CUX1 loss impairs EHMT2 recruitment to DNA breaks and disrupts DNA damage–induced histone methylation changes. (A) Model of GSD-FRET. Target proteins are identified with antibodies tagged with either a donor (D) or acceptor (A) fluorophore. An image is taken using the donor excitation maxima. If the donor fluorophore is not near an acceptor fluorophore, the donor will emit all energy at the donor emission wavelength (left panel, top). If the donor is colocalizing with the acceptor fluorophore, some of the donor emission energy will be transferred to the acceptor, in proportion to the proximity to the acceptor (right panel, top). The samples are then bleached at the acceptor wavelength, and the bleached acceptors are no longer able to absorb energy. The samples are then reimaged at the donor excitation wavelength. Any increase in donor brightness in the second image compared with the first image is proportional to the energy that was previously transferred to the acceptor, or FRET (bottom right). If the molecules were not colocalizing, there will be no change in donor emission intensity, and no FRET (bottom left). (B) GSD-FRET analysis of colocalization between anti-γH2AX (donor) and anti-CUX1 (acceptor) in K562 cells. Scale bar, 1 μm. Bottom row images are following depletion of the acceptor fluorophore. Scale bar, 0.25 μm. Inset, representative FRET. Scale bar, 0.25 μm. Images shown are representative of results from 3 independent experiments. (C) Representative immunoblot for CUX1 protein in gHPRT and gCUX1 K562 cells (n = 3). (D) Principal component analysis of the histone posttranslational matrix (PTM) generated with EpiProfile analysis of histone PTMs in irradiated and mock-irradiated gHPRT and gCUX1 K562 cells. Histones were extracted 1 hour following irradiation (6 Gy). Results shown are derived from analysis of 3 independent samples. (E) Immunofluorescence imaging for EHMT2 and γH2AX colocalization 1 hour after irradiation (6 Gy). Colocalization was quantified in gHPRT and gCUX1 K562 cells after irradiation with or without the addition of an EHMT2 inhibitor, UNC0642, 60 minutes prior to irradiation. The fraction of colocalized pixels was calculated per nucleus. Results shown are a composite of 4 independent experiments. Significance was determined with a Student t test between indicated samples. (F) EpiProfile analysis of H3K27 methylation in irradiated and mock-irradiated gHPRT and gCUX1 K562 cells. Histones were extracted 1 hour following irradiation (6 Gy). Significance was determined with a Student t test between indicated samples. Results shown are derived from analysis of 3 independent samples. (G) ChIP-seq of H3K27me3 in K562 cells. Volcano plot showing differentially occupied ChIP-seq sites (n = 2) between clonal gHPRT and gCUX1 K562 cell lines. Each point is the average of 2 replicates. DiffBind was used to identify significantly differentially occupied sites. Red points indicate significance ≤10% FDR. (H) The panels depict a smooth line fit to the average column-wise read density for all differentially bound sites across the 20-kb window. Top panel, all H3K27me3 sites; bottom, only H3K27me3 sites significantly decreased in gCUX1 cells (10% FDR) (binomial P value = 7.35 × 10⁻⁶⁰). (I) The normalized read density is quantified at all H3K27me3 sites and at H3K27me3 sites lost in gCUX1 cells. A Mann-Whitney test was used to determine significance. IR, irradiation RPKM, reads per kilobase mapped reads.