Investigation of changes in histone modifications and factor recruitment to milk protein genes during mammary gland differentiation and identification of novel STAT5A-DNA binding sites in mammary gland tissue [Elektronische Ressource] / von Stefanie Kliem

Nickname

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 35
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Investigation of changes in histone modifications and factor recruitment
to milk protein genes during mammary gland differentiation
and identification of novel STAT5A-DNA binding sites in mammary gland tissue




Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften




vorgelegt beim Fachbereich Biowissenschaften (15)
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main




von
Stefanie Kliem
aus Bad Homburg vor der Höhe




Frankfurt am Main 2008
D30









vom Fachbereich Biowissenschaften der Johann Wolfgang Goethe-Universität
als Dissertation angenommen.









Dekan: Prof. Dr. V. Müller
Gutachter: Prof. Dr. A. Starzinski-Powitz
PD Dr. E. Pfitzner


Datum der Disputation:















Eidesstattliche Erklärung

die selbstständige Anfertigung dieser Arbeit erkläre ich an Eides statt.


Frankfurt am Main, den 13 Juni 2008



(Stefanie Kliem)

Table of Contents


1. Introduction....................................................................................................... 1
1.1 Mouse mammary gland development........................................................... 1
1.2 STAT signal pathway activation and structure.............................................. 3
1.3 STAT5 .......................................................................................................... 5
1.3.1 In vivo function of STAT5A and STAT5B determined by gene
targeting deletion in mice .................................................................................6
1.3.2 Regulation and function of STAT5 in the mammary gland ...............................8
1.4 Regulation of transcription.......................................................................... 11
1.4.1 Basal transcription ......................................................................................... 11
1.4.2 Chromatin structure ....................................................................................... 12
1.4.3 Function of histone modifications in transcription........................................... 12
1.5 Aim of project.............................................................................................. 17

2. Materials and Methods ................................................................................... 18
2.1 Materials..................................................................................................... 18
2.1.1 General chemicals and Materials................................................................... 18
2.1.2 Solutions and Buffers..................................................................................... 18
2.1.3 Enzymes........................................................................................................ 19
2.1.4 Kits and molecular weight markers ................................................................ 19
2.1.5 Antibodies...................................................................................................... 19
2.1.6 Oligonucleotides ............................................................................................ 20
2.1.7 Plasmids........................................................................................................ 25
2.1.8 Bacteria and bacterial culture media.............................................................. 26
2.1.9 Tissue culture reagents and cell lines ............................................................ 27
2.1.10 Online Bioinformatic resources and tools ..................................................... 27
2.2 Methods...................................................................................................... 29
2.2.1 General molecular methods for cloning and working with nucleic acids ......... 29
2.2.2 Tissue culture methods.................................................................................. 36
2.2.3 Molecular Biology techniques ........................................................................ 38

3. Results............................................................................................................. 44
3.1 Factor recruitment and changes in histone modifications at the endogenous
ß-casein and WAP gene regions during mammary gland differentiation .... 44
3.1.1 Both STAT5a and STAT5b bind to the enhancer and promoters of
the ß-casein and WAP gene in mammary gland tissue.................................. 46
3.1.2 RNA polymerase II is recruited to the promoter, transcribed region
and 3´region of the ß-casein and WAP genes during pregnancy
and lactation ................................................................................................. 49
3.1.3 Distribution of histone modifications during different mammary gland stages. 49
3.1.4 Increased histone acetylation at the ß-casein and WAP genes
during pregnancy and lactation ...................................................................... 50
3.1.5 Local histone acetylation of specific lysine residues occurs on the
ß-casein and WAP genes during mammary gland differentiation ................... 53
3.1.6 Changes in histone methylation at the ß-casein and WAP genes
during mammary gland differentiation ............................................................ 53
3.1.7 The presence of tri-methylated (K4) histone H3 correlates with the active
transcriptional state of ß-casein and WAP gene during pregnancy and
lactation ......................................................................................................... 54
3.1.8 Tri-methylated lysine 27 of histone H3 is associated with the ß-casein
and the WAP gene in mammary gland tissue ................................................ 57
3.1.9 Histone H3 abundance .................................................................................. 57
3.1.10 Histone modifications and transcription factor binding at STAT5 responsive genes in mammary epithelial cell lines.......................................................... 58
3.1.11 Tri-methylated lysine 27 of histone H3 is also associated with
the ß-casein gene promoter in the mammary epithelial cell line .................. 60
3.1.12 Both STAT5A and STAT5B are recruited to the SOCS3 promoter
of differentiation competent HC11 cells........................................................ 62
3.1.13 RNA polymerase II and histone modifications are found at the
SOCS3 promoter, but not at the ß-casein promoter ..................................... 62
3.2 Identification of novel STAT5A-binding sites in mammary gland tissue
from a lactating mouse using ChIP............................................................. 63
3.2.1 Verification of STAT5A binding to known differentiation specific
STAT5-target genes ...................................................................................... 63
3.2.2 Approach used for ChIP based cloning and the identification of novel
STAT5-binding sites in cloned DNA regions .................................................. 65
3.2.3 Confirmation of STAT5A and STAT5B binding to novel target sequences ..... 70
3.2.4 Investigation into RNA polymerase II recruitment and
histone 2M(K4)H3 modifications at four of the novel STAT5-binding sites ..... 74
3.2.5 Comparative sequence analysis of the novel STAT5-binding sites
between different vertebrate species ............................................................. 76
3.2.5.1 The second STAT5 motif of the c20 clone shows homology between
the mouse and rat sequence.................................................................................................... 76
3.2.5.2 The c34 clone is located within the second intron of the Arhgef3 gene and all 3
STAT5 motifs show evolutionary conservation in at least 5 different mammals ................... 77
3.2.5.3 The c73 clone is located within the last intron of the Inpp4b gene and three out of
five STAT5-binding sites show homology between the mouse and rat sequence................ 79
3.2.5.4 The 339bp region of c153 clone is evolutionarily conserved and contains
one STAT5 consensus binding site and 3 STAT-like sites..................................................... 81
3.2.5.5 The c194 region contains two putative STAT5-binding sites which
show homology between the mouse and the rat genome ...................................................... 82
3.2.5.6 The c236 region contains two putative STAT5-binding sites which
are conserved between mouse and rat genome..................................................................... 83
3.2.5.7 The c398 clone is located in the promoter and within the first intron of the