Extended Data Fig. 8: The bromodomains of BRD1, BRD2 and BRD13 bind to acetylated histones and the second PHD domain of PMS2B binds to methylated H3 at lysine 4.
a, Sequence alignment of the bromodomains of HsBRD9, AtBRD1, AtBRD2, AtBRD13, HsBRM, ScSTH1, and AtBRM. The BRD1 bromodomain conserved residues Y207, N251 and Y258 marked with asterisks were subjected to point mutation. b, Coomassie blue staining of the wild-type BRD1, BRD2, and BRD13 bromodomains and of the mutated BRD1 bromodomain (BRD1-M) tagged by HIS. The fusion proteins were purified from E. coli. The experiments were repeated for at least two times with similar results. c,d, ITC binding assays measuring the binding affinity of the bromodomains of BRD1,BRD2, BRD13 and BRD1-M for acetylated and unmodified H3 peptides (c) and acetylated and unmodified H4 peptides (d). e, Schematic representations indicate the domain architecture of PMS1A and PMS2B and the truncated versions of PMS1A and PMS2B purified from E. coli. f, A phylogenetic tree of the PHD domains in PMS1A, PMS1B, PMS2A, PMS2B, and in the known PHD-containing H3K4me3 readers ARID5, SHL and EBS. The RING fingers of PMS2A and PMS2B that are closely related to the conserved PHD finger were included for analysis. g, Coomassie blue staining of the wild-type PMS1A and PMS2B PHD domains and of the mutated PMS2B PHD domain tagged by GST. The proteins were expressed and purified from E. coli. The experiments were repeated for at least two times with similar results. h, Determination of the binding of wild-type PMS1A and PMS2B PHD domains to methylated and unmethylated histone peptides by in vitro binding assays. i, Sequence alignment of the second PHD fingers of PMS2A and PMS2B (PMS2A-PHD2 and PMS2B-PHD2) and the PHD fingers of ARID5, SHL and EBS. Three conserved aromatic residues (Y148, Y155, and W170) in the PMS2B-PHD2 marked with asterisks were mutated to alanine in the mutated version of PMS2B (PMS2B-PHD-M). The experiment was repeated for two times with similar results. j, The effect of the PMS2B-PHD2 mutation on the binding ability of PMS2B for methylated H3 at lysine 4 as determined by in vitro binding assays. The experiment was repeated for two times with similar results.
