Inducible systems for the characterization of insulating and repressing motifs [Elektronische Ressource] / vorgelegt von Sabine Fischer

Inducible systems for the characterization of
insulating and repressing motifs
Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.
vorgelegt von
Sabine Fischer
aus RothAls Dissertation genehmigt von der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung: 22. Dezember 2009
Vorsitzender der Promotionskomission: Prof. Dr. Eberhard Bänsch
Erstberichterstatter/in: Prof. Dr. Wolfgang Hillen
Zweitberichterstatter/in: Prof. Dr. Uwe SonnewaldINDEX
1 Zusammenfassung ................................................................................................. 1
2 Summary ................................................................................................................ 2
3 Introduction ........................................................................................................... 3
3.1 Insulators................................................................................................................. 3
3.1.1 Transcriptional models for insulator function.................... 4
3.1.2 Structural models for insulator function............................................................. 7
3.1.2.1 Loop formation through interacting proteins ......................................... 8
3.1.2.2 Loop formation through interaction with the nuclear matrix................. 8
3.1.3 Blocking the spread of heterochromatin .......................................................... 10
3.2 The multivalent transcription factor CTCF...................... 11
3.2.1 Structure of CTCF............................................................................................ 11
3.2.2 CTCF as regulator of gene expression............................. 12
3.2.2.1 Insulator function ................................................................................. 13
3.2.2.2 Transcriptional repression.... 14
3.3 The minichromosome maintenance complex protein 7 – Mcm7 ..................... 15
3.4 The Tet-system...................................................................................................... 16
3.5 Scope of the thesis. 19
4 Results................................................................................................................... 20
4.1 Looping out of an enhancer in chromosomal environment.............................. 20
4.1.1 Retroviral integration of the reporter construct................................................ 20
4.1.2 Linear integration of the reporter construct...................... 22
4.1.3 Co-transfection introducing a meganuclease restriction site............................ 24
4.1.3.1 Southern Blot analysis.......................................................................... 26
4.1.3.2 Cassette-exchange to tk-neo with and without SceI............................. 28
4.1.3.3 Analysis of a single cross-over event................................................... 33
4.2 Transcriptional repression mediated by CTCF................ 37
4.2.1 Mapping of the N-terminal CTCF repression motif......................................... 37
4.2.1.1 Localization of the N-terminal repression motif.. 37INDEX
4.2.1.2 Alanine scanning to identify the residues important for repression ........
of a CMV-promoter-enhancer construct .............................................. 39
4.2.1.3 Alanine scanning to identify the residues important for repression.........
of an SV40-promoter-enhancer construct........... 41
4.2.1.4 Residues important for repression are also active in the context ............
of the entire CTCF N-terminal domain ................................................ 45
4.2.2 Analysis of CTCF alanine mutants in COS-7 cells.......... 47
4.2.3 The repression motif is active in stably transfected cell lines.......................... 49
4.3 CTCF interacts with itself ................................................................................... 51
4.3.1 Yeast two-hybrid screens with different CTCF domains................................. 51
4.3.2 Dimerization based on loop-forming systems.................. 54
4.4 Yeast two-hybrid Screen with a peptide library................................................ 61
4.4.1 Interaction with Mediator and NFATc4........................... 66
4.5 Yeast two-hybrid Screen with a cDNA library.................................................. 68
4.6 Co-Immunoprecipitation of CTCF-domains and Mcm7.................................. 72
4.6.1 Co-Immunoprecipitation of CTCF, Mcm7 and Smc1...... 73
5 Discussion ............................................................................................................. 75
5.1 Introduction of a loop-forming system into the chromosome .......................... 75
5.1.1 Introduction of a recombinase-based integration system................................. 75
5.1.1.1 Integration of a Cre/lox system based on gpt as ......
selection-counterselection marker........................................................ 76
5.1.1.2 Integration of tk-neo for selection by retroviral integration................. 76
5.1.1.3 Integration of tk-neo for selection in a combined vector ..................... 77
5.1.1.4 Co-transfection with the integrated meganuclease cleavage site SceI. 77
5.1.1.5 Cre-exchange with the selection/ counterselection system .................. 78
5.2 The multivalent transcription factor CTCF ...................................................... 79
5.2.1 Characterization of the N-terminal CTCF repression motif............................. 80
5.2.2 Sequence identity of the repression domain in different organisms ................ 81
5.3 Dimerization of CTCF ......................................................................................... 82
5.3.1 Yeast two-hybrid screen for the identification of self-interacting domains ........
of CTCF............................................................................................................ 83INDEX
5.3.2 Loop-forming experiments for the identification of self-interacting domains.....
of CTCF............................................................................................................ 83
5.4 Identification of protein partners of CTCF by Yeast two-hybrid ......................
analysis using a peptide library........... 84
5.4.1 Analysis of the interaction with NFATc4 and Med12 ..................................... 85
5.5 CTCF interacts with Mcm7................................................. 86
5.5.1 Co-immunprecipitation experiments with Mcm7 and CTCF .......................... 87
5.6 CTCF, Mcm7 and Smc1 ...................................................................................... 88
6 Materials and Methods ....................................................................................... 90
6.1 Materials................................................................................................................ 90
6.1.1 Chemicals........ 90
6.1.2 Auxiliary Materials .......................................................................................... 92
6.1.3 Instruments....................................... 92
6.1.4 Enzymes, Proteins and Size markers................................ 94
6.1.5 Commercially available systems (“kits”)......................... 94
6.1.6 Oligonucleotides............................................................... 94
6.1.7 Plasmids........................................................................... 99
6.1.8 Antibodies ...................................... 112
6.1.9 Bacteria........... 112
6.1.10 Yeast strains................................................................... 112
6.1.11 Cell lines......................................... 112
6.2 Media, Buffers and Solutions ............................................................................ 113
6.2.1 Bacterial growth media.................. 113
6.2.2 Yeast growth media........................................................................................ 114
6.2.3 Cell culture media.......................... 115
6.2.4 Buffers and solutions...................... 116
6.3 General Methods ................................................................................................ 119
6.3.1 General methods............................. 119
6.3.2 Cell culture ..................................................................................................... 119
6.3.2.1 Cultivation of HeLa, COS-7, HEK293 cell lines ............................... 119
6.3.2.2 Cell counts.......................................................................................... 119INDEX
6.3.2.3 Long-term storage of cell lines........................................................... 119
6.3.3 Methods for transfection and characterisation of cell lines............................ 120
6.3.3.1 Transient transfection of HeLa, COS-7, HEK293 cells ..........................
using Lipofectamine™ ....................................................................... 120
6.3.3.2 Harvesting of HeLa, COS-7, HEK293 cells...... 120
6.3.3.3 Determination of protein concentrations............ 120
6.3.3.4 Luciferase assay ................................................................................. 120
6.3.3.5 Stable transfection of HeLa cells....................... 121
6.3.3.