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.
|Qualification||Doctor of Philosophy|
|Award date||8 Apr 2009|
|Publication status||Published - 8 Apr 2008|