Ubiquitin-dependent recruitment of SLFN11 to chromatin is regulated by deubiquitinase (DUB) and RNF168.

The molecular mechanisms driving SLFN11 chromatin recruitment remain partially elucidated. Using high-throughput imaging of 162 oncology-focused compounds in U2OS cells with inducible SLFN11 expression, we discovered that deubiquitinase (DUB) inhibitors drive massive SLFN11 recruitment to chromatin, preferentially at promoter regions while concurrently suppressing transcription. DUB inhibitors such as VLX-1570 promote ubiquitin-dependent enrichment of SLFN11 without detectable DNA damage, distinct from the camptothecin-induced RPA-associated SLFN11 foci formed at stressed replication forks. Yet, SLFN11 chromatin recruitment both by DUB inhibitors and DNA damage are suppressed by TAK243 demonstrating their ubiquitylation dependency. RNF168 is required for SLFN11 ubiquitylation and its subsequent chromatin association, and ubiquitylation within SLFN11's middle linker domain (lysines 390, 391, and 429) with K27-linked polyubiquitin chains is essential for the chromatin recruitment of SLFN11. These findings suggest the importance of SLFN11 ubiquitylation by RNF168 for SLFN11 chromatin recruitment and SLFN11 transcriptional regulatory role at promoter regions.