DEK is an oncoprotein that is overexpressed in many forms of cancer and participates in numerous cellular pathways. Of these different pathways, relevant interacting partners and functions of DEK are well described in regard to the regulation of chromatin structure, epigenetic marks, and transcription. Most of this understanding was derived by investigating DNA-binding and chromatin processing capabilities of the oncoprotein. To facilitate the generation of mechanism-driven hypotheses regarding DEK activities in underexplored areas, we have developed the first DEK interactome model using tandem-affinity purification and mass spectrometry. With this approach, we identify IMPDH2, DDX21, and RPL7a as novel DEK binding partners, hinting at new roles for the oncogene in de novo nucleotide biosynthesis and ribosome formation. Additionally, a hydroxyurea-specific interaction with replication protein A (RPA) was observed, suggesting that a DEK-RPA complex may form in response to DNA replication fork stalling. Taken together, these findings highlight diverse activities for DEK across cellular pathways and support a model wherein this molecule performs a plethora of functions.DDX21, DEK, IMPDH2, interactome, mass spectrometry, ribosome, RPA | I N TR ODU C TI ONDEK is a DNA-binding and predominantly nuclear protein that was first identified as a DEK-NUP214 fusion protein in AML. 1 Since this discovery, DEK has been classified as an oncoprotein and shown to be overexpressed in many diverse tumor types, 2 wherein the degree of overexpression was linked to worse prognosis, advanced stage tumors, and chemotherapy resistance. 3-9 Cellular functions of the oncoprotein include activities in modifying chromatin structure, 2,10-13 histone chaperoning, 14,15 epigenetic modification and transcription regulation, [16][17][18][19]20,21 DNA repair,22,23 DNA replication fork restart, 24 mitotic non-disjunction events, 25 evasion of senescence and apoptosis, 19,26,27 proliferation 28-30 cancer stem cell fitness and invasion, 28,31 inflammation, 32,33 and metabolic reprogramming. 34 Precise molecular mechanisms whereby DEK regulates these cellular processes remain unclear in many cases. For instance, while DEK is necessary for optimal non-homologous end joining (NHEJ) DNA repair, the mechanism of action or interacting partners remain elusive. 23 For an oncogene that is widely implicated in human carcinogenesis and outcome, determining how DEK operates is imperative. However, this has proven challenging as the oncoprotein DEK has no known enzymatic activity or paralogs. 2 While significant progress has been made to understand molecular DEK activities in transcription via chromatin remodeling, 2,[10][11][12][13][16][17][18][19] there is a dearth of information regarding 88 | V C 2017 Wiley Periodicals, Inc.wileyonlinelibrary.com/journal/prot Proteins. 2018;86:88-97.

The DEK oncoprotein regulates cellular chromatin function via a number of protein–protein interactions. However, the biological relevance of its unique pseudo‐SAP/SAP‐box domain, which transmits DNA modulating activities in vitro, remains largely speculative. As hypothesis‐driven mutations failed to yield DNA‐binding null (DBN) mutants, we combined random mutagenesis with the Bacterial Growth Inhibition Screen (BGIS) to overcome this bottleneck. Re‐expression of a DEK‐DBN mutant in newly established human DEK knockout cells failed to reduce the increase in nuclear size as compared to wild type, indicating roles for DEK–DNA interactions in cellular chromatin organization. Our results extend the functional roles of DEK in metazoan chromatin and highlight the predictive ability of recombinant protein toxicity in E. coli for unbiased studies of eukaryotic DNA modulating protein domains.

In two proof‐of‐concept studies, we established and validated the Bacterial Growth Inhibition Screen (BGIS), which explores recombinant protein toxicity in Escherichia coli as a largely overlooked and alternative means for basic characterization of functional eukaryotic protein domains. By applying BGIS, we identified an unrecognized RNA‐interacting domain in the DEK oncoprotein (this study) and successfully combined BGIS with random mutagenesis as a screening tool for loss‐of‐function mutants of the DNA modulating domain of DEK [1]. Collectively, our findings shed new light on the phenomenon of recombinant protein toxicity in E. coli. Given the easy and rapid implementation and wide applicability, BGIS will extend the repertoire of basic methods for the identification, analysis and unbiased manipulation of proteins.