Ubiquitination is a post-translational modification that regulates essential cellular processes, including chromatin accessibility and DNA repair. UbKEKS, an emerging ubiquitin variant, has been linked to nucleolar organization1, but its broader nuclear roles and protein targets remain poorly understood. Emerging evidence suggests that UbKEKS may impact chromatin dynamics by modulating nuclear proteins and histones, potentially through cytoplasmic mediators such as BRCA1-associated protein (BRAP).
We investigated the role of UbKEKS in BRAP regulation, its connection to BRCA1 activity, and its possible involvement in chromatin compaction via linker histones. To identify direct UbKEKS targets, we used an AviTag-based proximity labeling strategy combined with mass spectrometry. To further explore UbKEKS function, we generated UbKEKS knockout (KO) U2OS cells using CRISPR-Cas9 and compared protein abundance and localization with wild-type cells through proteomics and immunofluorescence.
Our results identified BRAP, a multifunctional E3 ligase involved in nuclear import and BRCA1 regulation, as a direct target of UbKEKS. Co-immunoprecipitation assays revealed that BRAP is modified by UbKEKS via another E3 ligase rather than through self-ubiquitination. In UbKEKS KO cells, BRAP abnormally accumulated in both nucleus and cytoplasm, indicating that UbKEKS controls BRAP localization. This mislocalization may influence BRCA1 retention in the cytoplasm, implicating UbKEKS in DNA damage response, especially in breast cancer. Additionally, UbKEKS KO cells showed elevated levels of several Histone H1 variants, suggesting a role for UbKEKS in chromatin compaction.
Altogether, our findings position UbKEKS as a key regulator of BRAP and a potential modulator of chromatin architecture and DNA repair pathways.