An shRNA kinase screen identifies regulators of UHRF1 stability and activity in mouse embryonic stem cells

Propagation of DNA methylation through cell division relies on the recognition of methylated cytosines by UHRF1. In reprogramming of mouse embryonic stem cells to naive pluripotency (also known as ground state), despite high levels of <i>Uhrf1</i> transcript, the protein is targeted for degradation by the proteasome, leading to DNA methylation loss. We have undertaken an shRNA screen to identify the signalling pathways that converge upon UHRF1 and control its degradation, using UHRF1-GFP fluorescence as readout. Many candidates we identified are key enzymes in regulation of glucose metabolism, nucleotide metabolism and Pi3K/AKT/mTOR pathway. Unexpectedly, while downregulation of all candidates we selected for validation rescued UHRF1 protein levels, we found that in some of the cases this was not sufficient to maintain DNA methylation. This has implications for development, ageing and diseased conditions. Our study demonstrates two separate processes that regulate UHRF1 protein abundance and activity.