In situ genetic dissection of the boundary element Miscadestral pigmentation in the Bithorax complex of Drosophila melanogaster

Metzler, Mario. In situ genetic dissection of the boundary element Miscadestral pigmentation in the Bithorax complex of Drosophila melanogaster. 2016, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_12139

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In the past, dissection of cis-regulatory elements (enhancers, boundary elements, Polycomb Response Elements, etc.) was mostly done using transgenic assays. The study of such elements at the endogenous genomic location was, in many cases, not possible due to technical restrictions. Nowadays, the CRISPR/Cas9 method allows the precise modification, and hence dissection of cis-regulatory elements in their natural genomic environment, allowing an unbiased observation of their role in the regulation of their target gene. In this thesis I will describe the dissection of a well studied cis-regulatory element at its endogenous genomic location: the Miscadestral pigmentation (Mcp) boundary in the Drosophila melanogaster Bithorax Hox Complex (BX-C).
In Drosophila melanogaster the Miscadestral pigmentation boundary element and the adjacent Polycomb Response Element divide the abdominal-A (abd-A) from the Abdominal-B (Abd-B) gene. Deletion of this elements leads to spurious interaction of abdominal-A cis-regulatory elements with the Abdominal-B gene. Such incorrect interactions of enhancers have to be prevented not only in the Hox cluster but throughout the genome to allow precise gene regulation. The dissection of such an element as a model system can give insights on how the genome is organized in independent gene regions.
I used the CRISPR/Cas9 method in combination with both Non- Homologous-End-Joining and Homologous-Recombination DNA re- pair mechanisms to induce deletions in the Mcp region, and to establish a phiC31-integrase dependent landing site in the Mcp genomic location. The established landing site can be used to bring back into the Mcp locus modified genetic material, for example Mcp sequences with small deletions or completely unrelated boundary sequences from other genomic loci or species.
The endogenous Mcp boundary and the Polycomb Response Element are dissected in great detail, and the obtained results demonstrate that the function of the boundary depends on a single CCCTC-binding factor (CTCF, a known boundary associated factor) site and on four Pleiohomeotic (Pho, part of the Polycomb Protein Group) sites in the Polycomb Response Element. Those results are further refined using classical boundary element assays in transgenic flies. Furthermore, I study the role of the orientation of those regulatory DNA sequences in the endogenous genomic locus.
Surprisingly, some deletions in the Mcp region give rise to phenotypes not associated previously with this boundary, suggesting the presence of additional cis-regulatory elements other than the already known boundary element and PRE.
This study further demonstrates the versatility of the CRISPR/Cas9 method in studying cis-regulatory elements in their endogenous genomic position, a possibility that will enable more precise and clean investigation of gene regulation in the future.
Advisors:Affolter, Markus and Karch, Fran├žois
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Cell Biology (Affolter)
UniBasel Contributors:Affolter, Markus
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:12139
Thesis status:Complete
Number of Pages:1 Online-Ressource (ix, 116 Seiten)
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Last Modified:08 Feb 2020 14:37
Deposited On:17 May 2017 11:53

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