Figure 8. Nucleic acid binding and H3K9me3 binding both contribute to SUV39H1-mediated constitutive heterochromatin silencing.
(A) Sanger DNA sequencing traces showing point mutations in the endogenous SUV39H1 locus in DLD-1 cells, generated by CRISPR/Cas9 gene editing. (B) Representative western blot showing H3K9me3, histone H4, SUV39H1, and HP1α levels in normal unedited DLD-1 cells (far left), or SUV39H2 KO DLD-1 cells with the indicated mutations in SUV39H1. (C) Total H3K9me3 levels in mutant SUV39 DLD-1 cells, measured by quantitative western blot. Nuclear lysate was prepared and blotted with indicated antibodies, and H3K9me3 signal was normalized to histone H4 levels. Graphed are the means of 3 repeats, errors bars represent standard error. (D) Quantification of H3K9me3 localization at centromeres in mutant SUV39 DLD-1 cells. Cells were grown on coverslips, fixed and permeabilized, then stained with the indicated antibodies, imaged, and quantified using centromere finder software, using CREST staining as a centromere marker. Graphed are the means of 4 repeats, error bars represent standard error. Significance was determined using paired, two-tailed t-tests. *p<0.05, **p<0.005, ***p<0.0005. (E) Representative images of immunofluorescence staining of mutant SUV39 DLD-1 cells, used for quantification shown in 8D. Cells are stained for DNA (blue), H3K9me3 (green), and CREST centromere stain (red). (F) Quantification of α-satellite RNA in mutant SUV39 DLD-1 cells by RT-qPCR. Total α-satellite RNA levels are normalized to GAPDH RNA levels. Graphed are the means of 3 repeats, error bars represent standard error. Significance was determined using paired, two-tailed t-tests. *p<0.05, **p<0.005.