To establish an AlphaScreen assay, the PHD-BRD cassettes of TRIM24 and both TRIM33 isoforms were expressed with an N-terminal His 6 tag attached. Consistent with the X-ray crystallography data above, it has been established previously that methylation of the K4 position of histone H3 reduces affinity to the PHD finger: K d values for the TRIM24 PHD have been measured by ITC as 8.6 μM for H3 1-15 K4Me 0 , 41 μM for H3 1-15 K4Me 1 , 198 μM for H3 1-15 K4Me 2 , and >400 μM for H3 1-15 K4Me 3 . The presence of the four N-terminal residues of the peptide (H3 1- 4 ), which are recognized by the b-sheet surface of the PHD finger, are essential for affinity to TRIM33α. Consequently, we maintained K4Me 0 in most of the peptides investigated, to avoid affinities falling below detection limits. The peptide recognition profiles of TRIM24, TRIM33α and TRIM33β were investigated by determining affinity of their tandem BRD-PHD cassettes for H3-mimicking peptides containing combinations of acetylation at K14, K18 or K23, and methylation of K9 (Table ). The data from the AlphaScreen assay show that the TRIM24 PHD-BRD binds preferentially to H31-27K23Ac over the other monoacetylated peptides, while both TRIM33α and TRIM33β PHD-BRD bind preferentially to H31-27K18Ac, which is consistent with the literature (Table ). We note that the dual modified peptide H31-27K18AcK23Ac shows increased affinity for the TRIM24 PHD-BRD (Kd = 0.0768 ± 0.002 μM vs 0.182 ± 0.012 μM for H31-27K18Ac). Conversely, H31- To confirm the data obtained using our AlphaScreen assay, we investigated the affinity of the H31-27K9Me3, H31-27K18Ac, and H31-27K9Me3K18Ac peptides for the TRIM24, TRIM33α, and TRIM33β PHD-BRD using ITC (Table ). The data obtained show similar trends to the