FIG 9.

Heterochromatin establishment in a newly formed telomere is independent of its elongation. Clr4 is the only histone H3 lysine 9 methyltransferase and is required for heterochromatin. (A) Exponentially growing wild-type and clr4Δ cells bearing the 48-bp prototelomere and the I-SceI expression cassette were treated with ahTET (9 μM final concentration), and aliquots were taken either prior to treatment (0 h) or after treatment (1 to 32 h), following the approach used for Fig. 3A. Blots from the clr4Δ cells are shown. The wild-type cells gave results nearly identical to those in Fig. 3A. (B) Efficiency of ura4⁺::hph⁺ cleavage, as shown in Fig. 3C. The error bars show SEM from duplicate assays for clr4Δ and triplicate assays for the wild type (WT). (C) Telomere elongation is nearly identical in wild-type and clr4Δ backgrounds. The modal TRF sizes of the newly formed telomere after induction were measured as for Fig. 4C. Band sizes on these blots vary by approximately ±0.03 kb. WT sizes are from the formation experiment shown in Fig. 3A. clr4Δ sizes are from the formation experiment shown in panel A. (D) Doubling times of wild-type or clr4Δ cells bearing the 48-bp prototelomere and the I-SceI expression cassette prior to ahTET induction (uninduced), 0 to 8 h post induction, and 8 to 20 h postinduction (as determined by the OD₆₀₀). (E) The clr4Δ mutation eliminated telomeric silencing. Growth of wild-type cells (ura4⁻) or wild-type or clr4Δ cells bearing a 48-bp prototelomere before (−) and after (+) 20 h of induction was assessed by spotting 5-fold serial dilutions of cells onto rich-medium plates (YES) without and with hygromycin (hyg) or onto synthetic medium without (−ura) or with (+ura) uracil.