Photocrosslinking Probes Reveal Nonhistone Partners of the TRIM24 Bromodomain
- Genetic code expansion installed site-specific photocrosslinkers (AzF and BzF) at residue L922, within the acetyllysine-binding pocket of the TRIM24 bromodomain.
- Both crosslinker variants retained recognition of acetylated histones and crosslinked efficiently under UV, enabling interaction-based protein profiling (IBPP) in a native context.
- Quantitative proteomics of the two complementary probes gave differing capture profiles: AzF enriched for NPM1, BzF enriched for CDK9, with MAP4, RAN, and SND1 as additional candidates.
- ChIP-seq showed TRIM24, CDK9, and NPM1 co-occupying regions near transcription start sites of cancer-associated genes.
- Together the results suggest the TRIM24 bromodomain reads acetylation on nonhistone proteins as well as histones, broadening its reader function and informing chemical-probe design against this pocket.
The crosslinker sits inside the acetyllysine pocket itself, at L922 — that's not a fortuitous surface hit, it's evidence TRIM24 reads acetylated lysine on NPM1 and CDK9 through the same pocket it uses on histones, not a peripheral contact. That reframes TRIM24 as an acetyl-lysine reader with a broader substrate scope, and it means any inhibitor competing for that pocket is also competing off a nonhistone binding event, not just a histone mark. Practically, TRIM24 probe programs need NPM1/CDK9 engagement counter-screens now, and the TSS co-occupancy by ChIP-seq suggests that off-target isn't background noise but transcriptionally active. Worth flagging: this is GCE-based interactome mapping, not medicinal chemistry — no chemotype, no SAR, just a map of what a pocket-directed probe will also touch. BET programs learned this same lesson with nonhistone acetyl-lysine substrates years ago; TRIM24 is only now catching up to that complexity.