Functional characterization of mammalian Polycomb Response Elements
Tanja Drexel
2013
unpublished
During development it is the main aim of multicellular organisms to grow not only in size but also to specialize and form different cells and tissues. This is accomplished by changes in the transcriptional profile of the cells, which has to be maintained during several cell divisions. The highly conserved Polycomb (PcG) and Trithorax (TrxG) group proteins constitute a gene regulatory system that is essential for maintaining the correct identity of both stem cells and differentiated cells [1,
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... The PcG and TrxG proteins act through cis-regulatory elements called Polycomb/Trithorax response elements (PREs) which are crucial for epigenetic inheritance [2-4]. PREs are best characterized in flies, and can be predicted even genome wide via an algorithm specifically designed for Drosophila melanogaster [5]. However, although PcG and TrxG proteins are highly conserved, PREs are not, and therefore a computational approach to predict PREs in mammals has previously been lacking. A bioinformatician in the Ringrose group was able to adapt the fly algorithm to the mammalian system and could predict several putative PREs in the mouse genome. Since these predictions had to be confirmed it was my task to revalidate them experimentally. I integrated several PRE reporter constructs stably into ROSA-26 in mouse ESCs, to avoid genomic position effects and measured the luciferase expression. For this purpose I took advantage of an already established luciferase reporter assay system and tested the repressive effect of the PREs on the reporter in transient experiments and stable cell lines. In order to address the question whether the silencing of the reporter is PcG dependent, I furthermore depleted PcG proteins via siRNA knockdowns. In this thesis I could show that several PREs showed silencing of the reporter in transient experiments whereas they behaved differently in Rosa-26 in stable ES cell lines. The PRE Ptch1 was able to silence the reporter even in the stable cell lines. Furthermore I contributed in the improvement of the g [...]
doi:10.25365/thesis.28893
fatcat:redurd6wpfa33n4mmr5qubpyw4