FIG. 2.

Cellular abundance and dimer formation of Swi6 and Chp2. (A and B) Cellular abundance of Swi6 and Chp2 proteins. The indicated amounts of recombinant His₆-Swi6 or His₆-Chp2 proteins and of whole-cell extracts (WCE) prepared from the indicated number of wild-type cells were subjected to Western blot analyses using anti-Swi6 (A) and anti-Chp2 (B) antibodies. The total heterochromatic region of a G₂ phase cell is estimated to be ∼950 kb, consisting of ∼430 kb of centromeres, ∼40 kb of silent mating type loci, and ∼520 kb of telomeres. Given that every ∼200 bp of heterochromatic DNA is organized into a nucleosome with two H3 tails, the heterochromatin region contains ∼9,500 H3 tails. (C and D) The Chp2 dimer is less stable than the Swi6 dimer. (C) Chromatogram and elution profile of the Swi6 protein by size exclusion chromatography. Recombinant His₆-tagged Swi6 and Swi6^L315E proteins were loaded onto a Sephadex 200 pg column. Elution profiles for Swi6 (red) and Swi6^L315E (blue) are shown (top), and the eluted proteins were resolved by 10% SDS-PAGE and visualized by Coomassie staining (bottom). (D) Chromatogram and elution profile of Chp2 protein by size exclusion chromatography. Recombinant His₆-tagged Chp2 and Chp2^I370E proteins were resolved by a Sephadex 200 pg column. Elution profiles are shown as chromatograms (top) and 10% SDS-PAGE visualized by Coomassie staining (bottom). The positions of eluted marker proteins are indicated above each chromatogram. (E to H) The dimer configuration is critical for the in vivo function of Swi6 and Chp2. (E) The swi6⁺ or swi6^L315E gene was introduced into the ars1 locus of Δswi6 cells with the LEU2 gene and ectopically expressed from the swi6 promoter. Silencing of the Kint2::ura4⁺ reporter in control wild-type, Δswi6, and Δswi6-derivative cells was evaluated by spotting assay. (F) Western blot analysis of Swi6 in wild-type cells or cells expressing Swi6 or Swi6^L315E from the swi6 promoter at the ars1 locus on a Δswi6 background. (G) The chp2⁺ or chp2^I370E gene was introduced into the ars1 locus of Δchp2 cells with the LEU2 gene and ectopically expressed from the chp2 promoter. Silencing of the Kint2::ura4⁺ reporter in control wild-type, Δchp2, and Δchp2-derivative cells was evaluated by spotting assay. (H) Western blot analysis of Chp2 in wild-type cells or cells expressing Chp2 or Chp2^I370E from the chp2 promoter at the ars1 locus on a Δchp2 background. α, anti; AU, arbitrary units; N/S, nonselective medium.