The human chromatin protein DEK and its nuclear bodies in DNA replication

Abstract

Originally discovered as a fusion protein in a leukemia patient, the highly conserved human oncoprotein DEK has been shown to bind to chromatin in cells and alter the topology of DNA in vitro. DEK is highly post-translationally modified. Amongst others, it is a high affinity target of covalent and non-covalent poly(ADP-ribosyl)ation (PARylation). Besides regulating the transcription of many genes, DEK was implicated both in DNA repair and replication. Downregulation of DEK expression slows down replication forks and impairs proliferative capability, enhancing replication-associated DNA damage. These phenotypes are aggravated when the cells are treated with low concentrations of replication stress-inducing drugs. A role for DEK in DNA replication is corroborated by the formation of DEK protein clusters once per cell cycle during late S phase. These clusters, termed DEK bodies, were observed for the first time in a GFP-DEK knock-in U2-OS cell line previously established in the laboratory and correspond to replication sites, as indicated by PCNA colocalization studies. These findings led to the hypothesis that DEK or a variant of DEK is involved in the replication of DNA sequences which are typically duplicated late in S phase. Still, the molecular mechanisms through which DEK exerts its replication-promoting activity are not understood yet. This thesis presents a thorough and systematic investigation of DEK bodies with the aim to further explore DEK´s role in replication and narrow down potential mechanisms of action. Determination of DEK body fine structure via superresolution microscopy revealed that DEK binds in the vicinity of the replication fork and does not colocalize with nascent DNA. Similarly, iPOND (Isolation of Proteins on Nascent DNA) experiments showed an enrichment of DEK on maturing chromatin but not on nascent DNA in the context of global DNA replication. To identify regulators of DEK body formation, a microscopy-based high-throughput siRNA screening assay was performed. Over 300 genes were downregulated in U2-OS GFP-DEK cells and the dynamics of DEK body formation were investigated via time-lapse fluorescence microscopy. Automated image analysis revealed that siRNAs targeting histone acetyl- and methyltransferases and proteins involved in the SUMO and ubiquitin modification pathways upregulate DEK bodies. In a follow-up validation screen, several siRNAs targeting SUMOylation proteins SUMO1, SAE1 and UBE2I were confirmed as DEK body upregulators. A SUMOylation assay confirmed that DEK is indeed a target of SUMOylation in cells. The findings of this thesis pinpoint SUMO modification as a major regulatory pathway of the activity of DEK during replication, in particular in late S-phase. They also provide a rationale for future studies addressing the interplay of SUMOylation and PARylation in determining DEK´s role at the interface of DNA replication and repair.