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. 2008 Sep 22;28(23):6973–6988. doi: 10.1128/MCB.00791-08

FIG. 1.

FIG. 1.

Swi6 and Chp2 play distinct roles in heterochromatin assembly. (A) Schematic drawing of Swi6 and Chp2. Gray and black boxes represent the conserved CD and CSD, respectively. N-terminal (N) and hinge (H) regions are also indicated. The amino acid sequence of C-terminal Swi6 or Chp2 is shown beneath each drawing, and the positions of amino acids mutated in this study (L315E and I370E) are indicated by an arrow. (B) Schematic diagram of the mating-type (mat) locus. The position of the silencing reporter gene (Kint2::ura4+) is shown. The mat locus is not drawn to scale. (C) Both Swi6 and Chp2 are required for heterochromatin silencing. A 10-fold serially diluted culture of the indicated strain was spotted onto nonselective medium (N/S), −Ura medium, or medium containing 5-FOA (left). The expression level of the ura4+ transcript of the wild-type, Δswi6, or Δchp2 strain was evaluated by real-time PCR analysis and compared with the level in wild-type cells (right). Error bars represent the standard error of the mean. (D) Chp2 production fails to rescue the defective silencing in swi6-115 mutants. Swi6 or Chp2 protein was produced from the swi6 promoter (Pswi6) on an episomal plasmid (pRE), and silencing of the Kint2::ura4+ reporter in swi6-115 mutant cells was assayed by the plating efficiency on selective medium. Medium lacking leucine was used to select the cells harboring episomal plasmids. Empty pRE vector was used as the control. (E) Swi6 production does not fully rescue the silencing defect in Δchp2 cells. Each gene was introduced into the ars1 locus with the LEU2 gene and ectopically expressed from the chp2 promoter (Pchp2). Recovery of the Kint2::ura4+ silencing defect was evaluated by spotting assay (left) and RT-PCR analysis (right). (F and G) Western blot analysis of ectopically expressed Swi6 and Chp2. The level of Swi6 or Chp2 produced from Pswi6 on an episomal plasmid pRE on a swi6-115 background was compared with that of wild-type cells (F). The level of Swi6 or Chp2 expressed from Pchp2 at the ars1 locus on a Δchp2 background was compared with that of wild-type cells (G). α, anti; exp, exposure; N/S, nonselective medium.