GLI genes [Elektronische Ressource] : cis-acting regulatory elements / vorgelegt von Amir Ali Abbasi

Abbasi

Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 47
• Poids de l'ouvrage: 57 Mo

Extrait

GLI genes: Cis-Acting Regulatory Elements
____________________________________________________________


Dissertation
zur
Erlangung des Doktorgrades
der Humanbiologie
(Dr. hum. biol.)


dem
Fachbereich Medizin
der Philipps-Universität Marburg, Germany











vorgelegt von
Amir Ali Abbasi
aus Birote, Pakistan




Zentrum für Humangenetik
Philipps-Universität
Marburg, 2008



I





















IIGLI genes: Cis-Acting Regulatory Elements
_____________________________________________________________


Dissertation
zur
Erlangung des Doktorgrades
der Humanbiologie
(Dr. hum. biol.)


dem
Fachbereich Medizin
der Philipps-Universität Marburg, Germany











vorgelegt von
Amir Ali Abbasi
aus Birote, Pakistan



Zentrum für Humangenetik
Philipps-Universität
Marburg, 2008









III


























Angenommen vom Fachbereich Humanmedizin
Der Philipps-Universität Marburg am.11-6-2008 (Tag der disputation)

Gedruckt mit Genehmigung des Fachbereichs

Dekan: Prof. Dr. med. M. Rothmund

Referent: Prof. Dr. rer . nat . K.-H. Grzeschik

Korreferent: Prof. Dr. Guntram Suske


IV

CONTENTS

ZUSAMMENFASSUNG 1
SUMMARY 5
1 INTRODUCTION 8
1.1 9 Evolution from simple to complex organisms
1.2 11 Mechanisms to generate morphological complexity
1.3 12 How to identify regulatory sequences?
1.4 Sequence alignment tools 15
1.5 Motif finding 20
Innovation of limbs provides an insight into, how the vertebrates
1.6 22
achieved morphological complexity during their evolutionary history
1.6.1 Fin to limb transition involved cis-regulatory networks 28
1.7 Cis-regulatory modules in human disease 33
1.8 35 The GLI gene family, key developmental regulators
1.9 37 The role of GLI genes in limb development
Gli3 in conjunction with Shh imposes in the autopod
1.9.1 40
constraints on digit number and identity
1.9.2 Gli3 functions other than limb morphogenesis 42
1.10 The transcriptional regulation of the GLI3 gene 43
Paralogons, a mirror of chromosomal history or
1.11 43
composed by functional restraints?
Conservation of regulatory modules for 1.12 44
paralogous genes
1.13 47 Pathogenic effects of GLI mutations: GLI3 morphopathies
1.14 AIMS 48
50 2 MATERIALS & METHODS
2.1 Reporter constructs 50
2.2 Deletion mutants 50
2.3 Cell cultures 53
2.4 Transient transfection and dual luciferase assay 54
2.5 Zebrafish enhancer / GFP reporter Assay 54
2.5.1 Anti-GFP Immunostaining 55
Chicken in ovo electroporations and enhancer reporter expression 2.6 55
analysis
2.6.1 56 In situ hybridization
2.7 56 Generation of transgenic mice
2.7.1 Embryo staining and histological analysis 56
2.8 Sequence data and comparative analysis 57
In silico mapping of conserved transcription
2.9 57
factor binding sites within each CNE
Estimation of evolutionary constraints on GLI sequences in 2.10 58
vertebrates
Dataset for gene families linked with the human 2.11 59
HOX clusters
I

Alignment and phylogenetic analysis of gene families
2.12 62
linked with the human HOX clusters

3 RESULTS 63
Prioritization of intra-GLI3 CNEs
3.1 63
(conserved non-coding elements) for functional analysis
Computational analysis to unravel within GLI3-CNEs highly conserved 3.2 67
sequence patterns with potentially functional relevance
In-vitro functional analysis of intra-GLI3 conserved non-coding 3.3 75
elements
3.3.1 Transcriptional silencers 75
3.3.2 76 Context dependent dual nature (activator / silencer) elements
3.4 79 In-vitro deletion analysis of selected sub-set of CNEs
In-vivo functional analysis of CNEs with transiently 3.5 82
transfected zebrafish embryos
Generalized scheme of GFP expression domains in zebrafish
3.6 89
embryos at 26-33 hpf or 50-54 hpf
One out of four conserved non-coding elements from the intronic
3.7 region of GLI3 showed weak enhancer activity in the chicken 90
limb bud
Expression of a reporter construct in transgenic mice under the
3.8 92
control of CNEs
3.9 Evolution of GLI sequences in vertebrates 103
3.9.1 Estimation of sequence divergence among species 103
3.9.2 Estimation of functional constraints 104
3.9.3 Evolutionary distance between paralogs 105
An insight into the phylogenetic history of HOX linked
3.10 107
gene families in vertebrates
3.10.1 Phylogenetic analysis 107
3.10.1.1 Fibrillar Collagen Family ─ COL 107
3.10.1.2 ERBB Receptor Protein Tyrosine Kinase ─ ERBB 108
3.10.1.3 Insuline-like Growth Factor Binding Protein ─ IGFBP 109
3.10.1.4 110 Integrin β ─ ITGB
3.10.1.5 111 Myosin Light Chain ─ MYL
3.10.1.6 Sp1 c2h2-type Zinc-Finger Protein ─ SP 112
3.10.1.7 Zinc-Finger Protein-Subfamily 1A ─ ZNFN1A 112
3.10.1.8 Anion Exchanger ─ SLC4A (AE) 112
3.10.1.9 GLI Zinc-Finger protein ─ GLI 113
3.10.1.10 Hedgehog ─ HH 114
3.10.1.11 Inhibin ─ INHB 114
3.10.2 Estimation of co-duplication events 115

119 4 DISCUSSION
Comparison of genomic architecture in and around GLI3
4.1 119 reveals an ancient gene regulatory network (AGRN)
within its introns
In-vitro regulatory activity of intra-GLI3 CNEs
4.2 122
is cell type specific

II
In-vitro deletion analysis defines functional modules 4.3 122
within CNE1, 5 and 6
Can transcription factor binding sites within CNEs explain their 4.4 124
evolutionary conservation?
Intra-GLI3 CNEs show tissue specific regulatory
4.5 125
activity in zebrafish embryos
Only CNE 11 could evoke reporter gene expression in
4.6 126
chick limb-bud
The evolutionary conserved human cis-regulators are involved in the
4.7 mediation of spatiotemporally distinct sub-domains of Gli3 127
expression in mouse
4.8 Activity of a human GLI3 promoter proximal regions 134
Two distinct enhancers controls Gli3 expression 4.9 136
in the developing limbs
Filling in the gap: the crosstalk among limb 4.10 140
specific cis-regulators
Multiple independently acting regulatory sequences signal the
4.11 occurrence of higher levels of modularity in the body plans 141
of modern vertebrates
Intra-GLI3 enhancers depicts the preservation and divergence
4.12 143
of target site specificity during the course of evolution
Evolutionary patterns of GLI sequences within and
4.13 144
between species
4.14 Evolutionary history of map position of the GLI paralogs 145
HOX linked paralogous regions may not reflect the outcome
4.15 147
of ancient block or whole chromosome duplication events

5 ABBREVIATIONS 151

152 6 REFERENCES

163 7 PUBLICATIONS

8 ACADEMIC TEACHERS 164

165 9 ACKNOWLEDGEMENTS

10 DECLARATION 166

167 11 CURRICULUM VITAE






III
Zusammenfassung
______________________________________________________________
Hintergrund: Frühembryonale Musterbildung wird durch zahlreiche komplexe
Signalwege gesteuert. Funktionelle Interaktionen zwischen Komponenten einer bestimmten
Signalübertragungs-Kaskade erfolgen auf vielen Ebenen, darunter bei der Bindung
extrazellulärer Signalmoleküle an ihre Rezeptoren, beim Import der Signale von den
Rezeptoren zum Zellkern oder bei der Interpretation eines Signals durch Aktivierung oder
Repression der Expression von Zielgenen mittels Transkriptionsfaktoren. Präzise funktionelle
Querverbindungen zwischen den Schritten einer Kaskade sind essentiell für eine normale
Entwicklung.
Die Mitglieder der GLI-Genfamilie sind wichtige Vermittler der Signalinformation im
“SONIC HEDGEHOG (SHH)-Signalweg“. Während der letzten Jahrzehnte wurden die
Mitglieder der GLI-Familie von Transkriptionsfaktoren eingehend mit genetischen,
molekularen und biochemischen Methoden erforscht. Dadurch verfügen wir jetzt über reiche
Informationen zur intrazellulären Lokalisierung der GLI-Proteine, über ihre
Interaktionspartner, ihre Antwort auf SHH-Signale, über die Art ihres Transports in den
Zellkern und auch über einige ihrer Zielproteine. Zudem wurde reiches Wissen über
Entwicklungsstörungen beim Menschen und anderen Modellorganismen, die mit Mutationen
von Mitgliedern der GLI-Genfamilie