ABSTRACT
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.
Subject(s)
Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Oncogene Proteins/chemistry , Oncogene Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/chemistry , Poly-ADP-Ribose Binding Proteins/metabolism , Binding Sites , Chromatin/chemistry , Chromatin/metabolism , Chromatography, High Pressure Liquid/methods , DNA/chemistry , HEK293 Cells , HeLa Cells , Humans , Molecular Structure , Protein Binding , Protein Conformation , Structure-Activity Relationship , Tandem Mass Spectrometry/methodsABSTRACT
DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , DNA Breaks, Double-Stranded , DNA Repair , DNA-Binding Proteins/metabolism , Homologous Recombination , Oncogene Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Animals , Apoptosis/drug effects , Apoptosis/radiation effects , DNA Breaks, Double-Stranded/drug effects , DNA Breaks, Double-Stranded/radiation effects , DNA Repair/drug effects , DNA Repair/radiation effects , Female , HeLa Cells , Histones/metabolism , Homologous Recombination/drug effects , Homologous Recombination/radiation effects , Humans , Male , Mice, Knockout , Protein Binding/drug effects , Protein Binding/radiation effects , Protein Kinase Inhibitors/pharmacology , Rad51 Recombinase/metabolism , Radiation, Ionizing , Replication Protein A/metabolismABSTRACT
Monoubiquitination of FANCD2 is a key step in the DNA damage response pathway involving Fanconi anemia proteins and the breast cancer susceptibility gene products, BRCA1 and BRCA2. One critical unresolved issue is the identity of the ubiquitin ligase responsible for this reaction. Two proteins, BRCA1 and FANCL(PHF9), have been suggested to be this ligase. Here we found that FANCL, but not BRCA1, evolutionarily co-exists with FANCD2 in several species. Moreover, the proportion of FANCD2 in chromatin and nuclear matrix is drastically reduced in a cell line mutated in FANCL, but not in that mutated in BRCA1. This defective distribution of FANCD2 in the FANCL-mutant cell line is likely due to its defective monoubiquitination, because the monoubiquitinated FANCD2 preferentially associates with chromatin and nuclear matrix, whereas non-ubiquitinated FANCD2 largely resides in the soluble fraction. Our data support the notion that FANCL, but not BRCA1, is the likely ligase for FANCD2 monoubiquitination.
