Defects in components of the proteasome enhance transcriptional silencing at fission yeast centromeres and impair chromosome segregation

Abstract

Fission yeast centromeres are transcriptionally silent and form a heterochromatin-like structure essential for normal centromere function; this appears analogous to heterochromatin and position effect variegation in other eukaryotes. Conditional mutations in three genes designated cep (centromere enhancer of position effect) were found to enhance transcriptional silencing within centromeres. Cloning of the cep1(+) and cep2(+) genes by functional complementation revealed that they are identical to the previously described genes pad1(+) and mts2(+), respectively, which both encode subunits of the proteasome 19S cap. Like Mts2 and Mts4, epitope-tagged Cep1/Pad1 localizes to or near the nuclear envelope throughout the cell cycle. The cep mutants display a range of phenotypes depending on the temperature. Silencing within the central domain of centromeres is increased at 36 degrees C. This suggests that the proteasome is involved in regulating silencing and thus centromeric chromatin architecture, possibly by lowering the level of some chromatin-associated protein by ubiquitin-dependent degradation. This is the first report of defective proteasome function affecting heterochromatin-mediated transcriptional silencing. At 36 and 32 degrees C, the cep mutants lose chromosomes at an elevated rate, and at 18 degrees C, the mutants are cryosensitive for growth. Cytological analysis at 18 degrees C revealed a defect in sister chromatid separation while other mitotic events occurred normally, indicating that cep mutations might interfere specifically with the degradation of inhibitor(s) of sister chromatid separation. These observations suggest that 19S subunits confer a level of substrate specificity on the proteasome and raise the possibility of a link between components involved in centromere architecture and sister chromatid cohesion.

FIG. 1

Strategy utilized to screen for mutants which enhance transcriptional repression within the central domain of S. pombe centromeres. (A) Schematic representation of S. pombe cen1. (B) Screening procedure. Details are given in the text. wt, wild type; EMS, ethyl methanesulfonate.

FIG. 2

Cold sensitivity and MBC resistance of cep mutants. Cells grown at 32°C were harvested, and about 10³ cells were spotted onto complete medium (YEA) with or without MBC (20 mg/liter). Incubation times were 7 days at 18°C and 3 days at 32°C. WT, wild type.

FIG. 3

(Top and middle) Mutations in cep1, cep2, and cep3 genes enhance repression of the ura4 gene when placed within the central domain of cen1. (Bottom) cep mutations also enhance silencing within the outer (otr) and inner (imr) repeats of centromere 1. Cells grown at 32°C in N/S medium were serially diluted (one-fifth); spotted onto URA⁻, FOA, and N/S plates; and incubated at 32 or 36°C. About 2 × 10⁴ cells were plated in the highest-density spots. Strains and location of the ura4 gene are as indicated. wt, wild type.

FIG. 4

Phenotypes of cep1, cep2, and cep3 mutants at the restrictive temperature. (A to C) Wild-type, cep1-1, cep2-12, and cep3-16 cells were grown to early log phase at 32°C and transferred at 18°C, and samples were taken for analysis every 1.5 h as described in the text. When shifted to the cold, cells from wild-type and cep mutants stopped dividing (A), but after 4.5 h, growth resumed, and a wave of mitosis was observed. Unlike the wild-type control, cep mutant cells divided only once at 18°C (B) and lost viability (C). (D) Cytological analysis by DAPI (red, pseudocolor) and antitubulin staining (green). Row 1 shows that early mitotic cells (spindle length < 5 μm) were first observed 4.5 h after transfer at 18°C. Rows 2 and 3 show that wild-type cells proceeded normally through mitosis (leftmost panels) but that all three cep mutants experienced defective chromosome segregation. After 6 h (row 2), about 50% of anaphase cells displayed lagging and/or aberrantly segregated chromosomes, and later (9 h [row 3]) abnormal telophase and septated cells were observed. After 12 h (row 4), cep1 and cep2 mutants eventually arrested as interphase-like cells while cep3-16 cells attempted a second mitosis but arrested as metaphase-like cells (rightmost panel). (E) Centromere FISH analysis of anaphase cells. The leftmost panel shows a wild-type cell with the centromeres (red signal) separated at both ends of the anaphase spindle (green). In most cases, the FISH signals in cep mutant cells were clustered at only one pole or in the midzone of the anaphase spindle, indicating a defect in sister chromatid separation. (F) rDNA FISH analysis of anaphase cells showing the absence of separation of chromosome III sister chromatids in cep mutants. Bars, 5 μm.

FIG. 5

Western blot detection of HA-tagged Cep1 and Cep1^cs proteins. Equal amounts of total proteins from the indicated strains were loaded in the lanes. The blot was probed with monoclonal anti-HA antibody.

FIG. 6

Cellular localization of Cep1. Cells from strain FY1521 (cep1⁺-HA) were grown to early log phase and then fixed and stained with anti-HA (red), antitubulin (green), and DAPI (blue). (A) Montage showing the localization of Cep1 through the cell cycle. For each cell, the corresponding panels are placed one below the other. From left to right, an example is shown of cells at various stages of the cell cycle from interphase to cytokinesis. (B) Anti-HA signal alone. Bar, 5 μm.