Cellular, biochemical and phylogenomic analyses of the mouse Jumonji domain containing 6 protein provide new evidence for functions as a 2-oxoglutarate dependent dioxygenase [Elektronische Ressource] / von Phillip Holgar Heinrich Hahn

Dr. Phillip Hahn

Sujets

• Biologie

Informations

• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2008
• Nombre de lectures: 55
• Langue: English
• Poids de l'ouvrage: 17 Mo

Extrait

Cellular, biochemical and phylogenomic analyses of the
mouse Jumonji domain containing 6 protein provide new
evidence for functions as a 2-oxoglutarate dependent
dioxygenase






Von der Fakultät für Lebenswissenschaften

der Technischen Universität Carolo-Wilhelmina

zu Braunschweig

zur Erlangung des Grades eines

Doktors der Naturwissenschaften

(Dr. rer. nat.)

genehmigte

D i s s e r t a t i o n













von Phillip Holgar Heinrich Hahn
aus Georgsmarienhütte






















































1. Referentin oder Referent: Prof. Dr. Rudi Balling
2. Referentin oder Referent: Prof. Dr. Jürgen Wehland
eingereicht am: 07.11.2007
mündliche Prüfung (Disputation) am: 20.12.2007
Druckjahr: 2008 Table of contents 1

Table of contents
TABLE OF CONTENTS ............................................................................................. 1 
SUMMARY ................................................................................................................. 4 
1.  INTRODUCTION ................................................................................................ 5 
1.1  The phosphatidylserine receptor ............................................................................................... 5 
1.1.1  The phosphatidylserine receptor and its proposed function in apoptotic cell clearance ....... 5 
1.1.2  Phosphatidylserine receptor loss-of-function studies ............................................................ 6 
1.1.2.1  PSR-1 loss-of-function mutation in Caenorhabditis elegans ......................................... 6 
1.1.2.2  Knockdown of zfpsr in Danio rerio ................................................................................. 6 
1.1.2.3  Knockout and overexpression of dPSR in Drosophila melanogaster ........................... 7 
1.1.2.4  Knockout of Ptdsr in mice .............................................................................................. 8 
1.1.3  Biochemical properties, domains and motifs ....................................................................... 10 
1.1.3.1  Nuclear localization sites ............................................................................................. 11 
1.1.3.2  Putative AT-hook like DNA binding motif .................................................................... 11 
1.1.3.3  Poly-serine stretch ....................................................................................................... 12 
1.1.3.4  The Ptdsr JmjC domain ............................................................................................... 12 
1.1.4  Revision of Ptdsr gene nomenclature .................................................................................. 14 
1.2  The Histone code and histone lysine demethylation ............................................................. 14 
1.2.1  Lysine-specific demethylase 1 18 
1.2.2  JmjC domain-containing proteins ......................................................................................... 19 
1.2.2.1  Jontaining protein class of histone lysine demethylases ...................... 19 
1.2.2.1.1  Proposed catalytical mechanism ............................................................................ 20 
1.2.2.2  JmjC domain-containing proteins as transcription factors in development ................. 21 
1.2.2.3  Domain composition of JmjC domain-containing proteins .......................................... 22 
1.2.2.3.1  Complex JmjC domain-containing proteins ............................................................ 22 
1.2.2.3.2  JmjC-only domain-containing proteins ................................................................... 23 
1.2.2.4  JmjC domain-containing proteins in disease ............................................................... 24 
1.3  Nucleic acid methylation and oxidative repair ........................................................................ 24 
1.3.1  Demethylation of DNA and RNA by AlkB and homologues ................................................. 25 
1.4  Aim of the study ......................................................................................................................... 27 
2.  RESULTS ......................................................................................................... 28 
2.1  Genomic structure and expression of Jmjd6 and evolutionary analysis in the context of
related JmjC domain-containing proteins ............................................................................... 28 
2.1.1  Overview of bioinformatics analyses approaches ................................................................ 28 
2.1.2  Comparative analysis of Jmjd6 homologous loci in vertebrates .......................................... 31 
2.1.3  Phylogenetic analysis of the Jmjd6 protein .......................................................................... 42 
2.1.4  Comparative modelling and analysis of the putative catalytic core domain of Jmjd6 .......... 46 
2.1.5  Integration of Jmjd6 into the JmjC domain-containing protein family .................................. 51 Table of contents 2

2.2  A cellular and biochemical analysis indicates no function of Jmjd6 in histone
demethylation but provides evidence for RNA association .................................................. 58 
2.2.1  Overview on Jmjd6 protein analyses ................................................................................... 58 
2.2.2  Analysis of intracellular Jmjd6 localization using specific antibodies .................................. 59 
2.2.3  Characterization of Jmjd6 multimer formation ..................................................................... 65 
2.2.4  Jmjd6 is not involved in histone lysine demethylation ......................................................... 71 
2.2.5  Jmjd6 is associated with RNA but not with DNA in cell nuclei ............................................. 76 
3.  DISCUSSION ................................................................................................... 79 
3.1  Evolutional and structural features of the Jmjd6 protein ...................................................... 79 
3.1.1  Phylogeny of the Jmjd6 protein ............................................................................................ 79 
3.1.2  Functional 3D Jmjd6 conservation analysis a structural Jmjd6 model ................................ 80 
3.1.3  JmjC domain sequences are characteristic and allow protein family classification ............. 81 
3.2  Effects of intracellular Jmjd6 localization and Jmjd6 protein multimerization ................... 82 
3.2.1  Intracellular Jmjd6 localization ............................................................................................. 82 
3.2.2  Jmjd6 is linked to a ribonucleic matrix ................................................................................. 84 
3.2.3  Possible mechanism of Jmjd6 multimerization .................................................................... 84 
3.2.4  Identification of novel Jmjd6 transcripts and possible biological consequences ................. 86 
3.3  The biological functions of Jmjd6 ............................................................................................ 87 
3.3.1  Jmjd6 is not a phosphatidylserine receptor .......................................................................... 87 
3.3.2  Jmjd6 is not involved in histone lysine demethylation ......................................................... 88 
3.3.3  Jmjd6 is a H3R2 & H4R3 demethylase ................................................................................ 88 
3.4  Conclusive possible explanations for the Jmjd6 knockout phenotype ............................... 89 
4.  OUTLOOK ........................................................................................................ 90 
5.  MATERIAL AND METHODS ............................................................................ 92 
5.1.1  Chemicals, enzymes, medium and cells .............................................................................. 92 
5.1.2  Jmjd6 expression constructs and transfection ..................................................................... 92 
5.1.3  Monoclonal antibody generation and epitope mapping ....................................................... 93 
5.1.4