BTBBCL6 dimers as building blocks for reversible drug-induced protein oligomerization

• Published in: Cell reports methods 2022-04, Vol.2 (4), p.100193-100193, Article 100193
• Main Authors: Nitsch, Lena, Jensen, Patrizia, Yoon, Hojong, Koeppel, Jonas, Burman, Shourya Sonkar Roy, Fischer, Eric Sebastian, Scholl, Claudia, Fröhling, Stefan, Słabicki, Mikołaj
• Format: Article
• Language: English
• Subjects: BI-3802; BI-3812; BTB domain; BTB switch; polymerization switch; protein polymerization; reversibility
• ISSN: 2667-2375; 2667-2375
• DOI: 10.1016/j.crmeth.2022.100193

Here, we characterize the BTB domain of the transcription factor BCL6 (BTBBCL6) as a small-molecule-controlled, reversible oligomerization switch, which oligomerizes upon BI-3802 treatment and de-oligomerizes upon addition of BI-3812. We show that the magnitude of oligomerization can be controlled in vitro by BI-3802 concentration and exposure time. In cellular models, exposure to BI-3802/BI-3812 can drive multiple cycles of foci formation consisting of BTBBCL6 fused to EGFP, which are not degraded due to the lack of a degron. We generated an epidermal growth factor receptor (EGFR)-BTBBCL6 fusion. Treatment with BI-3802, as an ON switch, induced EGFR-BTBBCL6 phosphorylation and activation of downstream effectors, which could in part be reversed by the addition of BI-3812, as an OFF switch. Finally, BI-3802-induced oligomerization of the EGFR-BTBBCL6 fusion enhanced proliferation of an EGF-dependent cell line in absence of EGF. These results demonstrate the successful application of small-molecule-induced, reversible oligomerization as a switch for synthetic biology. [Display omitted] •The BTBBCL6 domain is a compound-triggered, reversible oligomerization switch•Length of BTBBCL6 oligomers is controlled by drug concentrations and treatment time•Oligomerization of EGFR-BTBBCL6 fusions induces downstream signaling in absence of EGF Molecular switches are employed as synthetic biology tools to transition between at least two states. Here, we aimed to develop a reversible, drug-induced oligomerization switch. We characterized a minimal BTBBCL6 fusion construct acting as a switch that can undergo multiple rounds of oligomerization/de-oligomerization by small molecules. When fused to EGFR, this system enables reversible activation of downstream signaling, resulting in enhanced cell proliferation. Protein oligomerization is a ubiquitous process in nature. Nitsch et al. characterize a minimal BTBBCL6 domain that can be fused to a protein of choice to drive reversible oligomerization modulated by small molecules.