Bacterial Growth Inhibition Screen (BGIS) identifies a loss‐of‐function mutant of the DEK oncogene, indicating DNA modulating activities of DEK in chromatin

Abstract

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.

Bacterial Growth Inhibition Screen (BGIS): harnessing recombinant protein toxicity for rapid and unbiased interrogation of protein function

“…Based on this study, and the accompanying manuscript [1], we provide new evidence indicating that the multifaceted phenomenon of recombinant protein toxicity in E. coli can be exploited as a thus far largely overlooked means for alternative approaches for identification, assessment and manipulation of protein function. Such effects, in particular for expression of nucleic acid-interacting proteins in E. coli, are well acknowledged and have been observed before [43,44,[73][74][75][76].…”

“…2A,B; Fig. 5A, and [1]), as well as with one of its orthologues from Drosophila melanogaster (Fig. 5A).…”

“…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.…”

“…As detailed in our accompanying manuscript [1], we employed BGIS as a streamlined biological screening tool in combination with random mutagenesis in order to quickly select loss‐of‐function DNA modulating mutants of DEK.…”

“…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. Keywords: DEK; Escherichia coli; oncogene; protein domain; protein function; recombinant protein toxicity; RNA-binding domain Heterologous protein expression in the enteric bacterium Escherichia coli or its many genetically engineered derivatives represents an essential and ubiquitously applied approach in modern molecular biosciences [1][2][3][4]. Taking advantage of the well-known biology and the ease-of-use of prokaryotes, bacterial protein expression typically represents the go-to choice, as it allows for rather simple, quick and lowcost-high-yield expression of a large array of factors non-native to E. coli.…”