Fig. 3. Structural analysis of BAHCC1^BAH reveals a unique H3K27me3-’reading’ pocket.

a, Overall structure of BAHCC1^BAH (aquamarine) bound to the H3K27me3 peptide (yellow). BAHCC1^BAH and peptide are shown in ribbon and stick representation, respectively.

b, Close-up view of the BAHCC1^BAH-H3K27me3 interactions. The H3K27me3-interacting residues of BAHCC1^BAH and peptide are shown as pink and yellow sticks, respectively. Dashed line indicates hydrogen bond.

c, Positioning of the H3K27me3 side chain (yellow) within the aromatic cage of BAHCC1^BAH. The cage residues (pink) of mouse and human BAHCC1^BAH are labeled in red, with the latter shown in parentheses.

d, Schematic diagrams of the BAHCC1^BAH (black)-H3K27me3 (magenta) interactions. Hydrogen bonding and electrostatic interactions are shown as black and green dashed lines, respectively. Hydrophobic interactions are colored in yellow.

e, ITC measures the binding affinity between BAHCC1^BAH, either WT or mutant, and H3K27me3 peptide.

f, Pulldown using biotinylated H3K27me3 peptide and GST-BAHCC1^BAH, either WT or BAH-mutated.

g, CRISPR/Cas9-mediated gene editing for introducing the Y2533A homozygous point mutation to BAHCC1 in JURKAT cells. Shown are Sanger sequencing results using cDNA as template.

h, CoIP for association between endogenous BAHCC1 and H3K27me3-containing histones in JURKAT cells that express the 3×Flag-tag knockin allele of BAHCC1, either WT or harboring a Y2533A (left, two independent lines) or W2554G (right) homozygous mutation at BAHCC1^BAH.