Figure 5. Direct H4 Tail Binding is Necessary for KAP1 Promoter Occupancy and Target Gene Activation.

(A) The ¹H, ¹⁵N HSQC spectra of KAP1PHD-BD collected upon titration with H4 peptide. Inset shows residues displaying chemical shifts. Spectra are color-coded according to the PHD-BD:H4 molar ratio.

(B) Histogram showing chemical shift perturbations. Vertical grey bars indicate Pro and unmapped residues. The dash line indicates a threshold value of average + 3×SD. Chemical shift assignments of the apo-PHD-BD state were from BMRB (ID 11036).

(C) Left: Predicted model depicting the frequency of residue specific contacts between KAP1_PHD and H4 in the production run of the MD simulations using backbone amide resonances in panel (A) as restraints. Middle: Residues exhibiting H4-induced resonance perturbations above threshold are mapped in orange onto the structure of the predicted complex. Right: The electrostatic surface potential of the predicted complex is colored blue and red for positive and negative charges, respectively.

(D-E) In vitro binding assay between recombinant proteins and (D) H4 peptide or (E) biotinylated mono-nucleosomes. Western blots probed with anti-GST. Quantitation relative to WT KAP1 of (E) are shown.

(F) Western blot of KAP1 proteins in the indicated HCT116 cell lines.

(G) Cell growth assay (cell counts ± SEM; n=3).

(H) RT-qPCR of KAP1 target genes after reconstitution of the indicated proteins in shKAP1 HCT116 (mean expression relative to shNT+GFP ± SEM; normalized to RPL19; n=3).

(I) FLAG ChIP-qPCR of the indicated proteins at KAP1 target gene promoters (mean % Input DNA ± SEM; n=3).