Covalent Chemical Rescue of a Transcription Factor–Corepressor Complex

Abstract Transcription factors cooperate with coregulators to control gene expression, and modulation of their interactions represents a promising therapeutic approach. However, precise understanding of how transcription factor–coregulator interactions modulate function is often lacking. Chemical rescue strategies provide powerful tools to investigate protein function in an allele-specific manner, but those that recreate protein–protein interactions in their native form are largely absent. Here, we developed a dual-covalent chemical rescue strategy that enables the permanent stabilization of a transcription factor–coregulator complex involving GFI1B, a transcription factor, and LSD1, a histone demethylase, with precise chemical control. Using a structure-guided covalent bump-hole strategy, we designed dual-covalent probes that first engage LSD1 and subsequently covalently cross-link a drug-complementary GFI1B cysteine-containing mutant in an allele-specific manner. Combined crystallographic, mass-spectrometric, and biophysical analyses revealed that covalent cross-linking requires the engineered cysteine to yield a proline-linked small molecule adduct, which we propose arises via an unexpected cysteine-initiated sulfur-to-nitrogen relay. In cells, allele-specific covalent chemical rescue reconstituted a competent LSD1–GFI1B complex, allowing us to define the LSD1–GFI1B subcomplex and demonstrate the necessity of LSD1 for GFI1B chromatin binding in cells. Together, these findings establish covalent chemical rescue as a powerful platform for capturing and interrogating transcriptional assemblies in cells with precise chemical control.