VITO proteins are essential new cofactors of the muscle regulatory network [Elektronische Ressource] / von Michał Mielcarek

Mmielca

Sujets

• Biologie

Informations

• Publié par: martin-luther-universitat_halle-wittenberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 19 Mo

Extrait

VITO proteins are essential new cofactors of the
muscle regulatory network


Doctoral Thesis

zur Erlangung des akademischen Grades Dr. rer. nat.

vorgelegt der

Mathematisch-Naturwissenschaftlich-Technischen Fakultät
der Martin-Luther-Universität Halle-Wittenberg


von
Micha ł Mielcarek
Geb. am: 21 September 1976
in Pozna ń, Poland


Reviewers:
1. Prof. Dr. T. Braun
2. Prof. Dr. R. Renkawitz-Pohl
3. Prof. Dr. E. Wahle

02.03.2007 Halle (Saale)
urn:nbn:de:gbv:3-000011638
[http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000011638] CONTENT
1. Introduction 1
1.1. Regulation of transcription in vertebrates 1
1.2. Transcription regulation in skeletal and cardiac muscles 3
1.3. Transcription Enhancer Factors (TEF) family 4
1.3.1. Evolution and structure of TEFs family 4
1.3.2. Expression and regulation of Transcription Enhancer Factor
members 6
1.3.2.1. TEF-1 6
1.3.2.2. TEF-3 8
1.3.2.3. TEF-4 9
1.3.2.4. TEF-5 10
1.3.3. Biological function of TEF gene family 11
1.3.3.1. Role of TEFs in skeletal muscles 13
1.3.3.2. Regulation of transcription in cardiac muscles by TEFs 14
1.3.4. Co-activators of TEFs 17
1.3.4.1. YAP65 and TAZ as co-activators of TEFs 17
1.3.4.2. p160 family as co-activators of TEFs 19
1.3.4.3. TONDU as co-activator of TEFs 19
1.4. Aim of the studies 20
2. Abbreviations 21
3. Materials and Methods 24
3.1. Materials 24
3.1.1. Basic materials 24
3.1.2. Chemicals 24
3.1.3. Radiochemicals 25
3.1.4. Specific Reagents 25
3.1.5. Enzymes 26
3.1.6. Kits 27
3.1.7. Oligonucleotides 27
3.1.7.1. Sequencing primers 27
3.1.7.2. Primers for RT-PCRs 28
3.1.7.3. Cloning primers 29
3.1.7.4. Oligonucleotides for RNAi 31
3.1.8. Vectors and Plasmids 32
I CONTENT
3.1.8.1. Plasmids for riboprobes synthesis 34
3.1.9. Bacterial strains 34
3.1.10. Cell lines 35
3.1.11. Antibodies 35
3.1.12. Mice strains 35
3.1.13. Buffers and solutions 36
3.2. Methods 37
3.2.1. Standard molecular biology methods 37
3.2.2. Cloning strategies 37
3.2.3. Expression and diagnostic plasmids 37
3.2.3.1. Expression plasmids 37
3.2.3.2. Plasmids for riboprobes synthesis 39
3.2.4. Identification of the VITO-1 genomic locus – screening
of cosmid libraries 40
3.2.5. In situ hybridization 42
3.2.5.1. Embryos preparation 42
3.2.5.2. Tissue preparation for paraffin embedding 42
3.2.5.3. Riboprobes synthesis 43
3.2.5.4. Whole mount in situ hybridization 43
3.2.5.5. In situ hybridization on paraffin embedded tissue slides 45
3.2.5.6. Eosin staining 46
3.2.6. Cell culture methods 46
3.2.6.1. Basic maintenance 46
3.2.6.2. Differentiation of C2C12 cells into myotubes 47
3.2.6.3. MyoD dependent conversion of fibroblast cell lines 48
3.2.6.4. Transient transfection 48
3.2.6.4.1. Calcium phosphate 48
3.2.6.4.2. Electroporation 49
3.2.6.5. Stable cell line generation 49
3.2.6.6. Fluorescence Activated Cell Sorting (FACS) 50
3.2.7. Immunocytochemistry 50
3.2.7.1. Immunoperoxidase detection 50
3.2.7.2. Immunocytochemistry with fluorescent labeled secondary antibody 51
3.2.8. Total RNA isolation from tissues and cells 51
II CONTENT
3.2.9. PCR and RT-PCR 51
323.2.10. P random primed labeling probes preparation 52
3.2.11. Southern blot analysis 52
3.2.12. Northern blot analysis 53
3.2.13. Western blot analysis 54
3.2.14. Overexpression of VITO-1 and VITO-2 proteins in E.coli 54
3.2.15. CAT assay 57
3.2.16. β-galactosidase activity 57
3.2.17. Sumoylation assay 58
3.2.18. Statistics 58
4. Results 59
4.1. VITO family of genes as new homologues of vestigial
and TONDU proteins 59
4.2. Expression pattern of VITO genes family 64
4.2.1. VITO-1 is specifically expressed in skeletal muscles 64
4.2.2. VITO-2 is not a muscle specific gene 69
4.3. Mouse Vgl-4 is ubiquitously expressed in the adult tissues 77
4.4. Expression of TEFs and their co-activators is modulated
during the course of C2C12 myoblasts differentiation 79
4.5. Intracellular localization of VITO1/2 proteins in the various cell lines 81
4.6. VITO-1 but not VITO-2 enhances MyoD-mediated
myogenic conversion of fibroblasts 84
4.7. Knockdown of VITO-1/2 genes by RNAi 87
4.7.1. VITO-1 and VITO-2 can be knocked-down efficiently using RNAi 88
4.7.2. Attenuation of VITO-1 but not VITO-2 inhibits MyoD
mediated conversion of 10T1/2 and 3T3 cells 91
4.7.3. Knockdown of VITO-1/2 genes in terminally differentiated
C2C12 cell line by RNAi 93
4.8. VITO-2 is not a direct transcriptional activator 100
4.9. Expression of VITO-1/2 genes in the Myf-5 and delta1
knockout mice 102
4.10. Posttranslational modification of VITO family members 106
5. Discussion 110
5.1. Identification of vestigial and TONDU related co-activators 110
III CONTENT
5.2. Tissue distribution of the VITO family of genes 112
5.3. Nuclear localization of VITO proteins 117
5.4. VITO family of genes lack a transactivation domain 117
5.5. MyoD mediated conversion of fibroblasts and role of VITO genes 118
5.6. VITO family of genes are required for C2C12
myoblasts differentiation 121
5.7. VITO genes are differently regulated by the Notch pathway 123
5.8. Lack of VITO-1/2 expression in Myf-5 mutant mice 124
5.9. VITO-1 but not VITO-2 is target of SUMO modifier 125
6. Summary 127
7. Zusammenfassung 130
8. Appendix 133
8.1. Sequences of mouse and human VITO-1/2 genes and their alignments 133
8.2. Curriculum Vitae 143
8.3. Publications and scientific activity in congresses during PhD studies 146
8.3.1. Publications 146
8.3.2. Presentation 146
9. Acknowledgements 147
10. References 149



IV INTRODUCTION
1. INTRODUCTION

1.1. Regulation of transcription in vertebrates

Much has been learned about the regulation of the eukaryotic genes transcription
over the past three decades. Transcriptional regulation is the framework responsible for
a cell specification and development of a complex tissues and organs. Basically,
transcription is a polymerisation reaction of single nucleotides leading to mRNA. This
reaction is catalysed by polymerase I, II or III depending on DNA in the presence of
2+ 2+Mg or Mn ions. Transcription might be divided in three steps: initiation, elongation
and termination.

11
CCOO--AACCTTIIVVAATTOORRSS
TRANS-ACTING
TRANSCRIPTION
FACTORS
PROMOTER GENE
DDNNAA
EENNHHAANNSSOONNSS

CIS-ELEMENTS
LCR
TRANSCRIPTION
GGEENNEE EENNHHAANNSSOONNSS
33’’UUTTRRCCIISS--EELLEEMMEENNTTSS mmiiRRNNAA
pre-mRNA

INHIBITION OF
TRANSLATIONor
SPLICING, EDITING, MODIFICATION

DDEEGGRRAADDAATTIIOONNSS

mRNA 2

TRANSLATION


PPRROTOTEEIINNSS

Fig. 1. A schematic drawing of enhancers and their co-activators in the regulation
of transcription. Two yellow boxes 1 and 2 show two control steps for protein
expression at the transcription level.

1 INTRODUCTION
From this point of view, transcription seems to be a simple enzymatic reaction.
However, the central of transcription requires many specific regulatory proteins, which
can interact with DNA via hydrogen and van der Waals bonds in the specific DNA
regions, so called promoters. At this point, it is assumed that transcription is regulated
by different bounding prop