Molekulare Funktion des Onkogens DEK in globaler Chromatinintegrität
Zusammenfassung der Projektergebnisse
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.
Projektbezogene Publikationen (Auswahl)
- (2017) Systems analysis identifies melanoma-enriched pro-oncogenic networks controlled by the RNA binding protein CELF1. Nature communications 8 (1) 2249
Cifdaloz, Metehan; Osterloh, Lisa; Graña, Osvaldo; Riveiro-Falkenbach, Erica; Ximénez-Embún, Pilar; Muñoz, Javier; Tejedo, Cristina; Calvo, Tonantzin G.; Karras, Panagiotis; Olmeda, David; Miñana, Belén; Gómez-López, Gonzalo; Cañon, Estela; Eyras, Eduardo
(Siehe online unter https://doi.org/10.1038/s41467-017-02353-y) - (2018) The nuclear DEK interactome supports multi-functionality. Proteins 86 (1) 88–97
Smith, Eric A.; Krumpelbeck, Eric F.; Jegga, Anil G.; Prell, Malte; Matrka, Marie M.; Kappes, Ferdinand; Greis, Kenneth D.; Ali, Abdullah M.; Meetei, Amom R.; Wells, Susanne I.
(Siehe online unter https://doi.org/10.1002/prot.25411) - (2013) Concise review: role of DEK in stem/progenitor cell biology. Stem Cells, 31, 1447-1453
Broxmeyer, H.E., Mor-Vaknin, N., Kappes, F., Legendre, M., Saha, A.K., Ou, X., O'Leary, H., Capitano, M., Cooper, S. and Markovitz, D.M.
(Siehe online unter https://doi.org/10.1002/stem.1443) - (2013) Intercellular trafficking of the nuclear oncoprotein DEK. Proc Natl Acad Sci USA, 110, 6847-6852
Saha, A.K., Kappes, F., Mundade, A., Deutzmann, A., Rosmarin, D.M., Legendre, M., Chatain, N., Al-Obaidi, Z., Adams, B.S., Ploegh, H.L. et al.
(Siehe online unter https://doi.org/10.1073/pnas.1220751110) - (2013) Stacking the DEK: from chromatin topology to cancer stem cells. Cell Cycle, 12, 51-66
Privette Vinnedge, L.M., Kappes, F., Nassar, N. and Wells, S.I.
(Siehe online unter https://doi.org/10.4161/cc.23121) - (2015) DEK oncogene expression during normal hematopoiesis and in Acute Myeloid Leukemia (AML). Blood Cells Mol Dis, 54, 123-131
Logan, G.E., Mor-Vaknin, N., Braunschweig, T., Jost, E., Schmidt, P.V., Markovitz, D.M., Mills, K.I., Kappes, F. and Percy, M.J.
(Siehe online unter https://doi.org/10.1016/j.bcmd.2014.07.009) - (2015) DEK over-expression promotes mitotic defects and micronucleus formation. Cell Cycle, 14, 3939-3953
Matrka, M.C., Hennigan, R.F., Kappes, F., DeLay, M.L., Lambert, P.F., Aronow, B.J. and Wells, S.I.
(Siehe online unter https://doi.org/10.1080/15384101.2015.1044177) - (2015) The human oncoprotein and chromatin architectural factor DEK counteracts DNA replication stress. Oncogene, 34, 4270-4277
Deutzmann, A., Ganz, M., Schonenberger, F., Vervoorts, J., Kappes, F. and Ferrando-May, E.
(Siehe online unter https://doi.org/10.1038/onc.2014.346) - (2017) DEK is required for homologous recombination repair of DNA breaks. Sci Rep, 7, 44662
Smith, E.A., Gole, B., Willis, N.A., Soria, R., Starnes, L.M., Krumpelbeck, E.F., Jegga, A.G., Ali, A.M., Guo, H., Meetei, A.R. et al.
(Siehe online unter https://doi.org/10.1038/srep44662)
