dubTAGs enable on-demand stabilization for tunable and reversible control of endogenous protein levels.

Precise and rapid control over cellular protein levels is essential to dissect complex biological systems. Chemical genetic approaches such as dTAG, in which a target is fused to a degron tag (FKBP12F36V) and degraded upon small molecule-mediated recruitment of E3 ligases, have enabled rapid and tunable control over protein abundance. However, no analogous tool exists to precisely increase protein levels and actively reverse dTAG-mediated degradation. Here, we developed heterobifunctional small molecules (dubTAGs) that stabilize FKBP12F36V-tagged proteins by recruiting endogenous deubiquitinases. Utilizing stem cell-derived cranial neural crest cells (CNCCs) in which the transcription factors SOX9 or TWIST1 are endogenously tagged with FKBP12F36V, we identified OTUB1- or USP7-recruiting heterobifunctional molecules that demonstrated effective target stabilization and ternary complex formation. We demonstrate that dubTAG-mediated protein stabilization is dependent on deubiquitinase recruitment, target-specific, and can tunably and rapidly reverse dTAG-mediated degradation. We applied dubTAGs to assess how stabilizing endogenous SOX9 impacts chromatin accessibility in CNCCs, finding both monotonic and non-monotonic regulatory element responses that are driven by distinct sequence features. dubTAGs are readily applicable tools for investigating the effects of elevated protein levels and tunably reversing targeted degradation, enabling new approaches to study protein dosage effects in development, disease, and therapeutic discovery.