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Publié par | friedrich-schiller-universitat_jena |
Publié le | 01 janvier 2012 |
Nombre de lectures | 148 |
Poids de l'ouvrage | 3 Mo |
Extrait
Epigenetic gene regulation
of differentiation and epithelial-mesenchymal transition
in mammary epithelial cells
Dissertation
zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.)
vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller-Universität Jena
von M.Sc. in Biochemistry Xin Fu
geboren am 26.05.1973 in Shandong, China
Gutachter:
PD Dr. Edith Pfitzner
Prof. Dr. Christoph Englert
Prof. Dr. Anna Starzinski-Powitz
Tag der öffentlichen Disputation: 13.07.2011
II Table of Contents
Table of Contents
Table of Contents ................................................................................................... III
Summary .............................................................................................................. VIII
Zusammenfassung ................................................................................................. X
List of Figures ....................................................................................................... XII
List of Tables ....................................................................................................... XIV
Abbreviations ....................................................................................................... XV
1 Introduction ....................................................................................................... 1
1.1 Epigenetic regulation of gene expression .............................................................. 1
1.1.1 Epigenetics and chromatin ................................................................................... 1
1.1.2 Histone modifications ........................................................................................... 2
1.1.3 Histone lysine methylation 2
1.1.3.1 . Trimethylated Lys 27 of histone 3 (H3K27me3) ............................................... 3
1.1.3.2 . Dimethylated Lys 9 of histone 3 (H3K9me2) .................................................... 3
1.1.4 Histone lysine demethylases ............................................................................... 4
1.2 Mammary epithelial cell differentiation ................................................................... 6
1.2.1 Mammary gland development and functional differentiation ................................ 6
1.2.2 A protective role of mammary epithelial differentiation in breast tumorigenesis .. 7
1.2.3 Regulation of mammary epithelial cell differentiation ........................................... 8
1.2.3.1 . Stat5a is required for mammary epithelial cells differentiation ......................... 8
1.2.3.2 . Transcription factors control the mammary luminal cell fate ............................ 9
1.2.4 Regulation of beta-casein gene expression in mammary epithelial cells ........... 10
1.2.5 The epigenetic status of beta-casein promoter during differentiation ................ 12
1.2.5.1 . DNA methylation at beta-casein promoter ...................................................... 12
1.2.5.2 . Histone modification at beta-casein promoter ................................................ 12
1.3 Mammary epithelial cell transition of mesenchymal cell aspect ........................ 13
1.3.1 Epithelial-mesenchymal transition (EMT) .......................................................... 13
1.3.2 EMT in physiological and pathological events ................................................... 15
1.3.2.1 . EMT in embryonic development and wound healing ...................................... 15
1.3.2.2 . EMT in fibrosis and cancer metastasis ........................................................... 15
1.3.2.3 . EMT plays a special role in breast cancer metastasis .................................... 15
III Table of Contents
1.3.3 Regulation of EMT ............................................................................................. 16
1.3.3.1 . EMT is regulated by molecular networks ........................................................ 16
1.3.3.2 . CBF-A/KAP-1/FTS-1 complex acts as EMT master regulator. ....................... 17
1.3.4 Epigenetic modifications involved in EMT .......................................................... 18
1.4 Aims ......................................................................................................................... 19
2 Materials and Methods .................................................................................... 20
2.1 Materials ................................................................................................................... 20
2.1.1 Antibodies .......................................................................................................... 20
2.1.1.1 . Primary antibodies .......................................................................................... 20
2.1.1.2 . Secondary antibodies ..................................................................................... 20
2.1.2 Primers for quantitative PCR.............................................................................. 20
2.1.3 Plasmids ............................................................................................................ 22
2.1.4 Buffers and solutions.......................................................................................... 22
2.1.5 Enzymes and kits ............................................................................................... 22
2.1.6 Epigenetic inhibitors ........................................................................................... 23
2.1.7 Hormones .......................................................................................................... 23
2.1.8 Bacteria .............................................................................................................. 23
2.1.9 Cell lines ............................................................................................................ 23
2.2 Methods ................................................................................................................... 24
2.2.1 Microbiology methods ........................................................................................ 24
2.2.1.1 . Transformation of bacteria .............................................................................. 24
2.2.1.2 . Isolation of plasmid DNA (Mini preparation) ................................................... 24
2.2.1.3 . (Maxi preparation) .................................................. 24
2.2.2 DNA methods ..................................................................................................... 24
2.2.2.1 . Measurement of DNA concentration .............................................................. 24
2.2.2.2 . Restriction digestion of DNA .......................................................................... 25
2.2.2.3 . 5’-dephosphorylation of DNA ......................................................................... 25
2.2.2.4 . Ligation of DNA .............................................................................................. 25
2.2.2.5 . PCR ................................................................................................................ 25
2.2.2.6 . Agarose gel electrophoresis of DNA .............................................................. 25
2.2.2.7 . Isolation of DNA from agarose gels ................................................................ 25
2.2.3 RNA methods ..................................................................................................... 26
2.2.3.1 . RNA extraction ............................................................................................... 26
2.2.3.2 . Measurement of RNA concentration 26
IV Table of Contents
2.2.3.3 . First-strand cDNA synthesis ........................................................................... 26
2.2.3.4 . Quantitative RT-PCR ...................................................................................... 26
2.2.3.5 . Analysis of the quantitative PCR Data ............................................................ 27
2.2.4 Tissue culture methods 27
2.2.4.1 . Cultivation and treatment of cells ................................................................... 27
2.2.4.2