Epigenetic gene regulation of differentiation and epithelial-mesenchymal transition in mammary epithelial cells [Elektronische Ressource] / Xin Fu. Gutachter: Edith Pfitzner ; Christoph Englert
97 pages

Epigenetic gene regulation of differentiation and epithelial-mesenchymal transition in mammary epithelial cells [Elektronische Ressource] / Xin Fu. Gutachter: Edith Pfitzner ; Christoph Englert

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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 ............................................................................

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Publié le 01 janvier 2012
Nombre de lectures 148
Poids de l'ouvrage 3 Mo

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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

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