Figure 3: Tethering the 1–167 domain of Suv39h1 to pericentric heterochromatin accelerates de novo targeting of HP1α.

(a) Experimental scheme. (b) De novo localization of endogenous HP1α (red) and H3K9me3 (white) in Suv39h double-null cells expressing TALE-Suv39h2, TALE-Suv39h1 or TALE-Suv39h1 domains tagged with HA analysed by immunofluorescence 5 h after transfection. Suv39-HA constructs are tethered to pericentric heterochromatin via TALE engineered to bind specifically to major satellite repeats in pericentric domains and revealed by HA staining (green). Scale bar, 10 μm. We examined 300 transfected cells and performed quantitative analysis of the percentage of transfected cells with endogenous HP1α or H3K9me3 enriched at pericentric heterochromatin domains (PHC). Error bars on the graph represent s.d. from three independent experiments. A comparison of transfected protein expression is shown on the Western blot. **P<0.01; ***P<0.001 (Student’s t-test). Scale bar, 10 μm. (c) Time course analysis of the de novo localization of endogenous HP1α in Suv39h double-null cells transfected with TALE-Suv39h1 domains tagged with HA. The percentage of cells with HP1α enriched at pericentric heterochromatin domains (PHC) as a function of time after transfection is represented. Error bars indicate s.d. of four independent experiments (400 transfected cells counted in each condition). A comparison of transfected protein expression is shown on the western blot. (d) De novo localization of endogenous HP1α in Suv39h double-null cells transfected with TALE-Suv39h1-1-167-WT, TALE-SUV39h1-1-167-KQRmut, TALE-SUV39h1-1-167-EQEmut or TALE-Suv39h1-1-114 tagged with HA, 5 h post transfection. We performed quantitative analysis of endogenous HP1α (left) and TALE-Suv39h1-HA constructs (right) enrichment at PHC on more than 100 transfected cells for each condition from three independent experiments. NS, not significant; **P<0.01; ***P<0.001 (Student’s t-test).