CRISPR-Cas9 Screening Identifies UBE3C as a Top Negative Regulator of THEMIS Protein Expression

Utilizing unbiased CRISPR/Cas9 screening integrated with affinity purification-mass spectrometry, we identify UBE3C as the physiological E3 ligase for THEMIS. Mechanistically, UBE3C interacts with the CABIT1 domain of THEMIS and catalyzes K48-linked polyubiquitination, primarily targeting the conserved Lysine 208 residue for proteasomal degradation.

UBE3C deletion in T cells in vivo results in normal thymic T cell development but causes an age-dependent defect in peripheral T cell homeostasis. Most critically, genetic epistasis analysis demonstrates that UBE3C deletion alleviates the developmental block in Themis+/- mice and rescues the peripheral homeostatic defects of UBE3C-deficient mice. Our findings establish the UBE3C-THEMIS axis as the core ubiquitin-dependent protein quality control module, revealing an unrecognized role for UBE3C in adaptive immunity and providing a molecular explanation for the exquisite dosage sensitivity of THEMIS.