Proteins that respond to DNA damage play critical roles in normal and diseased states in human biology. Studies have suggested that the S. cerevisiae protein CMR1/YDL156w is associated with histones and is possibly associated with DNA repair and replication processes. Through a quantitative proteomic analysis of affinity purifications here we show that the human homologue of this protein, WDR76, shares multiple protein associations with the histones H2A, H2B, and H4. Furthermore, our quantitative proteomic analysis of WDR76 associated proteins demonstrated links to proteins in the DNA damage response like PARP1 and XRCC5 and heterochromatin related proteins like CBX1, CBX3, and CBX5. Co-immunoprecipitation studies validated these interactions. Next, quantitative imaging studies demonstrated that WDR76 was recruited to laser induced DNA damage immediately after induction, and we compared the recruitment of WDR76 to laser induced DNA damage to known DNA damage proteins like PARP1, XRCC5, and RPA1. In addition, WDR76 co-localizes to puncta with the heterochromatin proteins CBX1 and CBX5, which are also recruited to DNA damage but much less intensely than WDR76. This work demonstrates the chromatin and DNA damage protein associations of WDR76 and demonstrates the rapid response of WDR76 to laser induced DNA damage.

A hub protein in protein interaction networks will typically have a large number of diverse interactions. Determining the core interactions and the function of such a hub protein remains a significant challenge in the study of networks. Proteins with WD40 repeats represent a large class of proteins that can be hub proteins. WDR76 is a poorly characterized WD40 repeat protein with possible involvement in DNA damage repair, cell cycle progression, apoptosis, gene expression regulation, and protein quality control. WDR76 has a large and diverse interaction network that has made its study challenging. Here, we rigorously carry out a series of affinity-purification coupled to mass spectrometry (AP-MS) to map out the WDR76 interactome through different biochemical conditions. We apply AP-MS analysis coupled to size exclusion chromatography to resolve WDR76-based protein complexes. Furthermore, we also show that WDR76 interacts with the CCT complex via its WD40 repeat domain and with DNA-PK-KU, PARP1, GAN, SIRT1, and histones outside of the WD40 domain. An evaluation of the stability of WDR76 interactions led to focused and streamlined reciprocal analyses that validate the interactions with GAN and SIRT1. Overall, the approaches used to study WDR76 would be valuable to study other proteins containing WD40 repeat domains, which are conserved in a large number of proteins in many organisms.

BRK phosphorylates and targets SMAD4 for proteasomal degradation to regulate aspects of the epithelial-mesenchymal transition.

A hub protein in protein interaction networks will typically have a large number of diverse interactions. Determining the core interactions and the function of such a hub protein remains a significant challenge in the study of networks. Proteins with WD40 repeats represent a large class of proteins that can be hub proteins. WDR76 is a poorly characterized WD40 repeat protein with possible involvement in DNA damage repair, cell cycle progression, apoptosis, gene expression regulation, and protein quality control. WDR76 has a large and diverse interaction network that has made its study challenging. Here, we rigorously carry out a series of affinitypurification coupled to mass spectrometry (AP-MS) to map out the WDR76 interactome through different biochemical conditions. We apply AP-MS analysis coupled to size exclusion chromatography to resolve WDR76-based protein complexes. Furthermore, we also show that WDR76 interacts with the CCT complex via its WD40 repeat domain and with DNA-PK-KU, PARP1, GAN, SIRT1, and histones outside of the WD40 domain. An evaluation of the stability of WDR76 interactions led to focused and streamlined reciprocal analyses that validate the interactions with GAN and SIRT1. Overall, the approaches used to study WDR76 would be valuable to study other proteins containing WD40 repeat domains, which are conserved in a large number of proteins in many organisms

Introduction: Chromatin remodeling complexes play important roles in the control of genome regulation in both normal and diseased states, and are therefore critical components for the regulation of epigenetic states in cells. Given the role epigenetics plays in cancer, for example, chromatin remodeling complexes are routinely targeted for therapeutic intervention. Areas covered: Protein mass spectrometry and proteomics are powerful technologies used to study and understand chromatin remodeling. While impressive progress has been made in this area, there remain significant challenges in the application of proteomic technologies to the study of chromatin remodeling. As parts of large multisubunit complexes that can be heavily modified with dynamic post-translational modifications, challenges in the study of chromatin remodeling complexes include defining the content, determining the regulation, and studying the dynamics of the complexes under different cellular states. Expert Commentary: Important considerations in the study of chromatin remodeling complexes include the complexity of sample preparation, the choice of proteomic methods for the analysis of samples, and data analysis challenges. Continued research in these three areas promise to yield even greater insights into the biology of chromatin remodeling and epigenetics and the dynamics of these systems in human health and cancer.