMICA ET BIOPHYSICA ACTA 2002; 1598:147-155. [PMID: 12147355 DOI: 10.1016/s0167-4838(02)00367-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Human upstream binding factor (hUBF) belonging to a family of protein containing DNA binding domain-HMG box, is important in the activation of rRNA gene transcription. It contains six tandemly arranged HMG box domains, each of which is thought to be as a basic architectural unit in the interaction of DNA and protein. Here the DNA binding domain of hUBF HMG box 1 was cloned and heterologously expressed in Escherichia coli. Through a single purification step using a Ni2+-chelating column, the highly purified recombinant protein could be obtained. This recombinant protein contains 99 amino acids with a hexahistidine tag added to the C-terminus. It was expressed as a monomer, which was determined by gel filtration. Circular dischroism studies show that it comprises approximately 54.3% alpha-helix and 43.6% random coil at pH 7. This result is in good agreement with that of FTIR, which are 59.9% alpha-helix and 40.1% random coil. There is no obvious change for the secondary structure of the recombinant protein as increasing pH from 5.0 to 12.0. But denaturation occurs at pH 3.0. Like many HMG box domains that were found in other proteins, it could bind to four-way DNA junction, a putative intermediate in DNA recombination, in a structure-specific manner. Magnesium ion has no effect on this binding activity, which is determined by both gel mobility shift assays and surface plasmon resonance (SPR). Since Mg2+ is present in the nucleus and RNA polymerase I is Mg2+-stimulated, we believe that this property is relevant for hUBF in vivo. SPR research shows that the recombinant hUBF HMG box 1 also has a strong binding ability to a GC-rich fragment within the rRNA gene core promoter.
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Affiliation(s)
- Wulin Yang
- Laboratory of Structure Biology, School of Life Science, University of Science and Technology of China, Anhui, Hefei, People's Republic of China
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Abstract
The general transcription factor TFIID facilitates recruitment of the transcription machinery to gene promoters and regulates initiation of transcription by RNA polymerase II. hTAF(II)130, a component of TFIID, interacts with and serves as a coactivator for multiple transcriptional regulatory proteins, including Sp1 and CREB. A yeast two-hybrid screen has identified an interaction between hTAF(II)130 and heterochromatin protein 1 (HP1), a chromatin-associated protein whose function has been implicated in gene silencing. We find that hTAF(II)130 associates with HP1 in an isoform-specific manner: HP1alpha and HP1gamma bind to hTAF(II)130, but not HP1beta. In addition, we show that endogenous hTAF(II)130 and components of TFIID in HeLa nuclear extracts associate with glutathione S-transferase-HP1alpha and -HP1gamma. hTAF(II)130 possesses a pentapeptide HP1-binding motif, and mutation of the hTAF(II)130 HP1 box compromises the interaction of hTAF(II)130 with HP1. We demonstrate that Gal4-HP1 proteins interfere with hTAF(II)130-mediated activation of transcription. Our results suggest that HP1alpha and HP1gamma associate with hTAF(II)130 to mediate repression of transcription, supporting a new model of transcriptional repression involving a specific interaction between a component of TFIID and chromatin.
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Affiliation(s)
- Milo F Vassallo
- Department of Microbiology and Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, NY 10016, USA
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43
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Al-Khouri AM, Paule MR. A novel RNA polymerase I transcription initiation factor, TIF-IE, commits rRNA genes by interaction with TIF-IB, not by DNA binding. Mol Cell Biol 2002; 22:750-61. [PMID: 11784852 PMCID: PMC133551 DOI: 10.1128/mcb.22.3.750-761.2002] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE.
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Affiliation(s)
- Anna Maria Al-Khouri
- Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA
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44
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45
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Stefanovsky VY, Pelletier G, Hannan R, Gagnon-Kugler T, Rothblum LI, Moss T. An immediate response of ribosomal transcription to growth factor stimulation in mammals is mediated by ERK phosphorylation of UBF. Mol Cell 2001; 8:1063-73. [PMID: 11741541 DOI: 10.1016/s1097-2765(01)00384-7] [Citation(s) in RCA: 189] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Ribosomal transcription in mammals is regulated in response to growth, differentiation, disease, and aging, but the mechanisms of this regulation have remained unresolved. We show that epidermal growth factor induces immediate, ERK1/2-dependent activation of endogenous ribosomal transcription, while inactivation of ERK1/2 causes an equally immediate reversion to the basal transcription level. ERK1/2 was found to phosphorylate the architectural transcription factor UBF at amino acids 117 and 201 within HMG boxes 1 and 2, preventing their interaction with DNA. Mutation of these sites inhibited transcription activation and abrogated the transcriptional response to ERK1/2. Thus, growth factor regulation of ribosomal transcription likely acts by a cyclic modulation of DNA architecture. The data suggest a central role for ribosome biogenesis in growth regulation.
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Affiliation(s)
- V Y Stefanovsky
- Cancer Research Centre and Department of Medical Biology, Laval University, Hôtel-Dieu de Québec, 11 côte du Palais, G1R 2J6, Québec, Canada
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46
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Dühr S, Torres-Montaner A, Astola A, García-Cozar FJ, Pendón C, Bolívar J, Valdivia MM. Molecular analysis of the 5' region of human ribosomal transcription factor UBF. DNA SEQUENCE : THE JOURNAL OF DNA SEQUENCING AND MAPPING 2001; 12:267-72. [PMID: 11916260 DOI: 10.3109/10425170109025001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Upstream binding factor, UBF, is a nucleolar autoantigen involved in the transcription of ribosomal DNA genes. Previously, human genomic clones served to demonstrate that an alternative pre-mRNA splicing of a single gene is used to form UBF1 and UBF2. Here, to complete characterizing the 5'end genomic organization of this nucleolar transcription factor, lambda clones containing the human UBF gene were isolated from a human placenta genomic library using a hamster UBF cDNA as a probe. An additional PCR product was isolated from HeLa genomic DNA to cover the first translated 60 nt of the gene containing the ATG initiation codon. We have also determined the transcription start site of the gene by primer extension analysis at nt 188 upstream from the start ATG codon. It served first, to identify an untranslated initial exon on the UBF gene covering the first 121 nt of human UBF cDNA, and also to establish the sequence of the proximal promoter. The human UBF promoter lacks a TATA and CAAT boxes but contains multiple binding sites for SP1, AP1, AP2, TFIID, NF-1 and a single site for NFAT-1. Consequently we have defined the first five exons of the human UBF gene covering 7.5kb. The complete gene now consists of 20 exons with intervening sequences and spans approximately 15kb of DNA.
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Affiliation(s)
- S Dühr
- Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Cádiz, Spain
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Hirschler-Laszkiewicz I, Cavanaugh A, Hu Q, Catania J, Avantaggiati ML, Rothblum LI. The role of acetylation in rDNA transcription. Nucleic Acids Res 2001; 29:4114-24. [PMID: 11600700 PMCID: PMC60214 DOI: 10.1093/nar/29.20.4114] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2001] [Accepted: 08/27/2001] [Indexed: 12/14/2022] Open
Abstract
Treatment of NIH 3T3 cells with trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), resulted in a dose-dependent increase in transcription from a rDNA reporter and from endogenous rRNA genes. Chromatin immunoprecipitation using anti-acetyl-histone H4 antibodies demonstrated a direct effect of TSA on the acetylation state of the ribosomal chromatin. TSA did not reverse inhibition of transcription from the rDNA reporter by retinoblastoma (Rb) protein, suggesting that the main mechanism by which Rb blocks rDNA transcription may not involve recruitment of deacetylases to rDNA chromatin. Overexpression of histone transacetylases p300, CBP and PCAF stimulated transcription in transfected NIH 3T3 cells. Recombinant p300, but not PCAF, stimulated rDNA transcription in vitro in the absence of nucleosomes, suggesting that the stimulation of rDNA transcription by TSA might have a chromatin-independent component. We found that the rDNA transcription factor UBF was acetylated in vivo. Finally, we also demonstrated the nucleolar localization of CBP. Our results suggest that the organization of ribosomal chromatin of higher eukaryotes is not static and that acetylation may be involved in affecting these dynamic changes directly through histone acetylation and/or through acetylation of UBF or one of the other components of rDNA transcription.
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Affiliation(s)
- I Hirschler-Laszkiewicz
- The Henry Hood Research Program, Sigfried and Janet Weis Center for Research, The Geisinger Clinic, 100 North Academy Avenue, Danville, PA 17822-2618, USA
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48
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Stefanovsky VY, Pelletier G, Bazett-Jones DP, Crane-Robinson C, Moss T. DNA looping in the RNA polymerase I enhancesome is the result of non-cooperative in-phase bending by two UBF molecules. Nucleic Acids Res 2001; 29:3241-7. [PMID: 11470882 PMCID: PMC55825 DOI: 10.1093/nar/29.15.3241] [Citation(s) in RCA: 75] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
The so-called upstream binding factor (UBF) is required for the initial step in formation of an RNA polymerase I initiation complex. This function of UBF correlates with its ability to induce the ribosomal enhancesome, a structure which resembles in its mass and DNA content the nucleosome of chromatin. DNA looping in the enhancesome is probably the result of six in-phase bends induced by the HMG boxes of a UBF dimer. Here we show that insertion/deletion mutations in the basic peptide linker lying between the N-terminal dimerisation domain and the first HMG box of Xenopus UBF prevent the DNA looping characteristic of the enhancesome. Using these mutants we demonstrate that (i) the enhancesome structure does not depend on tethering of the entering and exiting DNA duplexes, (ii) UBF monomers induce hemi-enhancesomes, bending the DNA by 175 +/- 24 degrees and (iii) two hemi-enhancesomes are precisely phased by UBF dimerisation. We use this and previous data to refine the existing enhancesome model and show that HMG boxes 1 and 2 of UBF lie head-to-head along the DNA.
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Affiliation(s)
- V Y Stefanovsky
- Cancer Research Centre and Department of Medical Biology, Laval University, Hôtel-Dieu de Québec, 11 Côte du Palais, Québec G1R 2J6, Canada
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Hosking BM, Wyeth JR, Pennisi DJ, Wang SC, Koopman P, Muscat GE. Cloning and functional analysis of the Sry-related HMG box gene, Sox18. Gene 2001; 262:239-47. [PMID: 11179689 DOI: 10.1016/s0378-1119(00)00525-4] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
The Sox gene family (Sry like HMG box gene) is characterised by a conserved DNA sequence encoding a domain of approximately 80 amino acids which is responsible for sequence specific DNA binding. We initially published the identification and partial cDNA sequence of murine Sox18, a new member of this gene family, isolated from a cardiac cDNA library. This sequence allowed us to classify Sox18 into the F sub-group of Sox proteins, along with Sox7 and Sox17. Recently, we demonstrated that mutations in the Sox18 activation domain underlie cardiovascular and hair follicle defects in the mouse mutation, ragged (Ra) (Pennisi et al., 2000. Mutations in Sox18 underlie cardiovascular and hair follicle defecs in ragged mice. Nat. Genet. 24, 434-437). Ra homozygotes lack vibrissae and coat hairs, have generalised oedema and an accumulation of chyle in the peritoneum. Here we have investigated the genomic sequences encoding Sox18. Screening of a mouse genomic phage library identified four overlapping clones, we sequenced a 3.25 kb XbaI fragment that defined the entire coding region and approximately 1.5 kb of 5' flanking sequences. This identified (i) an additional 91 amino acids upstream of the previously designated methionine start codon in the original cDNA, and (ii) an intron encoded within the HMG box/DNA binding domain in exactly the same position as that found in the Sox5, -13 and -17 genes. The Sox18 gene encodes a protein of 468 aa. We present evidence that suggests HAF-2, the human HMG-box activating factor -2 protein, is the orthologue of murine Sox18. HAF-2 has been implicated in the regulation of the Human IgH enhancer in a B cell context. Random mutagenesis coupled with GAL4 hybrid analysis in the activation domain between amino acids 252 and 346, of Sox18, implicated the phosphorylation motif, SARS, and the region between amino acid residues 313 and 346 as critical components of Sox18 mediated transactivation. Finally, we examined the expression of Sox18 in multiple adult mouse tissues using RT-PCR. Low-moderate expression was observed in spleen, stomach, kidney, intestine, skeletal muscle and heart. Very abundant expression was detected in lung tissue.
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Affiliation(s)
- B M Hosking
- University of Queensland, Institute for Molecular Bioscience, Centre for Molecular and Cellular Biology, Ritchie Research Laboratories, B402A, St Lucia, 4072, Queensland, Australia
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50
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Lee KB, Thomas JO. The effect of the acidic tail on the DNA-binding properties of the HMG1,2 class of proteins: insights from tail switching and tail removal. J Mol Biol 2000; 304:135-49. [PMID: 11080451 DOI: 10.1006/jmbi.2000.4206] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The high-mobility group (HMG) proteins HMG1, HMG2 and HMG2a are relatively abundant vertebrate DNA-binding and bending proteins that bind with structure specificity, rather than sequence specificity, and appear to play an architectural role in the assembly of nucleoprotein complexes. They have two homologous "HMG-box" DNA-binding domains (which show about 80 % homology) connected by a short basic linker to an acidic carboxy-terminal tail that differs in length between HMG1 and 2. To gain insights into the role of the acidic tail, we examined the DNA-binding properties of HMG1, HMG2b and HMG2a from chicken erythrocytes (corresponding to HMG1, HMG2 and HMG2a in other vertebrates). HMG1, with the longest acidic tail, is less effective than HMG2a and 2b (at a given molar input ratio) in supercoiling relaxed, closed circular DNA, in inducing ligase-mediated circularisation of an 88 bp DNA fragment, and in binding to four-way DNA junctions in a gel-shift assay. Removal of the acidic tail increases the affinity of the HMG boxes for DNA and largely abolishes the differences between the three species. Switching the acidic tail of HMG1 for that of HMG2a or 2b gives hybrid proteins with essentially the same DNA-binding properties as HMG2a, 2b. The length (and possibly sequence) of the acidic tail thus appears to be the dominant factor in mediating the differences in properties between HMG1, 2a and 2b and finely tunes the rather similar DNA-binding properties of the tandem HMG boxes, presumably to fulfill different cellular roles. The tail is essential for structure-selective DNA-binding of the HMG boxes to DNA minicircles in the presence of equimolar linear DNA, and has little effect on the affinity for this already highly distorted DNA ligand, in contrast to binding to linear and four-way junction DNA.
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Affiliation(s)
- K B Lee
- Cambridge Centre for Molecular Recognition and Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK
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