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.
Subject(s)
Chromatin/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA/metabolism , Escherichia coli/drug effects , Loss of Function Mutation , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Recombinant Proteins/toxicity , Bias , Cell Nucleus/drug effects , Cell Size/drug effects , Chromatin/chemistry , Chromatin/genetics , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/toxicity , Escherichia coli/genetics , Escherichia coli/growth & development , Gene Expression Regulation, Bacterial/drug effects , Genome, Bacterial/drug effects , Genome, Bacterial/genetics , Humans , Mutagenesis , Nucleosomes/chemistry , Nucleosomes/genetics , Nucleosomes/metabolism , Oncogene Proteins/chemistry , Oncogene Proteins/toxicity , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide Fragments/toxicity , Poly-ADP-Ribose Binding Proteins/chemistry , Poly-ADP-Ribose Binding Proteins/toxicity , Protein Domains/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Toxicity Tests/methodsABSTRACT
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.
Subject(s)
Escherichia coli/drug effects , Escherichia coli/growth & development , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/toxicity , Toxicity Tests/methods , Animals , Bias , Biocatalysis , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/toxicity , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/toxicity , Escherichia coli/genetics , Humans , Loss of Function Mutation , Oncogene Proteins/chemistry , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Oncogene Proteins/toxicity , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide Fragments/toxicity , Poly-ADP-Ribose Binding Proteins/chemistry , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/toxicity , Protein Domains/genetics , RNA/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/toxicity , Receptors, Eph Family/chemistry , Receptors, Eph Family/genetics , Receptors, Eph Family/metabolism , Receptors, Eph Family/toxicity , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Reproducibility of Results , Time Factors , Toxicity Tests/standardsABSTRACT
The mechanism of the cytotoxicity by natural killer (NK) cells is not known. It is speculated that there exist several positively regulated and negatively regulated target molecules expressed on the target cell surface. Although one of the latter is considered to be major histocompatibility complex antigen (MHC) class I, in this study we described a novel non-MHC class I molecule that may negatively regulate the NK cytotoxicity. This antigen is defined by monoclonal antibody Cho-1 and is composed of noncovalently associated antigens that are 40 and 200 kilodaltons in molecular size. The expression of this antigen is reduced along with the cell growth induced by growth factors and/or oncogenes. Thus, Cho-1-defined antigen appears to be involved as one of the resistant molecules in the cytotoxic mechanism of NK cells.
Subject(s)
Fibroblasts/cytology , Genes, MHC Class I/immunology , Killer Cells, Natural/immunology , Microtubule-Associated Proteins/immunology , Animals , Antibodies, Monoclonal/immunology , Antigen-Antibody Complex/genetics , Antigen-Antibody Complex/immunology , Cell Division/drug effects , Cell Division/immunology , Electrophoresis, Polyacrylamide Gel , Fibroblasts/immunology , Flow Cytometry , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Growth Substances/toxicity , Killer Cells, Natural/cytology , Microtubule-Associated Proteins/genetics , Molecular Weight , Oncogene Proteins/toxicity , RatsABSTRACT
Transformation of NIH3T3 cells with the ras, the sis, or the neu oncogene rendered cells less susceptible to cis-diamminedichloroplatinum(II). Since resistance to cis-diamminedichloroplatinum(II) is reported to be associated with increased levels of metallothionein, we examined effects of these oncogenes on metallothionein gene expression. NIH3T3 cells were first transfected with the lacZ gene whose transcription is under the control of mouse metallothionein I promoter and then with the ras, the sis, or the neu oncogene. The ras and the sis oncogenes increased beta-galactosidase activities which were induced either by metal (cadmium and zinc) or by glucocorticoid (dexamethasone), whereas the neu oncogene repressed its activity. When SV40 early promoter was used instead of metallothionein I promoter for the lacZ gene transcription, the beta-galactosidase activities were not affected by metal, dexamethasone, or any of these oncogenes. This result was coincident with that of reverse transcription polymerase chain reaction that metal-induced MT I mRNA was only detected in the sis- or the ras-transformed cells, whereas any of these oncogenes did not affect the metal-induced transcription of the MT II gene. These results demonstrate that the ras and the sis oncogenes upregulate the metal- or glucocorticoid-induced transcription from metallothionein I promoter, but the neu oncogene negatively regulates it. Thus, resistance to the chemotherapeutic agent by oncogenic transformation is partly associated with the metallothionein gene expression, and MT I and MT II gene expressions are differently controlled by different oncogenes.