The role of Men1 in pituitary gland tumourigenesis [Elektronische Ressource] / presented by Lars Gredsted

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• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2005
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 43 Mo

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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-Biochemist Lars Gredsted
Born in Copenhagen, Denmark
Oral examination:The Role of Men1 in Pituitary Gland Tumourigenesis
Referees: Priv. Doz. Jochen Wittbrodt
Prof. Dr. Günther SchützAcknowledgements
Acknowledgements
The work presented in this thesis would not have been possible without the help and
assistance from many people both inside and outside the lab.
First of all I would like to thank Mathias Treier for his advise and support throughout my four
years in his lab. I have really learned a lot during my time at EMBL.
I would also like to thank the other members of my TAC committee, Jochen Wittbrodt and
Walter Wittke and my second “Gutachter” from the university of Heidelberg Günther Schütz
for constructive discussions of my project.
I would like to thank all past and present members of the Treier lab; Vitor, Catherine, Katrin,
Anna Corinna, Uli, Eve, Sandra, Dirk, Maria, Henry and Thomas for making many of the
long working days entertaining through discussions about most things imaginable including
science. A special thanks to Katrin for helping with the ES cell tissue culture, Sandra for
helping with immunohistochemistry and Henry for helping with the Genespring analysis of
the microarray data.
A big thanks must go to everybody at the animal house for taking care of our mice and for
always being helpful even when I was late with requests. I would also like to thanks the
people past and present in the transgenic facility, without whom my mouse would still be a
model only.
Thanks to all the friends I have made at EMBL that have made these years some of the best of
my life. EMBL is a truly international place and I feel very fortunate to have been given the
chance to make friends from so many countries of the world.
I would also like to thank my parents who have always supported me whatever decision I
have made right or wrong.
Finally the biggest thanks must go to Rebecca, for always being there to support me and cheer
me up. I dedicate this these to youIndex
SUMMARY 1
1 INTRODUCTION 2
1.1 Growth control in the pituitary gland 2
1.1.1 The pituitary gland 2
1.1.2 Development of the pituitary gland 3
1.1.3 Control of pituitary gland growth 5
1.1.4 Pituitary gland tumours 6
1.2 Multiple endocrine neoplasia 9
1.2.1 Multiple endocrine neoplasias 9
1.2.2 Multiple Endocrine Neoplasia type 1 (MEN1) 11
1.2.3 MEN1 12
1.2.4 MENIN 13
1.3 Menin as a regulator of transcription 14
1.3.1 Menin interacting proteins 14
1.3.2 Menin and JunD 15
1.3.3 mSin3A and Menin 16
1.3.4 Menin and NF-κB 17
1.3.5 Smad transcription factors and Menin 19
1.3.6 TGF-β signalling and growth control 21
1.3.7 MLL2 Histone methylation complex and Menin 22
1.3.8 Menin regulation of telomerase activity 23
1.3.9 Menin regulated transcription and MEN1 24
1.4 AIMS OF STUDY. 26
2 RESULTS 28
2.1 Generation of Men1 deficient pituitary glands 28
2.1.1 Construction of the Men1 conditional knockout vector 28
LoxP-Neo2.1.2 Establishment of Men1 allele 30
2.1.3 FlpE and Cre mediated recombination 30
LoxP-Frt/Frt LoxP-Null/Null 2.1.4 Viability of Men1 mice and absence of Menin in Men1 embryos 34
2.1.5 Pituitary gland specific cre mouse lines 36
2.1.6 Pituitary gland specific deletion of Men1 38
2.1.7 Pituitary gland phenotype 38
2.1.8 Pituitary gland histology 41
2.2 Molecular characterisation of Men1 deficient pituitary glands 45
2.2.1 Analysis of Menin expression in Men1 deficient pituitary glands 45
2.2.2 Analysis of growth and apoptosis in Men1 47
2.2.3 Microarray analysis of expression pattern in Men1 deficient pituitary glands 50
2.2.4 Genes deregulated in Men1 deficient pituitary glands 54
2.2.7 VIP, Cdc2/cyclin B and IGF1 57
2.2.8 Analysis by in situ hybridisation of deregulated genes 62
2.2.9 Analysis of VIP expression by immunohistochemistry 65
2.3 Screening for novel Menin interaction partners 67
2.3.1 Yeast two hybrid screening for novel Menin interactors 67
2.3.2 Previously characterised genes as novel Menin interactors 69
Trip11/GMAP210/Trip230 69
Spectrin β2/ELF 69
Ldb1a 70Index
2.3.3 Novel proteins interacting with Menin 70
2.3.4 Expression pattern of Gad67 and cloning of cDNA 71
3 DISCUSSION 74
3.1 Men1 loss leads to pituitary gland hyperplasia and adenoma formation. 74
3.2 Gene expression profiling suggest novel mechanism of MEN1 tumourigenesis 77
3.3 VIP, IGF1 and Cdc2 are upregulated in Men1 deficient pituitary glands 78
3.4 VIP and pituitary gland proliferation 81
3.5 IGF and pituitary gland proliferation 82
3.6 VIP and IGF1 in Cancer 83
3.7 Regulation of VIP and IGF expression 83
3.8 Menin interacting proteins 84
3.9 VIP and IGF1 as mediators of MEN1 tumourigenesis 86
LIST OF ABBREVIATIONS 89
4 MATERIALS AND METHODS 91
4.1 Materials 91
4.1.1 Chemicals 91
4.1.2 Equipment, plastic ware and other materials 92
4.1.3 Enzymes 94
4.1.4 Molecular weight markers 94
4.1.5 Oligonucleotides 94
Targeting vector synthesis 94
Cloning of pGad67 95
Mouse genotyping: 95
In situ probes 95
4.1.6 Antibodies 96
Primary antibodies: 96
Secondary antibodies: 96
4.1.7 Plasmid vectors 96
4.1.8 Commercial kits 97
4.1.9 Generally used solutions 97
4.1.10 Generally used media for bacteria and yeast 98
4.1.11 Cells 98
Bacterial strains 98
Yeast strains 98
ES cells 99
4.2 Methods 99
4.2.1 DNA - Plasmids 99
Preparation of plasmid DNA from bacteria 99
Purification of supercoiled DNA by CsCl gradient centrifugation 99
Plasmid extraction from yeast 100
Spectrophotometric determination of DNA and RNA concentration 100
DNA restriction and Klenow treatment 100
Electrophoresis of DNA 100
Isolation and purification of DNA from preparative agarose gels 101
DNA ligation 101
Preparation of chemocompetent Escherichia coli XL-10 cells 101
Transformation of chemocompetent Escherichia coli XL-10 cells 101
Preparation and transformation of electrocompetent E.coli XL-10 cells 102
Transformation of yeast 102
4.2.2 DNA - λ phage 103
Culture and preparation of bacteria for infection with λ-phage 103
Infection with and plating of λ-phage 103Index
Detection of specific λ-phage plaques by southern blot 103
Picking λ-phage plaques 104
Extraction of λ-phage DNA 104
4.2.3 DNA - Genomic 104
Preparation of genomic DNA 104
Polymerase Chain Reaction (PCR) 105
Southern blot analysis 105
Radiolabelling of DNA probes for southern blot analysis 106
4.2.4 RNA 106
Microarray analysis of pituitary gland expression pattern 106
4.2.5 DNA constructs 107
Construction of the Men1 targeting vector 107
Cloning of full length Gad67 107
Generation of Pit-1-Cre transgene 108
DNA constructs for in situ probes 108
4.2.6 Cell culture methods 109
Culture conditions 109
Trypsinisation of cells 109
Mitomycin C treatment of Mouse Embryo Fibroblasts 109
Freezing and thawing cells 109
Electroporation of ES cells 110
Isolation of individual ES cell colonies 110
ES cell injection into blastocysts and chimera production 111
Establishment of MEFs 111
4.2.7 Tissue sectioning 111
Tissue preparation and fixation 111
Cryosectioning 112
Paraffin embedding and mounting 112
Vibrotome sectioning 112
4.2.8 Histochemistry and Immunohistochemistry 112
Hematoxylene and Eosin staining 112
Immunofluorescence 113
Immunohistochemistry 113
4.2.9 In situ hybridisation 115
Generation of in situ probes by in vitro transcription 115
Hybridisation 115
4.2.10 Mouse methods 116
4.2.11 Proteins 117
Protein concentration meassurements 116
SDS-PAGE 116
Western blotting 117
Purification of GST-Menin 117
REFERENCES 118Summary
Summary
The pituitary gland is a key regulator of growth, metabolism and sexual development. The
pituitary gland integrates signals from the hypothalamus and from peripheral endocrine glands
and responds to changing physiological needs by secreting a series of hormones that regulate
the activity of various endocrine glands as well as acting directly on many tissues. Tumours of
the pituitary gland are relatively frequent possibly due to the plasticity of the gland. Pituitary
gland tumours occur both sporadically and as part of inherited multiple endocrine neoplasia
(MEN) syndromes. MEN1 is one of these inherited syndromes. People suffering from MEN1
develop tumours of the pituitary gland, the parathyroid glands, the pancreatic islets and the
adrenal glands. MEN1 is caused by a loss of function mutation in the tumour suppressor gene
MEN1. Men1 expression is found in all tissues in the mouse and not only in the endocrine
system. Menin the protein encoded by Men1, shares no homology with any known proteins
and contains no recognisable protein domains. Menin is believed to function as a regulator of
transcription through binding to several specific transcription factors. These include Smad
transcription factors, JunD and members of the NF-kB family of proteins.
To investigate the phenotype of Men1 deficiency and to elu