Interplay of DNA Metabolic Pathways Involved in the Regulation of Gene Expression and Genomic Rearrangements

Abstract

Tightly-controlled cell division is of critical importance for multicellular organisms to be able to grow and survive over long periods of time. The most demanding step in this process is the faithful replication of the genome. If a cell fails to copy its DNA molecule in an error-free manner, the genetic changes in the DNA can lead to cell death or, in the worst case, to uncontrolled cell division, resulting in genome instability and cancer. The DNA is constantly challenged by exogenous and endogenous damaging agents, such as UV irradiation, water or reactive oxygen species that threaten the integrity of DNA. The resulting lesions are addressed by base excision repair (BER) and mismatch repair (MMR). The cell's repair pathways are, however, not only needed to correct damage in the DNA and mistakes arising during replication, but they are also essential for many physiological processes. BER and MMR were shown to play a role in antibody diversification during adaptive immune response and BER is implicated in DNA methylation dynamics to control gene expression. In the first part of the thesis, we set out to investigate the role of BER and MMR in class switch recombination (CSR), a process that enables B-cells to produce antibodies with different effector functions. Using DNA substrates containing defined nicks and uracils, we mimicked the intermediates arising during CSR by the action of activation induced cytidine deaminase (AID). We found that the creation of DNA double strand breaks, a step that is essential for CSR, heavily depends on the BER enzyme uracil DNA glycosylase (UNG) and on the MMR protein MutSα. In the second part of the thesis, I aimed to gain insights into the dynamic metabolism of DNA modifications that control gene expression by investigating the chromatin dynamics at the vitellogenin (VTG) gene regulatory region. The VTG gene represents a valuable model to study the events on chromatin leading to expression of the gene, as it is well-characterized and inducible by estradiol treatment. Demethylation events have been observed in the enhancer region in rooster DNA upon hormone treatment. In our cell line system, we saw no demethylation, but an increase in 5-hydroxymethylation, a product of oxidation mediated by the Ten-eleven Translocation proteins, implicated in DNA demethylation. In addition, the estrogen receptor α binding to its response element in the VTG enhancer was not altered by the presence of 5mC or 5hmC, but a shift of a methylation-sensitive factor was observed in nuclear extracts. In vitro methylation of a reporter plasmid under the control of the VTG regulatory region inhibited transcription, confirming an important role of methylation dynamics in the control of VTG expression. We found evidence that a CpG residing outside of the estrogen response element in an E-box in the VTG promoter is responsible for the methylation-sensitivity. The data presented in this thesis illustrate the diverse functions of eukaryotic repair pathways. They show the great potential of these versatile mechanisms, but also point out the danger they pose to cells and multicellular organisms. If they are not processed correctly, the DNA modifications and rearrangements can lead to tumorigenesis, highlighting the importance of a deep understanding of the mechanisms involved.