Ubiquitin-specific protease 7 (USP7) is a deubiquitylating enzyme that is involved in the regulation of multiple key cellular processes, including tumor suppression, transcription, epigenetics, the DNA damage response, and DNA replication. For debiquitinating enzymes, the regulatory mode of action is at the posttranslational level and proteomics tools are particularly suited to study this. Here, we took a multi-omics approach with a heavy targeted quantitative proteomics component to define the core USP7 network and to study its roles in regulation of the Polycomb system and in neurodevelopment.
We performed interactomics, in-depth global proteomics and ubiquitinomics to identify the USP7 interaction network and its regulation of neurodevelopment. Targeted proteomics using parallel reaction monitoring (PRM) was performed to accurately quantify relevant players of the Polycomb system and other suspected USP7 targets in a label free manner. Integrating unbiased proteomics, genomic, and functional molecular approaches, we determined the core USP7 regulatory circuitry. This multi-angle analysis establishes USP7 as a regulatory hub in a multinodal network involved in tumor biology, protein (de)ubiquitylation, genome regulation and neurodevelopment.
We found that USP7 modulates the ncPRC1 axis at the posttranslational level through stabilization of ncPRC1.6 and ncPRC1.1. At the transcriptional level, USP7 silences the autism related protein AUTS2, the subunit that suppresses H2A ubiquitylation by ncPRC1.3/5. Collectively, these USP7 activities increase the genomic deposition of H2AK119ub1. The connection of USP7 to the Polycomb system suggests that its role in cancer extends beyond regulation of p53. Our interactomics assay shows that USP7 has a remarkable range of interaction partners, of which only a portion appears to be stabilized by USP7. Next, we focused on the functions of these interaction partners and of USP7 itself in neurodevelopment. Neuronal cells in which USP7 was knocked out did not differentiate and no neurite outgrowth was observed, in contrast to WT cells. Global in-depth DIA-MS based proteomics suggested that proteins that were not expressed or only at lower levels in USP7 knockout compared to WT cells are involved in neuronal development and axon regulation. In contrast, expression of proteins involved in various metabolic processes was downregulated or switched off in differentiating WT cells. Current studies address the molecular mechanisms involved in the regulation of neurodevelopment by USP7.
Combined, our multi-omics results provide a resource for future studies on the role of USP7 in (neuro)development and cancer.