Precise temporal control of protein abundance is essential for dissecting cellular circuits. Existing chemical genetic methods such as dTAG enable rapid, tunable depletion of tagged proteins by recruiting E3 ligases to FKBP12F36V-tagged targets. The authors developed a complementary strategy—dubTAGs—heterobifunctional small molecules that instead recruit endogenous deubiquitinases to FKBP12F36V-tagged proteins, with the aim of stabilizing and increasing protein levels on demand and reversing degradation-based perturbations.
dubTAGs are heterobifunctional constructs that bridge an FKBP12F36V tag on a target protein and a cellular deubiquitinase. The authors report identifying molecules that recruit specific deubiquitinases and form ternary complexes with FKBP12F36V-tagged targets. Biochemical evidence presented in the source indicates that recruitment of deubiquitinases is required for the stabilization effect, supporting the intended mechanism of action: removal or prevention of ubiquitin modification to increase steady-state protein levels.
To test dubTAG activity in a biologically relevant context, the investigators used stem cell–derived cranial neural crest cells (CNCCs) in which endogenous transcription factors were genetically fused to FKBP12F36V. Specifically, the transcription factors SOX9 and TWIST1 were endogenously tagged with FKBP12F36V, providing physiologic expression contexts to evaluate whether dubTAGs can stabilize native proteins without overexpression artifacts.
From screening and characterization efforts, the authors identified heterobifunctional molecules that recruit either OTUB1 or USP7 to FKBP12F36V-tagged proteins. These dubTAGs demonstrated effective target stabilization and formation of ternary complexes between the deubiquitinase, the small molecule, and the tagged protein. The source emphasizes that stabilization depends on deubiquitinase recruitment, indicating mechanism-specific activity rather than nonspecific target effects.
The report describes several functional attributes of dubTAGs observed in the CNCC system and related assays. dubTAG-mediated protein stabilization was target-specific and dependent on engagement of the recruited deubiquitinase. The stabilization effect was tunable and rapid, permitting control over protein abundance levels. Importantly, dubTAGs could reversibly restore protein levels after dTAG-mediated degradation, enabling bidirectional control: dTAGs for rapid depletion and dubTAGs for on-demand restoration and elevation of the same FKBP12F36V-tagged proteins.
As a functional demonstration, the investigators applied dubTAGs to elevate endogenous SOX9 in CNCCs and assessed consequences on chromatin accessibility. The study reports that regulatory elements responded to increased SOX9 in both monotonic and non-monotonic ways. These distinct patterns of chromatin response were associated with underlying sequence features, indicating that graded or transient changes in a key transcription factor’s abundance can produce varied regulatory outcomes across the genome.
The authors position dubTAGs as broadly applicable tools for investigating the effects of elevated protein levels, complementing degradation-based approaches. Because dubTAGs enable tunable and reversible stabilization of endogenous proteins, they can be used to study protein dosage effects in development, disease modeling, and therapeutic discovery, and to interrogate dynamic relationships between protein concentration and downstream molecular or phenotypic readouts.
The source article is a preprint; experimental detail, data extent, and broader validation beyond the described CNCC examples are presented in the preprint but are not reproduced exhaustively here. Competing interests disclosed: the Jin laboratory has received research funds from multiple companies, J.J. holds equity and consultancy relationships with industry entities, and several institutional affiliations are noted. Funding sources declared include multiple NIH grants and private foundations.
Overall, dubTAGs are described as heterobifunctional small molecules that recruit endogenous deubiquitinases such as OTUB1 or USP7 to FKBP12F36V-tagged proteins, permitting on-demand, tunable, and reversible stabilization of endogenous protein levels. The approach was validated in stem cell–derived CNCCs tagged at endogenous loci (SOX9, TWIST1) and applied to study chromatin responses to increased SOX9, revealing both monotonic and non-monotonic regulatory element behavior linked to sequence features.