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Proteomic identification of the MYST domain histone acetyltransferase TIP60 as a coactivator of the myeloid transcription factor {C/EBPα [C-EBP-alpha] [Elektronische Ressource] / submitted by Deepak Bararia

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From the Clinical Cooperative Group “Leukemia” Department of Medicine III, University Hospital Ludwig-Maximilians-University, Munich Chair: Prof. Dr. med. Wolfgang Hiddemann Proteomic Identification of the MYST Domain Histone Acetyltransferase TIP60 as a Coactivator of the Myeloid Transcription Factor C/EBP α Thesis Submitted for a Doctoral degree in Human Biology at the Faculty of Medicine Ludwig-Maximilians-University, Munich, Germany Submitted by Deepak Bararia From Delhi, India 2007 Aus der Klinische Kooperations Gruppe ‚’’Leukämie’’ Der Medizinischen Klinik und Poliklinik III Großhadern der Ludwig-Maximilians-Universität München, Direktor: Prof. Dr. med. Wolfgang Hiddemann Proteomic Identification of the MYST Domain Histone Acetyltransferase TIP60 as a Coactivator of the Myeloid Transcription Factor C/EBP α Dissertation zum Erwerb des Doktorgrades der Humanbiologie an der Medizinischen Fakultät der Ludwig-Maximilians- Universität zu München, Germany Vorgelegt von Deepak Bararia Aus Dehli, Indien 2007 With permission from the Faculty of Medicine, University of Munich Supervisor/Examiner: Prof. Dr. med. Wolfgang Hiddemann Second Examiner: Prof. Dr. G. W. Bornkamm Co-Examiners: Prof. Dr. A. Roscher Priv. Doz. Dr. F. Oduncu Co-Supervisor: Prof. Dr. med. Stefan Bohlander Priv. Doz. Dr.

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Published 01 January 2007
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From the Clinical Cooperative Group “Leukemia”
Department of Medicine III, University Hospital
Ludwig-Maximilians-University, Munich
Chair: Prof. Dr. med. Wolfgang Hiddemann






Proteomic Identification of the MYST Domain Histone
Acetyltransferase TIP60 as a Coactivator of the
Myeloid Transcription Factor C/EBP α





Thesis Submitted for a Doctoral degree in Human Biology
at the Faculty of Medicine Ludwig-Maximilians-University,
Munich, Germany




Submitted by
Deepak Bararia



From
Delhi, India


2007

Aus der Klinische Kooperations Gruppe ‚’’Leukämie’’
Der Medizinischen Klinik und Poliklinik III Großhadern der Ludwig-
Maximilians-Universität München,
Direktor: Prof. Dr. med. Wolfgang Hiddemann





Proteomic Identification of the MYST Domain Histone
Acetyltransferase TIP60 as a Coactivator of the
Myeloid Transcription Factor C/EBP α



Dissertation zum Erwerb des Doktorgrades der Humanbiologie
an der Medizinischen Fakultät der Ludwig-Maximilians-
Universität zu München, Germany



Vorgelegt von
Deepak Bararia



Aus
Dehli, Indien


2007

With permission from the Faculty of Medicine,
University of Munich






Supervisor/Examiner: Prof. Dr. med. Wolfgang Hiddemann


Second Examiner: Prof. Dr. G. W. Bornkamm



Co-Examiners: Prof. Dr. A. Roscher
Priv. Doz. Dr. F. Oduncu




Co-Supervisor: Prof. Dr. med. Stefan Bohlander
Priv. Doz. Dr. Gerhard Behre



Dean: Prof. Dr. med D. Reinhardt





Date of Oral Examination: 01.08.2007

Mit Genehmigung der Medizinischen Fakultät
der Universität München







1. Berichterstatter: Prof. Dr. med. W. Hiddemann
2. Berichterstatter: Prof. Dr. G. W. Bornkamm



Mitberichterstatter: Prof. Dr. A. Roscher
Priv. Doz. Dr. F. Oduncu




Mitbetreuung durch
promovierte Mitarbeiter: Prof. Stefan Bohlander
Priv. Doz. Dr. Gerhard Behre



Dekan: Prof. Dr. med. D. Reinhardt



Tag der mündlichen Prüfung: 01.08.2007
Table of Contents


1 ABBREVIATIONS ...................................................................................................... 1
2 INTRODUCTION ........................................................................................................ 5
2.1 Hematopoiesis ................................................................................................................... 5
2.2 Transcription factors involved in normal hematopoiesis ............................................. 7
2.3 n factor C/EBP α in myeloid differentiation8
2.4 Acute Myeloid Leukemia (AML) .................................................................................. 11
2.5 C/EBP α and Cancer ....................................................................................................... 16
2.6 Importance of protein-protein interactions in AML: C/EBP α Network .................. 19
2.7 Proteomics of interacting proteins ................................................................................ 21
2.8 Protein identification ..................................................................................................... 22
2.8.1 By peptide mass fingerprinting (PMF) ..................................................................................... 22
2.8.2 By tandem mass spectrometry .................................................................................................. 23
2.9 TIP60 ............................................................................................................................... 24
2.10 MCM5 ............................................................................................................................. 25
3 MATERIALS ............................................................................................................. 26
3.1 Biological material ......................................................................................................... 26
3.1.1 Bacteria....... 26
3.1.2 Patient samples. ........................................................................................................................ 26
3.1.3 Mammalian Cell lines ............................................................................................................... 26
3.2 Cell culture media .......................................................................................................... 26
3.3 Chemicals Commercial solutions ................................................................................. 26
3.4 Kits .................................................................................................................................. 30
3.5 Radioactive Substances .................................................................................................. 30
3.6 Markers/Ladders ........................................................................................................... 30
3.7 Labwares ......................................................................................................................... 31
3.8 Antibodies ....................................................................................................................... 31
3.9 Plasmid Constructs ........................................................................................................ 32
3.10 Buffers ............................................................................................................................ 32
4 METHODS ................................................................................................................. 35
4.1 Cell Culture Techniques ................................................................................................ 35
4.1.1 Mammalian Cell Culture .......................................................................................................... 35
4.1.2 Transient Transfection of 293T Cells ....................................................................................... 35
4.1.3 ransfection of U937 Cells35
4.1.4 Treatment of U937 with Retionic acid ...................................................................................... 36
4.1.5 K562 ER-C/EBPα treatment with Estradiol ............................................................................. 36
4.2 Firefly and Renilla Luciferase Reporter Gene Assays ................................................ 36
4.3 Immunoblotting .............................................................................................................. 37
4.4 Preparation of Nuclear Extracts ................................................................................... 37
4.5 GST-Pull Down37
4.6 Co-Immunoprecipitation ............................................................................................... 38
4.7 GST-Purification ............................................................................................................ 39
4.8 Interaction assay with radiolabelled proteins .............................................................. 39
4.9 Histone Acetyltransferase (HAT) Assay ...................................................................... 39
4.10 Determination of DNA concentration .......................................................................... 40
4.11 FACS (flow activated cell sorting) analysis ................................................................. 40
4.12 Semi-quantitative RT-PCR ........................................................................................... 41
4.13 Expression analysis of TIP60 and C/ΕΒPα in leukemia samples .............................. 42
4.14 Chromatin Immunoprecipitation (ChIP Assay) ......................................................... 42
4.15 2D-Gel Electrophoresis ................................................................................................. 45
4.16 1-D SDS-PAGE .............................................................................................................. 46
4.17 Destaining protocol for Trypsin in-gel digestion ........................................................ 46
4.18 Trypsin In-Gel Digestion protocol ............................................................................... 47
4.19 Separation of Peptides by 1D nano-LC ....................................................................... 47
4.20 Sample Preparation for Mass Spectrometry ............................................................... 48
4.21 Mass Spectrometry ........................................................................................................ 48
5 AIM OF THE STUDY ............................................................................................... 50
6 RESULTS ................................................................................................................... 51
6.1 Purification of GST-tagged proteins ............................................................................ 51
6.2 Screening for C/EBP α interacting proteins in the myeloid cell line U937 ................ 52
6.2.1 2-D gel electrophoresis of interacting proteins ......................................................................... 52
6.2.2 1D SDS PAGE of differentially interacting proteins ................................................................ 53
6.3 List of C/EBP α interacting proteins identified by 2D MS or MS/MS ....................... 54
6.4 P αng proteins identified by 1D nano-LC-MS/MS .................. 55
6.5 Detailed analysis with Selected Putative Partner Proteins. ........................................ 56
6.5.1 MCM5 interacts with C/EBP α in vivo ...................................................................................... 56
6.5.2 TIP60 in α directly57
6.6 TIP60 increases the transactivation capacity of C/EBP α ........................................... 58
6.7 TIP60 HAT domain is required for the co-operativity with C/EBP α ....................... 60
6.8 YFPTIP60 and YFPTIP60(-HAT) expression ............................................................. 61
6.9 Coactivation by TIP60 depends on the DNA binding domain of C/EBP α ................ 62
6.10 TIP60 is found at the endogenous C/EBPα promoter in vivo .................................... 64