Regulation of estrogen responsive genes by human estrogen receptor alpha {(ERα) [(ER-Alpha)] [Elektronische Ressource] / presented by Nancy Bretschneider

Nancy Luce

Sujets

• Gesundheit

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 28
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences



























presented by

Diplom Naturwissenschaftlerin Nancy Bretschneider

Thesis submitted:
Oral examination:











Regulation of estrogen responsive genes by the human
Estrogen Receptor alpha (ERα)
































stReferees: 1 examiner Dr. Andreas Ladurner
nd2iner Prof. Dr. Günther Schütz
Member 1 Dr. Anne Ephrussi
Member 2 Prof. Dr. Christine Clayton
Acknowledgments
ACKNOWLEDGMENTS

First of all, I would like to thank Prof. Frank Gannon for giving me the opportunity of an interesting
PhD in his laboratory and for his advice during my time here. I am also especially grateful to my
supervisor Stefanie Denger for her constant interest, support and encouragement. Also, I would like to
thank all former and present members of the Gannon group for their help, support and inspiring
discussions: Heike Brand, Steven Johnsen, Sara Kangaspeska, Michael Hübner, Raphael Métivier,
Maria Polycarpou-Schwarz, George Reid, Brenda Stride, Jonathon Blake, Steffi Österreich. I have
enjoyed and benefited to work with them.

Furthermore, I would like to thank my Thesis Advisory Committee, as well as my Thesis Defence
Committee for their interest in my work and inspiring discussions: Prof. Christine Clayton, Dr. Anne
Ephrussi, Dr. Andreas Ladurner and Prof. Günther Schütz.

From all the excellent services provided by EMBL, I would especially like to thank Vladimir Benes
and the members of the GeneCore facility, the photolab and all the visible and invisible helpers in
EMBL.

Furthermore, I would like to thank Prof. Michael J. Kerins group (Galway, Ireland) for their
engagement in identification of BASE in breast tumour samples.

And last but not least, I thank my friends and family for their understanding, encouragement and the
great time we had. This thesis would not have been possible without you.

III
Table of contents
TABLE OF CONTENTS

ACKNOWLEDGMENTS..................................................................................................................... III
LIST OF FIGURES.............VIII
ZUSAMMENFASSUNG...... IX
SUMMARY ........................................................................................................................................... X
INTRODUCTION................................................................................................................................... 1
1. General introduction – estrogen receptor and disease......................................................................... 1
2. The nuclear receptor family ................................................................................................................ 1
2.1 The estrogen receptors .................................................................................................................. 2
2.1.1 The estrogen receptor alpha................................................................................................... 2
2.1.2 The A and B domains ............................................................................................................ 4
2.1.3 The DNA binding domain (C-domain).................................................................................. 4
2.1.4 The hinge region (D-domain).. 4
2.1.5 The ligand binding domain (E-domain)................................................................................. 5
2.1.6 The F-domain ........................................................................................................................ 6
2.1.2 ERα isoforms......................................................................................................................... 6
2.2 Posttranslational modifications of ERα ........................................................................................ 8
2.2.1 Phosphorylation ..................................................................................................................... 8
2.2.2 Acetylation............................................................................................................................. 9
2.2.3 Ubiquitination...................................................................................................................... 10
2.2.4 Sumoylation 10
3. Mechanisms of ERα mediated transcriptional activation ................................................................. 11
3.1 “Classical”: cyclic transactivation through estrogen responsive elements (EREs)..................... 11
3.2 Indirect transactivation through protein-protein interaction on non-ERE elements ................... 12
3.3 “Non-genomic activity” of ERα ................................................................................................. 13
4. Mechanism of hormone-induced gene repression............................................................................. 15
4.1 Repression of GATA-1 activity by estrogen............................................................................... 16
4.2 Passive repression through allosteric hindrance ......................................................................... 17
4.3 Recruitment of corepressors to a half ERE in the cyclin G2 promoter by ERα ......................... 17
4.4 DNA sequence as allosteric effector........................................................................................... 17
4.5 Enhancement of TNFα action on the TNFα promoter through apo-ERα .................................. 18
4.6 Crosstalk with NF-κB – an example of complexity.................................................................... 18
5. The estrogen receptor in breast cancer .............................................................................................. 20
6. Estrogen-stimulated gene activation – the impact of distal enhancers.............................................. 22
7. The BASE gene – what is known...................................................................................................... 23


IV Table of contents
RESULTS.............................................................................................................................................. 24
A Expression and regulation of the Breast Cancer and Salivary Gland Expressed gene 24
1. Validation of microarray analysis with qRT-PCR ............................................................................ 24
2. BASE regulation is ERα-dependent.................................................................................................. 24
2.1 BASE is rapidly down-regulated in response to E2................................................................... 25
2.2 Knockdown of ERα induces BASE expression......................................................................... 26
3. Cloning and testing of reporter constructs......................................................................................... 26
4. BASE expression in different cell lines............................................................................................. 27
5. BASE promoter analysis ................................................................................................................... 29
5.1 A 600 bp regulatory region is essential for BASE expression................................................... 29
5.2 Regulatory potential of the enhancer and identification of the core promoter............................ 30
5.3 Impact of the transcription start site............................................................................................ 31
5.4 Mutation of the EREs in enhancer and at transcription start site................................................ 32
5.5 BASE expression and regulation can be separated ..................................................................... 33
6. ERα can bind the enhancer in vitro and in vivo ............................................................................... 34
7. Bioinformatic analysis of segment C2 & identification of FoxA1 sites important for expression ... 35
8. FoxA1 is essential for BASE expression........................................................................................... 36
9. Bioinformatic analysis of the BASE promoter using MatInspector.................................................. 38
10. Validation of key roles for FoxA1 and ERα in BASE expression and regulation......................... 39
11. Further putative factors involved in BASE regulation .................................................................... 41
12. Introduction of ERα in SKBR3 cells allows estrogen-induced regulation ..................................... 43
13. BASE main tra