Domain-Selective BET Ligands Yield Next-Generation Synthetic Genome Readers/Regulators with Nonidentical Cellular Functions

SynTEF1, a prototype synthetic genome reader/regulator (SynGR), was designed to target GAA triplet repeats and restore the expression of frataxin (FXN) in Friedreich's ataxia patients. It achieves this complex task by recruiting BRD4, via a pan-BET ligand (JQ1), to the GAA repeats by using a sequence-selective DNA-binding polyamide. When bound to specific genomic loci in this way, JQ1 functions as a chemical prosthetic for acetyl-lysine residues that are natural targets of the two tandem bromodomains (BD1 and BD2) in bromo- and extra-terminal domain (BET) proteins. As next-generation BET ligands were disclosed, we tested a select set with improved physicochemical, pharmacological, and bromodomain-selective properties as substitutes for JQ1 in the SynGR design. Here, we report two unexpected findings: (1) SynGRs bearing pan-BET or BD2-selective ligands license transcription at the FXN locus, whereas those bearing BD1-selective ligands do not, and (2) rather than being neutral or inhibitory, an untethered BD1-selective ligand (GSK778) substantively enhances the activity of all active SynGRs. The failure of BD1-selective SynGRs to recruit BRD4/BET proteins suggests that rather than functioning as epigenetic/chromatin mimics, active SynGRs mimic the functions of natural transcription factors in engaging BET proteins through BD2 binding. Moreover, the enhanced activity of SynGRs upon cotreatment with the BD1-selective ligand suggests that natural transcription factors compete for a limited pool of nonchromatin-bound BET proteins, and blocking BD1 directs pan-BET ligands to more effectively engage BD2. Taken together, SynGRs as chemical probes provide unique insights into the molecular recognition principles utilized by natural factors to precisely regulate gene expression, and they guide the design of more sophisticated synthetic gene regulators with greater therapeutic potential.

Automated summary

Researchers have designed a synthetic genome reader/regulator (SynGR) to target and restore the expression of a specific gene in a genetic disorder. They found that certain ligands can activate gene transcription, while others cannot. In addition, blocking one specific ligand enhances the activity of all active gene regulators. These findings provide insights into the regulation of gene expression and have implications for the design of more effective gene regulators.