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. 1999 Oct;10(10):3171–3186. doi: 10.1091/mbc.10.10.3171

Figure 6.

Figure 6

S. pombe hst4+ is required for centromeric silencing. (A) Schematic representation of S. pombe centromere 1, which contains a central domain flanked by the imr and otr inverted repeats. (B) hst4Δ mutants derepress the otr locus. Fivefold dilutions were plated onto EMM plates as a growth control (complete), onto EMM plates lacking uracil (ura−), and onto 5-FOA plates (FOA) to assay silencing of the ura4+ reporter gene. Shown are the marked parent strain, four sister spore products plated at 32°C, and two additional isolates of marked hst4Δ strains. Strains in order of plating are: FY973, LPY3563, LPY3564, LPY3562, LPY3565, LPY3566, and LPY3570. The derepression seen was the same when plates were grown at 20°C (our unpublished results). (C) hst4+ is required for silencing within the imr repeat. As above, the parent strain, the marked and unmarked wild-type strains, and the marked and unmarked hst4Δ strains were plated onto EMM plates as a growth control (complete), onto EMM plates lacking uracil (ura−), and onto 5-FOA plates (FOA). Strains in order of plating are: FY498, LPY3993, LPY3994, LPY3992, and LPY3991. The derepression phenotype was the same when plates were grown at 20°C. (D) hst4+ is also required for silencing within the central domain of cen1 (TM1). The variable 5-FOA sensitivity seen at this locus is shown on the 5-FOA plate, where one isolate of the marked hst4Δ showed no sensitivity yet a second isolate had an ∼25-fold growth defect on 5-FOA at 32°C. When the plates were grown at 20°C rather than 32°C, the 5-FOA sensitivity increased to 625-fold in the marked hst4Δ strain. Strains in order of plating are: FY336, LPY3979, LPY3978, LPY3980, LPY3977, and LPY4387. (E) The hst4Δ mutant also shows temperature-dependent derepression within the central domain of cen3. The marked hst4Δ strain was ∼25-fold more 5-FOA sensitive than the unmarked strain at 32°C and ∼625-fold more sensitive at 20°C. This sensitivity at both temperatures was rescued by expression of N-terminally proA-tagged hst4+. Strains in order of plating are: LPY4379, LPY4380, LPY4381, LPY4384, and LPY4385. Note that, as in Figure 5B, all strains assayed in panels B–E are ura4− at the chromosomal locus (see Table 1).