DOCSLIB.ORG

Protein-Protein and Protein-DNA Interactions of the Human C2H2 Zinc Finger Proteins

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Protein-Protein and Protein-DNA Interactions of the Human C2H2 Zinc Finger Proteins Protein-protein and Protein-DNA Interactions of the Human C2H2 Zinc Finger Proteins by Ernest Radovani A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Molecular Genetics University of Toronto © Copyright 2019 by Ernest Radovani Protein-protein and protein-DNA interactions of the human C2H2 zinc finger proteins Ernest Radovani Doctor of Philosophy in the Graduate Department of Molecular Genetics University of Toronto 2019 Abstract TFs (transcription factors) bind DNA in a sequence specific manner and regulate transcription. Humans encode ~1600 TFs, which have been identified almost entirely on the basis of a putative DNA-binding domain. Knowledge of the mechanisms by which they regulate transcription is sparse. TFs sometimes contain effector domains, but often utilize uncharacterized regions to recruit various cofactors, including chromatin modifiers, components of the general transcription machinery, and other TFs. This thesis focuses on describing the interactome of C2H2-ZNFs (C2H2 zinc finger proteins) which have been classified as TFs due to their ability to bind DNA and regulate transcription in some studied cases. With ~750 proteins, C2H2-ZNFs have greatly expanded in mammals and are the largest and least well characterized subfamily of DNA-binding proteins in humans. Through AP/MS (affinity purification and mass spectrometry), we have identified PPIs (protein-protein interactions) for 345 C2H2-ZNFs and, through ChIP-seq (chromatin immunoprecipitation followed by next generation sequencing), we have identified genomic binding sites for 217 C2H2-ZNFs. From the AP/MS data it appears that, overall, the C2H2-ZNFs exhibit a diverse set of interactions that may give them the ability to perform dual functions in transcription activation and repression, therefore suggesting that they may act as TFs. However, they also exhibit PPIs suggestive of roles additional to what the conventional definition of a TF entails, such as in AS (alternative splicing), which provides evidence that they are multifunctional proteins. Furthermore, C2H2-ZNFs are enriched for ii binding to AS exons and 3'-ends of genes, providing additional evidence for involvement in co- transcriptional processes. Strikingly, from the AP/MS data, C2H2-ZNFs extensively associate with each other and, based on their DNA-binding patterns, I show that interacting pairs may co-bind DNA, since they bind in closer proximity to each other in the genome compared to pairs that were not found to interact. Lastly, by integrating the PPI data with ChIP-seq, ChIA-PET (chromatin interaction analysis by paired-end tag sequencing), and HiC data, I show that C2H2-ZNF interacting pairs may mediate long range DNA interactions and thus organize chromatin architecture. Altogether, this work provides a snapshot of the C2H2-ZNF interactome and its potential to regulate gene expression. iii Table of Contents Chapter 1 -- Introduction......................................1 1.1 Overview of the regulation of gene expression..............2 1.2 Compaction of the genome by histones.......................4 1.2.1 Histones............................................4 1.2.2 The histone code....................................5 1.2.3 Epigenetic states...................................5 1.3 The Transcription cycle of RNA Polymerase II...............6 1.4 Chromatin architecture and gene expression................10 1.4.1 Topologically Associated Domains...................10 1.4.2 Long range interactions and transcription..........11 1.4.3 Long range interactions involving exons............12 1.4.4 Long range interactions involving 3'-ends of genes....................................................12 1.4.5 Nuclear compartmentalization.......................13 1.5 Transcription factors and mechanisms by which they regulate transcription............................................16 1.5.1 Overview of human transcription factors............16 1.5.2 Transcription regulation through PPIs of TFs with chromatin-related proteins...............................18 1.5.3 Transcription regulation through PPIs of TFs with general transcription factors............................20 1.5.4 Transcription regulation through PPIs of TFs with other transcription factors..............................20 1.6 C2H2-ZNF transcription factors............................21 1.6.1 Organization of DNA-binding domains................21 1.6.2 Auxiliary domains associated with human C2H2-ZNFs..23 1.6.3 RNA-binding by C2H2-ZNFs...........................24 1.6.4 RNA-DNA hybrid binding.............................25 1.6.5 Transcriptional regulation of endogenous retroelements............................................25 1.6.6 Transcriptional regulation of protein coding genes.27 1.6.7 Regulation of alternative splicing.................28 1.6.8 Regulation of 3'-end formation.....................29 1.6.9 Roles in genome organization.......................30 1.7 Summary and thesis outline................................31 Chapter 2 -- Protein-protein interactions of the C2H2-ZNF proteins......................................................34 iv 2.1 Introduction..............................................36 2.2 Results...................................................38 2.2.1 Optimization of the AP/MS method...................38 2.2.2 Overview of analyzed C2H2-ZNF proteins.............43 2.2.3 Overview of the SAINT analysis of the PPI data.....44 2.2.4 Diversity of PPIs for C2H2-ZNFs....................59 2.2.5 Frequently occurring prey proteins in the C2H2-ZNF PPI network..............................................67 2.2.6 Annotation of the preys according to function......71 2.2.7 Interaction of C2H2-ZNFs with metabolic proteins...72 2.2.8 Interaction of C2H2-ZNFs with RNA-related proteins.76 2.2.9 Interaction of C2H2-ZNFs with transcription factors..................................................81 2.2.10 Interaction of C2H2-ZNFs with transcription related effector proteins........................................85 2.2.11 Interaction of C2H2-ZNFs with histones and proteins involved in post- translational modifications and DNA replication/repair.......................................88 2.2.12 Interaction of C2H2-ZNFs with virus related proteins.................................................88 2.2.13 Roles of prey proteins in transcription activation and repression...........................................89 2.2.14 Sub-cellular localization of prey proteins in the network..................................................94 2.3 Discussion................................................97 2.4 Chapter 2 supplementary figures..........................100 Chapter 3 -- DNA-binding landscape of C2H2-ZNF proteins across the human genome.............................................107 3.1 Introduction.................................................108 3.2 Results......................................................109 3.2.1 ChIP-seq workflow for C2H2-ZNFs...................109 3.2.2 Comparison of C2H2-ZNF ChIP-seq experiments to previously published datasets...........................110 3.2.3 Distribution of C2H2-ZNF peaks in open and closed chromatin...............................................116 3.2.4 Motif analysis....................................120 3.2.5 Overlap of C2H2-ZNF binding sites across the genome..................................................130 v 3.2.6 Potential cooperative DNA-binding between C2H2- ZNFs....................................................137 3.2.7 Binding of C2H2-ZNFs at functional elements.......141 3.2.8 C2H2-ZNFs and R-loops.............................143 3.3 Discussion...............................................148 3.4 Chapter 3 supplementary figures..........................151 Chapter 4 -- C2H2-ZNF proteins and organization of chromatin architecture.................................................163 4.1 Introduction.............................................164 4.2 Results..................................................166 4.2.1 RNA Polymerase II ChIA-PET........................166 4.2.2 Overlap of C2H2-ZNF PPIs with ChIA-PET data.......174 4.2.3 Overlap of C2H2-ZNF PPIs with HiC data............185 4.2.4 Overlap of C2H2-ZNF binding sites at LRIs with mutations in cancer cells...............................188 4.3 Discussion...............................................192 4.4 Chapter 4 supplementary figures..........................195 Chapter 5 -- Discussion, conclusions, and future directions..197 5.1 C2H2-ZNFs: not just TFs, but Jacks of all trades?........198 5.2 C2H2-ZNFs and the regulation of chromatin architecture...202 5.3 C2H2-ZNFs and RNA........................................207 5.4 Summary and conclusions..................................213 Chapter 6--Methods...........................................215 6.1 Generation of stable cell lines using the Flp-IN TREx system to express inducible GFP-tagged C2H2-ZNFs....................216 6.2 Generation of stable cell lines using the MAPLE system to express VA-tagged C2H2-ZNFs..................................217 6.3 AP/MS workflows..........................................217 6.3.1 Workflow 1 (Two step purification)................217 6.3.2 Workflow 2 (One step FLAG purification)...........219 6.3.3 Workflow 3 (One-step GFP purification)............220 vi 6.3.4 Data accession for optimization of AP/MS experiments.............................................220 6.4 SAINT analysis...........................................221 6.5 Overlap of binding sites
Load more

Recommended publications
  • Small Cell Ovarian Carcinoma: Genomic Stability and Responsiveness to Therapeutics
    Gamwell et al. Orphanet Journal of Rare Diseases 2013, 8:33 http://www.ojrd.com/content/8/1/33 RESEARCH Open Access Small cell ovarian carcinoma: genomic stability and responsiveness to therapeutics Lisa F Gamwell1,2, Karen Gambaro3, Maria Merziotis2, Colleen Crane2, Suzanna L Arcand4, Valerie Bourada1,2, Christopher Davis2, Jeremy A Squire6, David G Huntsman7,8, Patricia N Tonin3,4,5 and Barbara C Vanderhyden1,2* Abstract Background: The biology of small cell ovarian carcinoma of the hypercalcemic type (SCCOHT), which is a rare and aggressive form of ovarian cancer, is poorly understood. Tumourigenicity, in vitro growth characteristics, genetic and genomic anomalies, and sensitivity to standard and novel chemotherapeutic treatments were investigated in the unique SCCOHT cell line, BIN-67, to provide further insight in the biology of this rare type of ovarian cancer. Method: The tumourigenic potential of BIN-67 cells was determined and the tumours formed in a xenograft model was compared to human SCCOHT. DNA sequencing, spectral karyotyping and high density SNP array analysis was performed. The sensitivity of the BIN-67 cells to standard chemotherapeutic agents and to vesicular stomatitis virus (VSV) and the JX-594 vaccinia virus was tested. Results: BIN-67 cells were capable of forming spheroids in hanging drop cultures. When xenografted into immunodeficient mice, BIN-67 cells developed into tumours that reflected the hypercalcemia and histology of human SCCOHT, notably intense expression of WT-1 and vimentin, and lack of expression of inhibin. Somatic mutations in TP53 and the most common activating mutations in KRAS and BRAF were not found in BIN-67 cells by DNA sequencing.
    [Show full text]
  • Identification of the Binding Partners for Hspb2 and Cryab Reveals
    Brigham Young University BYU ScholarsArchive Theses and Dissertations 2013-12-12 Identification of the Binding arP tners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non- Redundant Roles for Small Heat Shock Proteins Kelsey Murphey Langston Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Microbiology Commons BYU ScholarsArchive Citation Langston, Kelsey Murphey, "Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins" (2013). Theses and Dissertations. 3822. https://scholarsarchive.byu.edu/etd/3822 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science Julianne H. Grose, Chair William R. McCleary Brian Poole Department of Microbiology and Molecular Biology Brigham Young University December 2013 Copyright © 2013 Kelsey Langston All Rights Reserved ABSTRACT Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactors and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston Department of Microbiology and Molecular Biology, BYU Master of Science Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity.
    [Show full text]
  • Genetic Basis of Simple and Complex Traits with Relevance to Avian Evolution
    Genetic basis of simple and complex traits with relevance to avian evolution Małgorzata Anna Gazda Doctoral Program in Biodiversity, Genetics and Evolution D Faculdade de Ciências da Universidade do Porto 2019 Supervisor Miguel Jorge Pinto Carneiro, Auxiliary Researcher, CIBIO/InBIO, Laboratório Associado, Universidade do Porto Co-supervisor Ricardo Lopes, CIBIO/InBIO Leif Andersson, Uppsala University FCUP Genetic basis of avian traits Nota Previa Na elaboração desta tese, e nos termos do número 2 do Artigo 4º do Regulamento Geral dos Terceiros Ciclos de Estudos da Universidade do Porto e do Artigo 31º do D.L.74/2006, de 24 de Março, com a nova redação introduzida pelo D.L. 230/2009, de 14 de Setembro, foi efetuado o aproveitamento total de um conjunto coerente de trabalhos de investigação já publicados ou submetidos para publicação em revistas internacionais indexadas e com arbitragem científica, os quais integram alguns dos capítulos da presente tese. Tendo em conta que os referidos trabalhos foram realizados com a colaboração de outros autores, o candidato esclarece que, em todos eles, participou ativamente na sua conceção, na obtenção, análise e discussão de resultados, bem como na elaboração da sua forma publicada. Este trabalho foi apoiado pela Fundação para a Ciência e Tecnologia (FCT) através da atribuição de uma bolsa de doutoramento (PD/BD/114042/2015) no âmbito do programa doutoral em Biodiversidade, Genética e Evolução (BIODIV). 2 FCUP Genetic basis of avian traits Acknowledgements Firstly, I would like to thank to my all supervisors Miguel Carneiro, Ricardo Lopes and Leif Andersson, for the demanding task of supervising myself last four years.
    [Show full text]
  • Environmental Influences on Endothelial Gene Expression
    ENDOTHELIAL CELL GENE EXPRESSION John Matthew Jeff Herbert Supervisors: Prof. Roy Bicknell and Dr. Victoria Heath PhD thesis University of Birmingham August 2012 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Tumour angiogenesis is a vital process in the pathology of tumour development and metastasis. Targeting markers of tumour endothelium provide a means of targeted destruction of a tumours oxygen and nutrient supply via destruction of tumour vasculature, which in turn ultimately leads to beneficial consequences to patients. Although current anti -angiogenic and vascular targeting strategies help patients, more potently in combination with chemo therapy, there is still a need for more tumour endothelial marker discoveries as current treatments have cardiovascular and other side effects. For the first time, the analyses of in-vivo biotinylation of an embryonic system is performed to obtain putative vascular targets. Also for the first time, deep sequencing is applied to freshly isolated tumour and normal endothelial cells from lung, colon and bladder tissues for the identification of pan-vascular-targets. Integration of the proteomic, deep sequencing, public cDNA libraries and microarrays, delivers 5,892 putative vascular targets to the science community.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Location Analysis of Estrogen Receptor Target Promoters Reveals That
    Location analysis of estrogen receptor ␣ target promoters reveals that FOXA1 defines a domain of the estrogen response Jose´ e Laganie` re*†, Genevie` ve Deblois*, Ce´ line Lefebvre*, Alain R. Bataille‡, Franc¸ois Robert‡, and Vincent Gigue` re*†§ *Molecular Oncology Group, Departments of Medicine and Oncology, McGill University Health Centre, Montreal, QC, Canada H3A 1A1; †Department of Biochemistry, McGill University, Montreal, QC, Canada H3G 1Y6; and ‡Laboratory of Chromatin and Genomic Expression, Institut de Recherches Cliniques de Montre´al, Montreal, QC, Canada H2W 1R7 Communicated by Ronald M. Evans, The Salk Institute for Biological Studies, La Jolla, CA, July 1, 2005 (received for review June 3, 2005) Nuclear receptors can activate diverse biological pathways within general absence of large scale functional data linking these putative a target cell in response to their cognate ligands, but how this binding sites with gene expression in specific cell types. compartmentalization is achieved at the level of gene regulation is Recently, chromatin immunoprecipitation (ChIP) has been used poorly understood. We used a genome-wide analysis of promoter in combination with promoter or genomic DNA microarrays to occupancy by the estrogen receptor ␣ (ER␣) in MCF-7 cells to identify loci recognized by transcription factors in a genome-wide investigate the molecular mechanisms underlying the action of manner in mammalian cells (20–24). This technology, termed 17␤-estradiol (E2) in controlling the growth of breast cancer cells. ChIP-on-chip or location analysis, can therefore be used to deter- We identified 153 promoters bound by ER␣ in the presence of E2. mine the global gene expression program that characterize the Motif-finding algorithms demonstrated that the estrogen re- action of a nuclear receptor in response to its natural ligand.
    [Show full text]
  • Ingenuity Pathway Analysis of Differentially Expressed Genes Involved in Signaling Pathways and Molecular Networks in Rhoe Gene‑Edited Cardiomyocytes
    INTERNATIONAL JOURNAL OF MOleCular meDICine 46: 1225-1238, 2020 Ingenuity pathway analysis of differentially expressed genes involved in signaling pathways and molecular networks in RhoE gene‑edited cardiomyocytes ZHONGMING SHAO1*, KEKE WANG1*, SHUYA ZHANG2, JIANLING YUAN1, XIAOMING LIAO1, CAIXIA WU1, YUAN ZOU1, YANPING HA1, ZHIHUA SHEN1, JUNLI GUO2 and WEI JIE1,2 1Department of Pathology, School of Basic Medicine Sciences, Guangdong Medical University, Zhanjiang, Guangdong 524023; 2Hainan Provincial Key Laboratory for Tropical Cardiovascular Diseases Research and Key Laboratory of Emergency and Trauma of Ministry of Education, Institute of Cardiovascular Research of The First Affiliated Hospital, Hainan Medical University, Haikou, Hainan 571199, P.R. China Received January 7, 2020; Accepted May 20, 2020 DOI: 10.3892/ijmm.2020.4661 Abstract. RhoE/Rnd3 is an atypical member of the Rho super- injury and abnormalities, cell‑to‑cell signaling and interaction, family of proteins, However, the global biological function and molecular transport. In addition, 885 upstream regulators profile of this protein remains unsolved. In the present study, a were enriched, including 59 molecules that were predicated RhoE‑knockout H9C2 cardiomyocyte cell line was established to be strongly activated (Z‑score >2) and 60 molecules that using CRISPR/Cas9 technology, following which differentially were predicated to be significantly inhibited (Z‑scores <‑2). In expressed genes (DEGs) between the knockout and wild‑type particular, 33 regulatory effects and 25 networks were revealed cell lines were screened using whole genome expression gene to be associated with the DEGs. Among them, the most signifi- chips. A total of 829 DEGs, including 417 upregulated and cant regulatory effects were ‘adhesion of endothelial cells’ and 412 downregulated, were identified using the threshold of ‘recruitment of myeloid cells’ and the top network was ‘neuro- fold changes ≥1.2 and P<0.05.
    [Show full text]
  • Solution Structure of the GUCT Domain from Human RNA Helicase II/Gu[Beta]
    proteins STRUCTURE O FUNCTION O BIOINFORMATICS Solution structure of the GUCT domain from human RNA helicase II/Gub reveals the RRM fold, but implausible RNA interactions Satoshi Ohnishi,1 Kimmo Pa¨a¨kko¨nen,1 Seizo Koshiba,1 Naoya Tochio,1 Manami Sato,1 Naohiro Kobayashi,1 Takushi Harada,1 Satoru Watanabe,1 Yutaka Muto,1 Peter Gu¨ntert,1 Akiko Tanaka,1 Takanori Kigawa,1,2 and Shigeyuki Yokoyama1,3* 1 Systems and Structural Biology Center, RIKEN, Tsurumi, Yokohama 230-0045, Japan 2 Department of Computational Intelligence and Systems Science, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8503, Japan 3 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan INTRODUCTION ABSTRACT a a a a Human RNA helicase II/Gu (RH-II/Gu or Deadbox Human RNA helicase II/Gu (RH-II/Gu ) and RNA helicase protein 21) is a multifunctional enzyme that unwinds dou- II/Gub (RH-II/Gub) are paralogues that share the same ble-stranded RNA in the 50 to 30 direction and folds single- domain structure, consisting of the DEAD box helicase 1–5 domain (DEAD), the helicase conserved C-terminal domain stranded RNA in an ATP-dependent manner. These (helicase_C), and the GUCT domain. The N-terminal regions RNA-unwinding and RNA-folding activities are independ- of the RH-II/Gu proteins, including the DEAD domain and ent, and they reside in distinct regions of the protein. The the helicase_C domain, unwind double-stranded RNAs. The RNA helicase activity is catalyzed by the N-terminal three- 1 C-terminal tail of RH-II/Gua, which follows the GUCT do- quarters of the molecule in the presence of Mg2 , where as main, folds a single RNA strand, while that of RH-II/Gub the RNA-foldase activity is located in the C-terminal region 1 does not, and the GUCT domain is not essential for either and functions in a Mg2 independent manner.2 As shown the RNA helicase or foldase activity.
    [Show full text]
  • Nucleolin and Its Role in Ribosomal Biogenesis
    NUCLEOLIN: A NUCLEOLAR RNA-BINDING PROTEIN INVOLVED IN RIBOSOME BIOGENESIS Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Julia Fremerey aus Hamburg Düsseldorf, April 2016 2 Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. A. Borkhardt Korreferent: Prof. Dr. H. Schwender Tag der mündlichen Prüfung: 20.07.2016 3 Die vorgelegte Arbeit wurde von Juli 2012 bis März 2016 in der Klinik für Kinder- Onkologie, -Hämatologie und Klinische Immunologie des Universitätsklinikums Düsseldorf unter Anleitung von Prof. Dr. A. Borkhardt und in Kooperation mit dem ‚Laboratory of RNA Molecular Biology‘ an der Rockefeller Universität unter Anleitung von Prof. Dr. T. Tuschl angefertigt. 4 Dedicated to my family TABLE OF CONTENTS 5 TABLE OF CONTENTS TABLE OF CONTENTS ............................................................................................... 5 LIST OF FIGURES ......................................................................................................10 LIST OF TABLES .......................................................................................................12 ABBREVIATION .........................................................................................................13 ABSTRACT ................................................................................................................19 ZUSAMMENFASSUNG
    [Show full text]
  • Acetyl-Histone H2A-K5 Rabbit Pab
    Leader in Biomolecular Solutions for Life Science Acetyl-Histone H2A-K5 Rabbit pAb Catalog No.: A15620 Basic Information Background Catalog No. Histones are basic nuclear proteins that are responsible for the nucleosome structure of A15620 the chromosomal fiber in eukaryotes. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer, around which approximately 146 bp of DNA is Observed MW wrapped in repeating units, called nucleosomes. The linker histone, H1, interacts with 14kDa linker DNA between nucleosomes and functions in the compaction of chromatin into higher order structures. This gene is intronless and encodes a replication-dependent Calculated MW histone that is a member of the histone H2A family. Transcripts from this gene lack polyA 14kDa tails but instead contain a palindromic termination element. This gene is found in the small histone gene cluster on chromosome 6p22-p21.3. Category Primary antibody Applications WB, IHC, IF Cross-Reactivity Human, Mouse, Rat, Other (Wide Range) Recommended Dilutions Immunogen Information WB 1:500 - 1:2000 Gene ID Swiss Prot 8329 P0C0S8 IHC 1:50 - 1:100 Immunogen IF 1:50 - 1:100 A synthetic acetylated peptide around K5 of human Histone H2A (NP_003508.1). Synonyms HIST1H2AI;H2A/c;H2AFC Contact Product Information Source Isotype Purification www.abclonal.com Rabbit IgG Affinity purification Storage Store at -20℃. Avoid freeze / thaw cycles. Buffer: PBS with 0.02% sodium azide,50% glycerol,pH7.3. Validation Data Western blot analysis of extracts of various cell lines, using Acetyl-Histone H2A-K5 antibody (A15620) at 1:1000 dilution.C2C12 cells and C6 cells were treated by TSA (1 uM) at 37℃ for 18 hours.
    [Show full text]
  • Comparison of Orthologs Across Multiple Species by Various Strategies
    COMPARISON OF ORTHOLOGS ACROSS MULTIPLE SPECIES BY VARIOUS STRATEGIES BY HUI LIU DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biophysics and Computational Biology in the Graduate College of the University of Illinois at Urbana-Champaign, 2014 Urbana, Illinois Doctoral Committee: Professor Eric Jakobsson, Chair, Director of Research Professor Gene E. Robinson Associate Professor Saurabh Sinha Assistant Professor Jian Ma Abstract Thanks to the improvement of genome sequencing technology, abundant multi-species genomic data now became available and comparative genomics continues to be a fast prospering filed of biological research. Through the comparison of genomes of different organisms, we can understand what, at the molecular level, distinguishes different life forms from each other. It shed light on revealing the evolution of biology. And it also helps to refine the annotations and functions of individual genomes. For example, through comparisons across mammalian genomes, we can give an estimate of the conserved set of genes across mammals and correspondingly, find the species-specific sets of genes or functions. However, comparative genomics can be feasible only if a meaningful classification of genes exists. A natural way to do so is to delineate sets of orthologous genes. However, debates exist about the appropriate way to define orthologs. It is originally defined as genes in different species which derive from speciation events. But such definition is not sufficient to derive orthologous genes due to the complexity of evolutionary events such as gene duplication and gene loss. While it is possible to correctly figure out all the evolutionary events with the true phylogenetic tree, the true phylogenetic tree itself is impractical to be inferred.
    [Show full text]
  • Long First Exons and Epigenetic Marks Distinguish Conserved Pachytene
    ARTICLE https://doi.org/10.1038/s41467-020-20345-3 OPEN Long first exons and epigenetic marks distinguish conserved pachytene piRNA clusters from other mammalian genes ✉ Tianxiong Yu 1,2,7, Kaili Fan 1,2,7, Deniz M. Özata 3, Gen Zhang4,YuFu 2,5,6, William E. Theurkauf4 , ✉ ✉ Phillip D. Zamore 3 & Zhiping Weng 1,2 – 1234567890():,; In the male germ cells of placental mammals, 26 30-nt-long PIWI-interacting RNAs (piR- NAs) emerge when spermatocytes enter the pachytene phase of meiosis. In mice, pachytene piRNAs derive from ~100 discrete autosomal loci that produce canonical RNA polymerase II transcripts. These piRNA clusters bear 5′ caps and 3′ poly(A) tails, and often contain introns that are removed before nuclear export and processing into piRNAs. What marks pachytene piRNA clusters to produce piRNAs, and what confines their expression to the germline? We report that an unusually long first exon (≥ 10 kb) or a long, unspliced transcript correlates with germline-specific transcription and piRNA production. Our integrative analysis of tran- scriptome, piRNA, and epigenome datasets across multiple species reveals that a long first exon is an evolutionarily conserved feature of pachytene piRNA clusters. Furthermore, a highly methylated promoter, often containing a low or intermediate level of CG dinucleotides, correlates with germline expression and somatic silencing of pachytene piRNA clusters. Pachytene piRNA precursor transcripts bind THOC1 and THOC2, THO complex subunits known to promote transcriptional elongation and mRNA nuclear export. Together, these features may explain why the major sources of pachytene piRNA clusters specifically gen- erate these unique small RNAs in the male germline of placental mammals.
    [Show full text]