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Establishment and Maintenance of 35S Rrna Gene Chromatin States in Saccharomyces Cerevisiae

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Establishment and Maintenance of 35S Rrna Gene Chromatin States in Saccharomyces Cerevisiae Establishment and maintenance of 35S rRNA gene chromatin states in Saccharomyces cerevisiae DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER NATURWISSENSCHAFTEN (DR. RER. NAT.) DER FAKULTÄT FÜR BIOLOGIE UND VORKLINISCHE MEDIZIN DER UNIVERSITÄT REGENSBURG vorgelegt von Manuel Wittner aus Ingolstadt im Februar 2012 Das Promotionsgesuch wurde eingereicht am: 07. Februar 2012 Die Arbeit wurde angeleitet von: PD Dr. Joachim Griesenbeck Prüfungsausschuss: Vorsitzender: Prof. Dr. Herbert Tschochner 1. Prüfer: PD Dr. Joachim Griesenbeck 2. Prüfer: Prof. Dr. Michael Rehli 3. Prüfer: Prof. Dr. Wolfgang Seufert Die vorliegende Arbeit wurde in der Zeit von Dezember 2008 bis Februar 2012 am Lehrstuhl Biochemie III des Institutes für Biochemie, Genetik und Mikrobiologie der Naturwissenschaftlichen Fakultät III der Universität Regensburg unter Anleitung von PD Dr. Joachim Griesenbeck im Labor von Prof. Dr. Herbert Tschochner angefertigt. Ich erkläre hiermit, dass ich diese Arbeit selbst verfasst und keine anderen als die angegebenen Quellen und Hilfsmittel verwendet habe. Diese Arbeit war bisher noch nicht Bestandteil eines Prüfungsverfahrens. Andere Promotionsversuche wurden nicht unternommen. Manuel Wittner Regensburg, 07. Februar 2012 Table of Contents Table of Contents 1 Summary...................................................................................................................1 2 Introduction ..............................................................................................................3 2.1 Eukaryotic chromatin structure..............................................................................3 2.1.1 Histone molecules represent the main components of chromatin............3 2.1.2 Chromatin dynamics and transcription .....................................................5 2.1.3 Chromatin dynamics and DNA replication ................................................6 2.1.4 Chromatin dynamics and DNA repair .......................................................8 2.2 The ribosomal gene locus of Saccharomyces cerevisiae ................................12 2.2.1 Cellular localisation and structure of the yeast rRNA gene locus...........12 2.2.2 Chromatin structure at the yeast rDNA locus .........................................16 2.2.3 rRNA gene chromatin dynamics and RNA polymerase I transcription ...21 2.2.4 rRNA gene chromatin dynamics and DNA repair ...................................23 2.3 Objectives ...............................................................................................................25 3 Results ....................................................................................................................27 3.1 Establishment and maintenance of alternative 35S rRNA gene chromatin states.......................................................................................................................27 3.1.1 The ratio of open and closed 35S rRNA gene chromatin states varies during the cell cycle ................................................................................27 3.1.2 35S rRNA gene chromatin continuously opens in the absence of replication................................................................................................35 3.1.3 Opening of 35S rRNA genes leads to histone depletion and Hmo1 recruitment ..............................................................................................37 3.1.4 Maintenance of open rRNA gene chromatin requires Pol I transcription in replicating cells .......................................................................................42 3.1.5 RNA polymerase I transcription is required to establish open 35S rRNA gene chromatin .......................................................................................44 3.1.6 Hmo1 is a component of open 35S rRNA gene chromatin in the absence of Pol I transcription and replication........................................................46 3.1.7 Hmo1 prevents replication-independent nucleosome assembly at open 35S rRNA genes .....................................................................................47 I Table of Contents 3.2 Chromatin dynamics at 35S rRNA genes after UV irradiation...........................49 3.2.1 Opening of 35S rRNA gene chromatin after UV irradiation occurs gradually from the 5’ to the 3’ end of the gene .......................................49 3.2.2 Histones associate with the open rRNA genes after UV irradiation .......53 3.2.3 All 35S rRNA genes are associated with histones after UV irradiation in the absence of NER................................................................................55 3.2.4 RNA polymerase I and Hmo1 remain associated with 35S rRNA genes during UV induced nucleosome assembly..............................................57 3.2.5 RNA polymerase I and Hmo1 stay part of 35S rRNA gene chromatin after UV induced nucleosome assembly in the absence of NER ...........61 4 Discussion..............................................................................................................65 4.1 Establishment and maintenance of alternative chromatin states at the 35S rRNA genes.............................................................................................................65 4.1.1 DNA replication and Pol I transcription are required for the establishment of 35S rRNA chromatin states ................................................................66 4.1.2 Maintenance of the open 35S rRNA gene chromatin state by Pol I and Hmo1 ......................................................................................................68 4.2 Changes in 35S rRNA gene chromatin after UV induced DNA damage...........71 4.2.1 Nucleosome assembly at open 35S rRNA genes after UV irradiation leads to a chromatin structure with mixed protein composition ..............71 4.2.2 Nucleosome deposition after UV irradiation and nucleosome removal during NER occurs in a 5’-3’-gradient at the 35S rRNA genes...............74 5 Material and Methods ...........................................................................................77 5.1 Material...................................................................................................................77 5.1.1 Chemicals ...............................................................................................77 5.1.2 Media and buffers ...................................................................................77 5.1.3 Nucleic acids...........................................................................................80 5.1.4 Enzymes and Polypeptides ....................................................................85 5.1.5 Antibodies ...............................................................................................85 5.1.6 Organisms...............................................................................................85 5.1.7 Equipment...............................................................................................88 5.1.8 Consumables..........................................................................................89 II Table of Contents 5.2 Methods ..................................................................................................................89 5.2.1 Enzymatic manipulation of DNA .............................................................89 5.2.2 Purification of nucleic acids.....................................................................90 5.2.3 Quantitative and qualitative analysis of nucleic acids.............................92 5.2.4 Yeast cultures and formaldehyde crosslinking (FA-X)............................95 5.2.5 Ethanol fixation of yeast cells..................................................................95 5.2.6 Cell cycle analysis...................................................................................96 5.2.7 UV irradiation ..........................................................................................96 5.2.8 Flow cytometry........................................................................................96 5.2.9 Preparation of nuclei...............................................................................97 5.2.10 Chromatin Endogenous Cleavage (ChEC).............................................97 5.2.11 Psoralen crosslinking..............................................................................98 5.2.12 Chromatin Endogenous Cleavage Psoralen Photocrosslinking Assay (ChEC/psoralen) .....................................................................................99 5.2.13 DNA workup of ChEC and ChEC/psoralen samples ..............................99 5.2.14 Restriction digest and agarose gel electrophoresis of ChEC and ChEC/psoralen samples .........................................................................99 5.2.15 Chromatin Immunoprecipitation (ChIP) ................................................100 5.2.16 Manipulation of Escherichia coli............................................................101 5.2.17 Manipulation of Saccharomyces cerevisiae..........................................102 5.2.18 Protein biochemical methods................................................................103 6 References............................................................................................................107 7 Abbreviations .......................................................................................................129 8 Publications..........................................................................................................131
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