| dc.description.abstract |
Myc is an oncogenic transcription factor (TF), promoting the growth and aggressiveness of many different cancer types, including liver cancer. Despite its tumorigenic function, Myc activation can induce DNA damage and replicative stress (RS), thereby promoting the apoptotic response. Hence, Myc would be an attractive target for cancer therapy. However, Myc does not have enzymatic activity or defined binding pockets, which makes it difficult to target it with small molecule inhibitors. Taken together, there is an urgent need to find new indirect mechanisms regulating Myc, to be able to design novel therapeutic strategies. Previous data from our working group showed that the ubiquitin E3 ligase Trim33 is a regulator of Myc-induced apoptosis (Popov et al., 2007). I could show during this thesis that Myc is not a substrate of Trim33 in liver cancer cells and that Trim33 does not influence Myc levels through its already known connections to the Wnt and TGFβ pathways. Furthermore, I demonstrated that Trim33 knockout (Trim33KO) cells can replicate DNA more efficiently, reenter cell cycle faster and proliferate more upon RS compared to control cells. Additionally, upon Trim33KO, DNA damage is induced later after induction of RS and is repaired faster during recovery from RS. These effects could be seen when RS was induced by Myc overexpression or by the treatment with the genotoxic drugs hydroxyurea (HU) and etoposide (Etop). Hence, the function of Trim33 is not limited to the regulation of Myc-induced apoptosis, but it is rather more generally modulating the response of cells to RS. Subsequently, RNA-Sequencing analysis revealed that the target genes of the TF E2F4 were strongly upregulated in Trim33KO cells compared to control cells. E2F4 is known to be involved in DNA replication and cell cycle control, making it a very promising candidate. I could show that E2F4 is a novel substrate of Trim33 and that its overexpression in control cells mimicked the RS phenotype observed in Trim33KO cells, demonstrating the E2F4 dependency of the mechanism. Finally, mass spectrometry analysis revealed that the binding of E2F4 to the helicase RecQL was enhanced upon Trim33KO, which could be validated by proximity ligation assays (PLAs). RecQL is known to stabilize replication forks and to be involved in DNA damage repair. Hence, RecQL could strongly simplify the handling of RS for cells. I could demonstrate that RecQL as well as E2F4 binding to chromatin is enhanced upon Trim33KO, suggesting that E2F4 recruits RecQL to DNA. Moreover, the overexpression of RecQL in control cells mimicked the enhanced fork rate observed in Trim33KO cells, whereas an inactive RecQL mutant (K119A) overexpressed in Trim33KO cells significantly impaired replication fork progression under RS. Two E2F4 mutants, one which cannot bind to DNA (E2F4-DB) and one which lacks a part of its C-terminus (E2F4-ΔC), cannot recruit RecQL to chromatin and revert the RS phenotype of Trim33KO cells. Taken together, our data suggest a model in which E2F4 recruits RecQL to chromatin, which in turn makes cells less sensitive to RS. |
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