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. 2012 Aug 15;26(16):1811–1824. doi: 10.1101/gad.190272.112

Figure 5.

Figure 5.

The TML domain of Dsh1 is essential for perinuclear and heterochromatin localization and function in siRNA-directed heterochromatin formation. (A) Schematic representation of the Dsh1 protein. The position of the TML domain (tml) is indicated by a gray box. A schematic representation of a tml deletion mutant is also shown. (B) Indirect immunofluorescence analysis of the localization of Dsh1-13myc and Cut11-GFP (top panels) or Lem2-GFP (bottom panels). White bars, 5 μm. (C) Indirect immunofluorescence analysis of the localization of Dsh1-13myc or Dsh1-tmlΔ-13myc. Strains expressing Dsh1-13myc (or Dsh1-tmΔ-13myc) and Lem2-GFP were used. Cells growing exponentially were stained with DAPI, an antibody against myc, and an antibody against GFP to visualize DNA, Dsh1-13myc, and Lem2-GFP, respectively. Lem2-GFP was stained to visualize the position of the nuclear periphery and spindle pole body. White bars, 5 μm. (D) ChIP analysis of Dsh1-13myc at otr1R∷ade6+ and dh in indicated strains. Enrichment relative to fbp1+ is shown on the Y-axis. The positions of the PCR products are shown in Figure 1A. (E) ChIP analysis of H3K9me2 (left) and Swi6 (right) in dsh1 mutants. The primers are the same as outlined in Figure 1E. Enrichment relative to fbp1+ is shown on the Y-axis. (F) siRNA analysis in dsh1 mutants. The probes used were the same as outlined in Figure 2A. (G) Coimmunoprecipitation assay for Rdp1-5Flag and Dcr1-3HA in dsh1 mutants. Immunoprecipitates prepared using an antibody against Flag were analyzed by Western blotting with antibodies against Flag and HA.