Aberrant association of chromatin with nuclear periphery induced by Rif1 leads to mitotic defect

(A) Spot tests of Rif1 overexpression in the PP1 mutants dis2∆ (KYP1762), dis2-11 (KYP1760), or sds21∆ (KYP1764) cells were conducted as described in Figs 1C and D. Rif1 overexpression inhibited the growth of mutant cells similar to WT cells. (B) Mutations introduced at the PP1-binding sites (RVxF and SILK motif) of Rif1. (C) Using the extracts made from the cells expressing both Flag-tagged Rif1 and Myc-tagged PP1 (Dis2 or Sds21) (KYP1769, KYP1770, KYP1772, and KYP1773), PP1 were immunoprecipitated by anti-Myc antibody, and co-immunoprecipitated Rif1 was detected. The PP1bs mutant of Rif1 does not interact with either PP1. (D) Time course of overexpression of rif1PP1bsmut–Flag₃ protein expressed on pREP41 plasmid under the nmt41 promoter after transfer to a medium lacking thiamine (lanes 3–9) (KYP1839). Lanes 1 and 2 (KYP1827), rif1PP1bsmut–Flag₃ is expressed at the endogenous rif1 locus under its own promoter in the presence or absence of thiamine in the medium. (E) Spot tests of the WT (KYP025, KYP015, and KYP1774) and rif1∆ (KYP1804, KYP1805, and KYP1839) cells overexpressing the WT or a PP1bs mutant. Overexpression of the PP1bs mutant Rif1 inhibited the growth of fission yeast cells in a manner similar to or slightly better than the WT Rif1.

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(A) The nda3-KM311 cold-sensitive mutant cells with WT rif1⁺ (KYP1268) or those expressing the WT Rif1 (Pnmt41–Rif1) (MS733) or PP1bs mutant Rif1 (Pnmt41–rif1PP1bsmut) (KYP1283) at the endogenous rif1 locus under nmt41 promoter were arrested at the M-phase by incubation at 20°C for 6 h with concomitant depletion of thiamine. The cells were released into the cell cycle at 30°C. The cell cycle progression was monitored by flow cytometry. The cells with Pnmt41–rif1PP1bsmut entered the S-phase at 30 min (at 18.5 h in FACS chart) after release from M-phase arrest, similar to the rif1⁺ cells, whereas those with Pnmt41-Rif1 entered the S-phase later (>60 min after release). (A, B) The level of Rif1 in the samples from (A) was examined by Western blotting. (C) The cells harboring Rif1 (wt or rif1PP1bsmut)-expressing plasmid or vector, as indicated, were starved for thiamine for the time indicated. The whole cell extracts were prepared and were run on SDS–PAGE containing MBP (Myelin Basic Protein) in the gel. In-gel kinase assays were conducted as described in the “Materials and Methods section.” HU, treated with 2 mM HU for the time indicated as a positive control of Cds1 activation. (C, D) Quantification of the results in (C).

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(A) Chromosomes are visualized by hht2+ (histone H3 h3.2)-GFP (right) and the chromosome segregation was assessed in Rif1-overexpressing yeast cells (KYP1776). Cells with unequally segregated chromosomes (indicated by mazenta arrowheads) or entangled chromosomes (indicated by blue arrowheads) increased at 24 h after Rif1 overexpression (left). (B) Rif1 was overexpressed in cells expressing Rad52–EGFP by depletion of thiamine for 24, 48, and 72 h. Rad52–EGFP foci in the cells were observed under fluorescent microscopy (KYP1777, KYP1778, KYP1860, and KYP1861). The numbers of Rad52 foci (representing DNA damages) were counted, and cells containing 0, 1, 2, or >3 foci were quantified. The extent of DNA damages increased with the duration of Rif1 overexpression.

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(A) Rif1 was overexpressed in cells expressing GFP-α2tub and cells with short or long spindle microtubules were counted (KYP1779 and KYP1780). In the upper panels, the photos of cells with short mitotic spindles and those with long spindles are shown. (B) Rif1 was overexpressed in the spindle assembly checkpoint activation mutants, mad2∆ or bub1∆, and cells with spindle microtubules were counted (KYP1815, KYP1816, KYP1817, and KYP1818). (C) SAC is induced in Rif1-overproducing cells. Cells expressing Sad1–GFP (spindle pole body) and Cut2-GFP (securin) were monitored under a fluorescent microscope starting from the time when spindle pole bodies started to separate (t = 0). Mitotic spindles between the two SPB disappear and the nuclear Cut2 signal disappear in non-overproducing cells by 14 min (KYP1836), whereas those in Rif1-overexpressing cells stay as late as for 38 min (KYP1837). White arrowheads indicate Sad1. The drawing shows the nuclear signals of Cut2 (pale green) and two dots of Sad2 and connecting microtubules. The strong green signals indicating by * in Rif1wt OE samples represent a dead cell. (D) Spot tests of SAC mutant cells overexpressing Rif1 (KYP025, KYP1805, KYP1815, KYP1816, KYP1817, and KYP1818). (E) Fractions of cells with aberrant morphology (indicated by arrowheads) are scored in the WT, mad2∆ or bub1∆ cells overproducing the WT Rif1 (KYP025, KYP1805, KYP1815, KYP1816, KYP1817, and KYP1818). Cells with aberrant morphology include multi-septated cells, cells with misplaced septum, enlarged cells, septated dead cells, and cells with breached morphology. Left, phase contrast images of the cells; right, quantification of cells with aberrant morphology. Rif1 OE, Rif1 overexpression. In (A, B, E), cells were grown in a medium lacking thiamine for 18 h.

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(A) Rif1 mutants were overexpressed in the WT (KYP1781 and KYP1782) and hsk1-89 cells (KYP1783 and KYP1784), and spot tests were conducted. The R236H mutant binds to chromatin but the L848S mutant does not (Kobayashi et al, 2019). (B) KYP1268 (nda3–KM311, Rif1–His₆–Flag₁₀; blue) and MS733 (nda3–KM311, nmt1–Rif1–His₆–Flag₁₀; red) were cultured in PMG medium containing 15 µM thiamine. The cells were washed with fresh PMG medium without thiamine and grown at 30°C for 12 h. The cells were arrested at the M-phase by shifting to 19.5°C for 6 h, and then were released from the M-phase by addition of an equal volume of fresh PMG medium pre-warmed at 43°C. At 20 min after release, the cells were analyzed by ChIP-seq. Two known Rif1bs are indicated by arrowheads. (B, C) Motif Logo of the conserved sequence motif identified by MEME suites from the sequences of the Rif1-binding segments determined by ChIP-seq in (B), and distribution of motif position probability determined by STREME (provided from MEME suites) on the 300-bp sequences centered on the Rif1-binding summits at the 128 and 169 peaks of “Rif1 no OE” and “Rif1 OE,” respectively. (D) Binding of Rif1 to Rif1bs_I:2663kb, Rif1bs_II:4255kb, telomere-associated sequences (telomere of chromosome II) and ars2004 (non-Rif1bs) were measured in the WT cells harboring vector, pREP41–Rif1–Flag₃, or pREP41–rif1PP1bsmut–Flag₃ by ChIP-qRT-PCR. Cells were grown in the medium lacking thiamine for 18 h before harvest. The IP efficiency was normalized by the level of input DNA.

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Cells expressing GFP-fused Histone H3 (h3.2-GFP) were observed under a fluorescent microscope after induction of Rif1 protein for 10 h or 18 h, as indicated. (A, B) Overexpression of WT (KYP1776) or PP1bs mutant (KYP1842). (C, D) Overexpression of L848S (chromatin binding-deficient) (KYP1844) or R236H (chromatin binding-proficient) (KYP1843) mutant. (A, C) Phase contrast and fluorescent images of the cells are presented. (A, B, C, D) Fractions of the cells with chromatin relocated at the nuclear periphery (indicated by arrowheads in (A, C)) are calculated and presented.

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(A) Rif1–mKO2 cells (KYP1866 and KYP1867), in which the endogenous Rif1 was tagged with mKO2, harboring pREP41–Flag₃ vector (upper) or pREP41–Rif1–Flag₃ (lower) were grown in the absence of thiamine for 20 h, and were extracted by Triton X-100 and DNase I and remaining endogenous Rif1–mKO2 signals (mazenta) were observed. The nuclear envelope was stained with Nup98 antibody (green). (B, C) The numbers (B) and the intensities (C) of nuclear foci were quantified in Rif1–mKO2 cells harboring pREP41–Flag₃ (Vector) or pREP41–Rif1–Flag₃ (Rif1OE) grown as in (A).

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