Project Details
Projekt Print View

Molecular function of the DEK oncoprotein in global chromatin integrity

Subject Area General Genetics and Functional Genome Biology
Term from 2012 to 2017
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 227390135
 
Final Report Year 2017

Final Report Abstract

Chromatin is a highly-ordered, yet dynamic structure that is essential in the regulation of all DNA-dependent processes, with the coordinated interplay of non-histone chromosomal factors playing important roles in its regulation. One such factor is the unique oncoprotein DEK, which has been implicated in the maintenance of heterochromatin via protein-protein-mediated mechanisms. In order to gain insights into the biology and regulation of DEK functions we first detailed the cellular networks DEK is involved in. Given that multiple previously applied strategies yielded only limited information, a Localization and Affinity Purification (LAP) approach was successfully devised and indeed produced reliable cellular DEK-interactome data. Mass spectrometric analyses derived from this proteome-wide search subsequently identified ~ 300 DEK-specific interaction partners, which confirmed already established DEK interactors, however, also revealed a large number of yet unrecognized factors. Analysis of this data set by means of functional association via Gene Ontology (GO) databanks revealed implications for DEK in a high, yet well-defined number of cellular processes, thus further confirming the multifunctional nature of this unique factor. To be able to illuminate DEK’s role in some of these identified processes, novel DEK knockout cell lines were established via the TALEN system, and their respective transcriptomes as well as global changes to cellular splicing patterns were analyzed via microarray analyses. Based on the observed biological deficits in the knockout cells and the newly obtained data from the DEK interactome and transcriptome, three fields with potential functional associations for DEK were chosen for downstream analyses: i) ribosome biology, ii) alternative splicing, and iii) interaction with the mRNA transport factor ALY. We further investigated the role of DEK in chromatin and found that DEK binds to the entry/exit site of nucleosomal DNA in vitro, and that knocking down DEK expression in cells leads to general alterations in the nucleosomal repeat length of cellular chromatin. As this aspect has not been studied before, multiple hypothesis-driven strategies were chosen to create “DNA-bindingdead” DEK mutants by site-directed mutagenesis. Though these initial approaches proved unsuccessful we found that expression of DEK in bacteria resulted in massive compaction of the bacterial genome due to DEK’s DNA-folding activity which resulted in a complete halt in bacterial growth. This fact in combination with random mutagenesis led to the development of a novel screening procedure, now termed bacterial growth inhibition screen (BGIS) that allowed for the selection of loss-of-function mutants, which were subsequently verified by biochemical analyses. By re-expression of one DNA-binding-dead mutant in newly created DEK knockout cells, first evidence was provided that the DNA binding and folding activities of DEK indeed are involved in the regulation of global chromatin structure. By subjecting other DEK domains with previously unidentified functions to the BGIS, we were able to identify and verify a hitherto unrecognized RNA-binding domain in DEK. By assessing the DEK-HP1a interplay in metastatic melanoma cell lines, we identified phosphorylation as a critical regulator of this interaction and suggesting phosphorylation as an a switch between heterochromatic and euchromatic functions. Taken together, we were able to obtain deeper insights of DEK functions in metazoan chromatin. These results will serve as critical basis for further functional studies with the goal to identify strategies to target this conserved nuclear oncoprotein for tumor intervention.

Publications

 
 

Additional Information

Textvergrößerung und Kontrastanpassung