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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:

Éva Kalmár
born in: Budapest, Hungary
Oral-examination: 05.03.2009.





Analysis of the cis-regulatory structure of developmentally regulated
genes in zebrafish embryo





















Referees: Prof. Dr.Uwe Strähle
. Prof. Dr.Jochen Wittbrodt

Abstract

Transcription regulation during vertebrate embryonic development is tightly
regulated by cis-regulatory elements and respective transcription factor complexes,
which bind to them. The interaction of these elements, followed by the recruitment of
the RNA polymerase II machinery, leads to transcription initiation, which is one of
the major regulatory steps in gene expression regulation. In this thesis I study three
aspects of cis regulatory function in the zebrafish embryo:

1. Non-coding genomic sequences, in some cases with extreme evolutionary
conservation, were shown to harbour enhancer function. After the completion of
several mammalian and vertebrate genomes, phylogenetic footprinting became
frequently used methods for cis-regulatory element identification. I present the
identification of conserved noncoding sequences in the pax2 locus and their in vivo
test for enhancer activity in transient transgenic zebrafish embryos.
2. Conserved non-protein coding sequences working as enhancers were
significantly enriched in and or around developmental regulators and/or transcription
factor genes. In the second part of this thesis I present the application of a combined
global and local alignment tool, which could identify higher number of conserved
noncoding elements with enhancer activity, then any of the previous methods. Two
thirds of the identified elements were shuffled during evolution. Although the
majority of these shuffled conserved elements were still assigned to gene classes of
transcription factors and developmental regulators, there were high enrichment in
genes belonging to the extracellular regions and behavioural Gene Ontology classes.
3. The assignment of identified enhancers to their target gene promoters is often
problematic, because of the potentially very large sequence distances separating them.
Furthermore, based on recent results, promoters show an unexpected diversity. As
promoter-enhancer interaction is mediated through multiprotein complexes, the
composition of these complexes is likely dependent on the properties of the cis-
regulatory elements involved and may result in interaction specificities. To investigate
whether the DNA sequence of core promoters and enhancers define the specificity of
their interaction, we have performed a high throughout screen, where 20 core
promoters and 13 enhancers were used to generate 260 combinations. Data analysis
after the automated image acquisition and processing revealed that enhancer function
is clearly promoter-specific.



I


II
Table of content
Abstract.......................................................................................................................... I
Table of content ...........................................................................................................III
Abbreviations................................................................................................................V
1) Introduction............................................................................................................1
1.1 Gene expression regulation in eukaryotes .....................................................1
1.2 Cis-regulatory elements .................................................................................2
1.3 Transcription factors ......................................................................................9
1.4 Genomic organisation of cis-regulatory elements .......................................12
1.5 Evolutionary aspects of cis-regulation.........................................................14
1.6 Medical aspects of cis-regulation.................................................................16
1.7 Mechanism of interaction between cis-regulatory elements........................19
1.8 Promoter-enhancer interaction specificity ...................................................21
1.9 Identification of novel cis-regulatory elements ...........................................23
1.10 Experimental approaches to verify cis-regulatory elements........................31
1.11 Zebrafish as a model organism ....................................................................32
2) Objectives ............................................................................................................37
3) Materials and methods .........................................................................................39
3.1 Standard molecular cloning .........................................................................39
3.2 The Multisite Gateway cloning....................................................................45
3.3 DNA injection into zebrafish embryos ........................................................48
3.4 Fish husbandry and care...............................................................................49
3.5 Staining methods..........................................................................................49
3.6 High throughput screening...........................................................................51
4) Results and discussion .........................................................................................54
III
4.1 Evolutionary conserved regions around the pax2 locus show differential
enhancer activity with different promoter constructs ..............................................54
4.2 Shuffled conserved sequences show enhancer activity, even if not related to
transcription factor or developmental regulator genes.............................................64
4.3 A high throughput screen to investigate promoter-enhancer specificity .....80
5) Conclusions........................................................................................................124
5.1 Four conserved non-coding elements form the pax2 locus show eye
enhancer activity ....................................................................................................124
5.2 Combined alignment approach reveals in increased number and variety of
conserved non-coding sequences with enhancer function.....................................124
5.3 Promoter-specific differences in enhancer action......................................125
6) Publications related to the thesis........................................................................126
7) Acknowledgements............................................................................................127
8) References..........................................................................................................128

IV
Abbreviations

ACH active chromatin hub
AP-1 activation protein 1
BRE TFIIB recognition elements
CAGE cap analysis of gene expression
CBF CAAAT-box binding factor
CFP cyan fluorescent protein
CNE conserved non-coding element
CNS central nervous system
CRM cis-regulatory module
CTCF CCCTC-binding factor
CTF CAAAT-binding transcriptional factor
DCE downstream core element
DPE downstream promoter element
ER estrogen receptor
EST expressed sequence tag
ETS erythroblastosis virus E26 oncogene homolog 1
GFP green fluorescent protein
GO gene ontology
GR glucocorticoid receptor
GRB genomic regulatory block
GTF general transcription factor
HMG1 high mobility group 1 protein
hpf hours post- fertilisation
Inr initiator
ISH in situ hybridisation
LacZ beta-galactosidase
LCR locus control region
MCS multispecies constrained sequence
MHB midbrain-hindbrain boundary
MTE motif ten element
NF-κB nuclear factor kappa B
V
PIC pre-initiation complex
PolII RNA polymerase II
pTRR putative transcriptional regulatory regions
PTS promoter targeting sequence
RACE rapid amplification of cDNA ends
rCNE regionally conserved element
SCE shuffled element
SCP super core promoter
SINE short interspersed element
SNP single nucleotide polymorphism
TAF TBP associated factor
TBP TATA binding protein
TF transcription factor
TFBS ription factor binding site
TLF TBP-like factor
TRF1 TBP-related 1
TSS transcription starts site
UCE ultra-conserved element
UCR ultra-conserved non-coding region
UTR untranslated region
XCPE1 X core promoter element 1
YFP yellow fluorescent protein
YSL yolk syntitial layer

VI

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