The gene encoding human SCGB 2A1 is under indirect androgen control operating through an Sp family binding site in prostate cells [Elektronische Ressource] / vorgelegt von Fei Xiao

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Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2004
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Aus dem Institut für Molekularbiologie und Tumorforschung
der Philipps-Universität Marburg
Geschäftsführender Direktor: Professor Dr. R. Müller







The Gene Encoding Human SCGB 2A1 is under Indirect
Androgen Control Operating through an Sp
Family Binding Site in Prostate Cells





Inaugural Dissertation
zur Erlangung des Doktorgrades der Humanbiologie

dem Fachbereich Medizin der
Philipps-Universität Marburg
vorgelegt von



Fei Xiao
aus JiangSu, V.R.China


Marburg 2003



Angenommen vom Fachbereich Humanbiologie
der Philipps-Universität Marburg am 01.10.2003
Gedruckt mit Genehmigung des Fachbereichs
Dekan: Professor Dr. Bernhard Maisch
Referent: Professor Dr. Miguel Beato
Correferent: Professor Dr.Gerhard Aumüller




2Contents
CONTENTS
CONTENTS ...................................................................................................................I
1. ABBREVIATIONS.................................................................................1
2. INTRODUCTION...................................................................................3
2.1 Transcriptional regulation in eukaryotic cells...........................................3
2.1.1 Transcription factors.................................................................................4
2.1.2 Chromatin structure and the regulation of transcription..........................7
2.2 Steroid hormones and their receptors .......................................................9
2.2.1 Structure and distribution of SHRs ...........................................................9
2.2.2 Interaction between SHRs and other factors ..........................................11
2.2.3 Androgens and androgen receptor..........................................................13
2.2.4 Non-genomic effects of androgens..........................................................16
2.3 Secretoglobins.........................................................................................17
2.3.1 The secretoglobin family of proteins.......................................................17
2.3.2 Human members of the secretoglobin family..........................................20
2.4 Aim of the Project...................................................................................22
3. MATERIALS AND METHODS .........................................................23
3.1 Materials .................................................................................................23
3.1.1 Chemicals and equipment.......................................................................23
3.1.2 Cell lines .................................................................................................25
3.1.3 Buffers and solutions...............................................................................26
3.1.4 Enzymes and Antibodies .........................................................................27
3.1.5 Oligonucleotides .....................................................................................28
3.1.6 Plasmids..................................................................................................29
3.2 Methods...................................................................................................31
3.2.1 Cell culture and preparation of charcoal treated FCS ...........................31
3.2.2 Purification of nucleic acids ...................................................................31
3.2.2.1 Preparation of high molecular weight DNA from cultured cells ............31
3.2.2.2 Preparation of total RNA from cultured cells .........................................32
3.2.3 Gel electrophoresis .................................................................................32
3.2.3.1 DNA agarose gel electrophoresis............................................................32

IContents
3.2.3.2 SDS polyacrylamide gel electrophoresis and Coomassie Blue
staining....................................................................................................32
3.2.4 Northern blotting analysis ......................................................................34
3.2.4.1 Electrophoresis of glyoxylated RNA through agarose gels.....................34
3.2.4.2 Transfer and fixation of denatured RNA to membranes..........................35
3.2.4.3 Northern hybridization............................................................................35
3.2.5 Southern blotting analysis.......................................................................36
3.2.5.1 Electrophoresis of DNA through agarose gels........................................36
3.2.5.2 Transfer and fixation of denatured DNA to membranes .........................36
3.2.5.3 Southern hybridization............................................................................37
3.2.6 Mapping of DNase I hypersensitive sites................................................37
3.2.7 Cloning and subcloning ..........................................................................38
3.2.8 PCR-mediated mutagenesis ....................................................................39
3.2.9 Preparation of competent E. coli and transformation ............................40
3.2.10 Transfections and reporter gene assays..................................................42
3.2.10.1 Transfection of LNCaP and HeLa cells with the calcium phosphate
method.....................................................................................................42
3.2.10.2 Luciferase assay......................................................................................43
3.2.10.3 β-Galactosidase assay ............................................................................43
3.2.11 Preparation of nuclear extracts ..............................................................44
3.2.12 DNase I footprinting ...............................................................................45
3.2.12.1 End-labeling of the DNA probe...............................................................45
3.2.12.2 Maxam-Gilbert sequencing reactions.....................................................46
3.2.12.3 DNase I digestion....................................................................................46
3.2.13 Oligonucleotides and EMSA...................................................................47
3.2.14 Protein expression and purification........................................................48
3.2.14.1 Preparation of the expression construct .................................................48
3.2.14.2 Expression of SCGB 2A1 in E. coli.........................................................49
3.2.14.3 Purification of histidine-tagged SCGB 2A1 protein ...............................50
3.2.15 Antibody preparation ..............................................................................50
3.2.16 Western blotting ......................................................................................51
3.2.17 Immunostaining.......................................................................................51
4. RESULTS ..............................................................................................53
4.1 SCGB 2A1 is expressed in the prostate ..................................................53
4.1.1 SCGB 2A1 expression is localized to the glandular epithelium .............53
4.1.2 Expression of SCGB 2A1 in LNCaP cells is induced by androgen.........53
4.2 A hormone dependent chromatin alteration occurs in the SCGB
2A1 promoter..........................................................................................56
IIContents
4.3 The proximal promoter (-136/+50) of the SCGB 2A1 gene is
sufficient for both constitutive and androgen-induced expression .........58
4.4 The dim-IR-GC box of the SCGB 2A1 promoter specifically
responds to androgens but not to glucocorticoids...................................60
4.5 DNase I footprints with nuclear extracts from LNCaP and HeLa
cells identify three functional elements ..................................................62
4.6 The ubiquitous proteins NF-Y, NF1 and Sp family factors bind to
the SCGB 2A1 promoter in vitro ............................................................65
4.6.1 NF-Y binds to the CCAAT-box of the SCGB 2A1 promoter....................65
4.6.2 NF1 recognizes a binding site with one substitution in each
canonical half site...................................................................................67
4.6.3 Sp family proteins recognize a dimeric inverted repeat type GC box.....69
4.7 NF-Y is functionally important for basal promoter activity whereas
binding of NF1 participates in and binding of Sp factors mediates
the androgen response.............................................................................73
4.8 Expression and purification of SCGB 2A1 and preparation of a
rabbit antiserum ......................................................................................75
4.9 Localization of SCGB 2A1 in prostate tissue.........................................77
5. DI