Probing tandem epigenetic reader domains

Thesis

Epigenetic signals include chemical modifications on DNA and post-translational modifications to amino acids in histone proteins. One of the most common sites of modification are lysine residues: addition of acetyl or methyl groups are recognised by "reader" domains including bromodomains and plant homeodomains (PHDs). These domains mediate protein-protein interactions, affecting downstream events including gene transcription. PHDs and bromodomains are found in close proximity within some proteins, raising the possibility of combinatorial signalling eliciting a range of responses. Tripartite motif-containing proteins 24 and 33 (TRIM24 and TRIM33) contain an adjacent PHD and bromodomain which recognise methylation and acetylation states of lysine residues, respectively (Figure 1). These interact with histone-mimicking peptides that display a specific combination of unmodified, trimethylated and acetylated lysine residues. Work has been carried out towards inhibition of these epigenetic readers, through target analysis, assay development, and screening of putative binding partners. Refinement of expression systems for TRIM24 and TRIM33 enabled structural investigations, and development of biophysical assays to provide screening capacity. These assays were used to further characterise interactions through profiling varyingly modified histone-like peptides. Based on structural information and computational modelling, an in silico screen was performed to identify putative binding partners, and generate a chemical pharmacophore. In vitro screening of a bespoke collection of compounds based on these results, was supplemented with a broad screen targeting protein-protein interactions. Subsequent rounds of validation identified four compounds which bind to either TRIM24 or TRIM33 (Figure 2). Prompted by the reliance of competition based assays on knowledge of a high affinity interaction with a ligand or peptide, a dimethylisoxazole unit was used as an acetyl lysine mimicking residue. Incorporation in a histone-like peptide, via selective alkylation, resulted in increased affinity of the peptide to the BRD4 bromodomain. Application to epigenetic reader systems with unknown cognate binding partners would improve assay capacity for ligand identification. This approach will facilitate synthesis and screening of novel probe molecules, to elucidate the function of tandem recognition domains in interpreting the "epigenetic code".

Authors: Measures, A

• DOI: 10.5287/ora-zbdy7xd9a
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford