The role of HP1β [HP1-beta] on genomic stability and cellular senescence [Elektronische Ressource] / vorgelegt von Mustafa Billur

Aus dem Forschungszentrum Borstel
Leibniz-Zentrum für Medizin und Biowissenschaften
Laborgruppe Immunepigenetik (Laborgruppenleiter: Dr. Prim B. Singh)
Abteilung Immunologie und Zellbiologie (Direktorin: Prof. Dr. Dr. Silvia Bulfone-Paus)

The role of HP1β on genomic stability
and cellular senescence

DISSERTATION
zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Christian-Albrechts-Universität
eliu Kz vorgelegt von
Mustafa Billur
Kiel, Oktober 2010

Referent/in:

Korreferent/in:

Prof. Dr. Dr. S……………………………………ilvia Bulfone-Paus

Prof. Dr. Thoma……………………………………s Roeder

Tag der mündlichen Prüfung: 08/11/2010 ……………………………………

Zum Druck genehmigt:

……………………………………

"It is, in fact, nothing short of a miracle that modern methods of teaching have not yet entirely
strangled that sacred spirit of curiosity and inquiry, for this delicate plant needs freedom no less than
stimulation."
Albert Einstein

Contents

Contents
List of abbreviations ........................................................................................................................................................ 1
List of figures ...................................................................................................................................................................... 3
List of tables ......................................................................................................................................................................... 5
Chapter 1. Introduction ................................................................................................................................................ 6
1.1 Heterochromatin and Epigenetics........................................................................................................................................ 7
1.1.1 Heterochromatin ................................................................................................................................................................ 7
1.1.2 Epigenetics ......................................................................................................................................................................... 8
1.1.2.1 Noncoding RNAs ........................................................................................................................................................... 9
1.1.2.2 Chromatin remodelling and histone variants .................................................................................................................. 9
1.1.2.3 DNA methylation ......................................................................................................................................................... 10
1.1.2.4 Histone modifications ................................................................................................................................................... 10
1.2 Heterochromatin protein 1 (HP1) ..................................................................................................................................... 12
1.2.1 HP1β ............................................................................................................................................................................... 14
1.2.2 HP1β, centromeres and sister chromatid cohesion .......................................................................................................... 17
1.2.3 HP1β and telomeres ........................................................................................................................................................ 20
1.2.4 HP1β and oncogene-induced senescence (OIS) .............................................................................................................. 22
1.2.5 HP1β knockout mice ....................................................................................................................................................... 24
1.3 The goal of the study........................................................................................................................................................... 25
Chapter 2. Materials and Methods ........................................................................................................................ 27
2.1 Materials ............................................................................................................................................................................. 28
2.1.1 Primers ............................................................................................................................................................................. 28
2.1.2 Plasmids ........................................................................................................................................................................... 28
2.1.3 Antibodies ........................................................................................................................................................................ 28
2.1.3.1 Primary antibodies ........................................................................................................................................................ 29
2.1.3.2 Secondary antibodies ..................................................................................................................................................... 30
2.1.4 Buffers, media and solutions ............................................................................................................................................ 30
2.1.4.1 Standard media and solutions ....................................................................................................................................... 30
2.1.4.2 Flow-FISH solutions .................................................................................................................................................... 31
2.1.4.3 Telomere-FISH and Giemsa staining solutions ........................................................................................................... 31
2.1.4.4 ChIP solutions .............................................................................................................................................................. 32
2.1.4.5 GST pull down solutions .............................................................................................................................................. 32
2.1.4.6 Protein purification solutions ........................................................................................................................................ 33
2.1.4.7 Coomassie stain solutions ............................................................................................................................................. 33
2.1.4.8 SDS-PAGE and Western blotting solutions ................................................................................................................ 33
2.1.4.9 Metaphase spread preparation solutions ....................................................................................................................... 34
2.1.5 Bacterial strains ................................................................................................................................................................ 34
2.1.6 Kits ................................................................................................................................................................................... 34

Contents

2.2 Methods .............................................................................................................................................................................. 35
2.2.1 Molecular biology ............................................................................................................................................................ 35
2.2.1.1 Preparation of plasmid DNA ........................................................................................................................................ 35
2.2.1.2 Isolation of total RNA from tissue ................................................................................................................................ 35
2.2.1.3 Measuring concentration of nucleic acids ..................................................................................................................... 35
2.2.1.4 Reverse transcription of RNA (cDNA synthesis) ......................................................................................................... 35
2.2.1.5 Transformation of plasmid DNA into E.coli ................................................................................................................ 35
2.2.1.6 Agarose gel electrophoresis ........................................................................................................................................... 36
2.2.1.7 Restriction digest .......................................................................................................................................................... 36