Role of the human LIN complex in DNA damage induced regulation of gene expression [Elektronische Ressource] / vorgelegt von Mirijam Mannefeld

Jirimam

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• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 26
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Role of the human LIN complex in DNA
damage induced regulation of gene
expression

Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg



vorgelegt von
Mirijam Mannefeld
aus
Offenburg

Würzburg, 2009




















Eingereicht am:
………………………………………………………………………….

Mitglieder der Prüfungskommission:
Vorsitzender:
1. Gutachter: Prof. Dr. Stefan Gaubatz
2. Gutachter: PD Dr. Alsheimer

Tag des Promotionskolloqiums:
………………………………………………………………………….

Doktorurkunde ausgehändigt am:
…………………………………………………………………………. CONTENT
1  Introduction ............................................................................................. - 1 - 
1.1  Human cell cycle ............................................................................... - 1 - 
1.2  DNA damage response ..................................................................... - 3 - 
1.2.1  G1 checkpoint ............................................................................ - 4 - 
1.2.2  G2/M checkpoint ........................................................................ - 6 - 
1.2.3 kpoint maintenance ................................................... - 6 - 
1.2.4  Checkpoint recovery and adaptation .......................................... - 9 - 
1.3  LINC – a human multiprotein complex ............................................ - 10 - 
1.3.1  LINC in target gene activation .................................................. - 13 - 
1.3.1.1  B-MYB ............................................................................. - 16 - 
1.3.2  LINC in target gene repression ................................................. - 17 - 
1.4  Aim of this project ........................................................................... - 17 - 
2  Materials and methods .......................................................................... - 19 - 
2.1  Materials ......................................................................................... - 19 - 
2.1.1  Chemical stocks ....................................................................... - 19 - 
2.1.2  Buffers ...................................................................................... - 20 - 
2.1.2.1  General buffers ................................................................ - 20 - 
2.1.2.2  Buffers for whole-cell lysates ........................................... - 21 - 
2.1.2.3  Buffers for immunoprecipitation and immunoblotting ....... - 21 - 
2.1.2.4  Buffers for chromatin immunoprecipitation ...................... - 23 - 
2.1.2.5  Buffers for flow cytometry (FACS) analysis ..................... - 24 - 
2.1.3  Antibodies ................................................................................. - 24 - 
2.1.3.1  Primary antibodies ........................................................... - 24 - 
2.1.3.2  Secondary antibodies ...................................................... - 26 - 
2.1.4  Primers ..................................................................................... - 26 - 
2.1.4.1  Primers for quantitative real time PCR ............................ - 26 - 
2.1.4.2  Primers for chromatin immunoprecipitation ..................... - 27 - 
I
CONTENT
2.1.5  siRNA sequences ..................................................................... - 27 - 
2.1.6  Cell lines, cell culture media and treatment .............................. - 28 - 
2.1.7  Markers .................................................................................... - 28 - 
2.1.8  Kits ........................................................................................... - 28 - 
2.1.9  Beads ....................................................................................... - 28 - 
2.2  Methods .......................................................................................... - 29 - 
2.2.1  Cell culture ............................................................................... - 29 - 
2.2.1.1  Passageing of cells .......................................................... - 29 - 
2.2.1.2  Transient transfection ...................................................... - 29 - 
2.2.1.3  Synchronization of U2OS cells by thymidine ................... - 29 - 
2.2.1.4  Treatment ........................................................................ - 29 - 
2.2.1.5  Cell cycle phases: flow cytometry (FACS) ....................... - 30 - 
2.2.2  Expression analysis .................................................................. - 30 - 
2.2.2.1  RNA Isolation .................................................................. - 30 - 
2.2.2.2  Reverse transcription ....................................................... - 31 - 
2.2.2.3  qRT-PCR ......................................................................... - 31 - 
2.2.3  Biochemical methods ............................................................... - 32 - 
2.2.3.1  Whole cell lysates ............................................................ - 32 - 
2.2.3.2  Protein concentration ....................................................... - 32 - 
2.2.3.3  Immunoprecipitation ........................................................ - 32 - 
2.2.3.4  SDS-polyacrylamide gel electrophoresis ......................... - 33 - 
2.2.3.5  Immunoblotting ................................................................ - 33 - 
2.2.3.6  Affinity purification of polyclonal antisera ......................... - 34 - 
2.2.3.7  Chromatin immunoprecipitation ....................................... - 34 - 
2.2.4  Molecular biology ..................................................................... - 36 - 
2.2.4.1  Isolation of plasmid DNA from bacteria ........................... - 36 - 
3  Results .................................................................................................. - 37 - 
II
CONTENT
3.1  LINC composition after DNA damage induction .............................. - 37 - 
3.1.1  Binding studies in p53 wt cells .................................................. - 37 - 
3.1.2  p130 and B-MYB do not bind to LINC simultaneously .............. - 40 - 
3.1.3  Change in LINC composition is a direct effect of DNA damage
induction ............................................................................................... - 41 - 
3.1.4  p130 phosphorylation status is crucial for binding to LINC ....... - 43 - 
3.2  Pathways leading to LINC switch .................................................... - 43 - 
3.2.1  Binding studies in p21 negative cells after DNA damage
induction… ............................................................................................ - 44 - 
3.2.2  Binding studies in p53 negative cells after DNA damage
induction… - 45 - 
3.2.3  Binding studies in p53 wt cells after Nutlin-3 treatment ............ - 47 - 
3.3  Regulation of G2/M gene expression .............................................. - 48 - 
3.3.1  G2/M gene expression in p53 wt and p53 negative cells ......... - 48 - 
3.3.2 ne expression is a direct effect ............. - 51 - 
3.3.3  Deregulated G2/M gene expression in p53 negative cells after DNA
damage induction is partially rescued by depletion of LIN-9 and/or B-
MYB..... ................................................................................................. - 58 - 
3.4  Premature mitotic entry of p53 negative cells after DNA damage
partially depends on B-MYB and LIN-9 .................................................... - 61 - 
3.5  B-MYB in primary breast tumors ..................................................... - 63 - 
4  Discussion ............................................................................................ - 65 - 
4.1  How does the composition of LINC switch in response to DNA-
damage? .................................................................................................. - 66 - 
4.2  LINC function in DNA damage response ........................................ - 67 - 
4.3  LINC function in tumors .................................................................. - 72 - 
5  Summary .............................................................................................. - 80 - 
6  Zusammenfassung ............................................................................... - 81 - 
III
CONTENT
7  Appendix ............................................................................................... - 82 -