Mediator promotes CENP-a incorporation at fission yeast centromeres

Abstract

At Schizosaccharomyces pombe centromeres, heterochromatin formation is required for de novo incorporation of the histone H3 variant CENP-A(Cnp1), which in turn directs kinetochore assembly and ultimately chromosome segregation during mitosis. Noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (Pol II) directs heterochromatin formation through not only the RNA interference (RNAi) machinery but also RNAi-independent RNA processing factors. Control of centromeric ncRNA transcription is therefore a key factor for proper centromere function. We here demonstrate that Mediator directs ncRNA transcription and regulates centromeric heterochromatin formation in fission yeast. Mediator colocalizes with Pol II at centromeres, and loss of the Mediator subunit Med20 causes a dramatic increase in pericentromeric transcription and desilencing of the core centromere. As a consequence, heterochromatin formation is impaired via both the RNAi-dependent and -independent pathways, resulting in loss of CENP-A(Cnp1) from the core centromere, a defect in kinetochore function, and a severe chromosome segregation defect. Interestingly, the increased centromeric transcription observed in med20Δ cells appears to directly block CENP-A(Cnp1) incorporation since inhibition of Pol II transcription can suppress the observed phenotypes. Our data thus identify Mediator as a crucial regulator of ncRNA transcription at fission yeast centromeres and add another crucial layer of regulation to centromere function.

Fig 1

Mediator components are required for chromosome segregation. (A) TBZ sensitivity test of Mediator mutants. Tenfold dilution series of the indicated strains were spotted on YEA plates with and without TBZ followed by incubation at 25°C. (B) H3K9me and Swi6 occupancy in the centromeric repeat regions (dh and imr) of chromosome I. (C) Images showing DNA content and spindle microtubules in anaphase cells of wt, med20Δ, dcr1Δ, and clr4Δ cells at 30°C. Immunofluorescent staining of tubulin, 4′,6′-diamidino-2-phenylindole (DAPI) staining, and merged images are shown as indicated. Scale bar, 5 μm. (D) Diagram showing the percentage of lagging chromosomes in anaphase cells of wt, med20Δ, dcr1Δ, and clr4Δ strains at 30°C (n = 100 to 125 per strain). (E) Rad21 occupancy in the core centromeric regions (cnt) of chromosome I and in a control locus (the fbp1 gene). (F) Northern blot showing siRNA levels using dh and dg probes. U6 RNA is used as a loading control. Relative siRNA fold changes are indicated below each lane. The locations of probes are indicated as black dots in Fig. 3C.

Fig 2

Mediator occupancy and transcription at centromeres during cell cycle progression. (A and B) Occupancy profiles for Pol II and Mediator (Med7-Myc) at centromere 1. ChIP-seq data are shown as reads per million (RPM). The centromere structure is shown aligned to the x axis. In panel A, Pol II occupancy in wt cells (red profile) are compared to that in a med20Δ mutant strain (black profile). (C and D) ChIP analyses of Med7-myc (C) and Pol II (D) occupancy in the otr (dh) region. ChIP ratios were normalized to no-antibody controls. (E) Septation index for the experiments shown in panels C and D. Peak indicating S phase. (F and G) Quantification of transcript levels by qPCR from the dh and imr regions in synchronized cells. Samples were normalized to act1⁺. Rel, relative. (H) Septation index for the experiments shown in panels F and G. Error bars indicate standard deviations from at least three independent experiments.

Fig 3

Loss of Med20 causes a defect in centromeric silencing. (A) Tenfold dilutions of the indicated strains were spotted on YEA plates, with and without 5-FOA. The locations of the introduced ura4⁺ markers are indicated after the strain names. (B) RT-PCR showing transcript levels of the ura4⁺ marker and a mutated version of ura4⁺ (ura4-DSE) transcribed from the endogenous locus. Reactions performed without reverse transcriptase (−RT) are shown as controls. (C) A Venn diagram showing how genes involved in centromere function and chromosome segregation overlap genome-wide transcription changes (cutoff, 1.5-fold) in the med20Δ strain. (D) RT-PCR analysis of the ura4⁺ marker located in otr, imr, and cnt regions. cdc20⁺ and sir2⁺ are expressed from a pREP81X vector. Minus (−) indicates without induction and plus (+) indicates with gene induction. The empty vector is shown as negative control. (E) Tiling array analysis shows transcription changes in med20Δ cells normalized to the wt level and med20Δ dcr1Δ cells normalized to the dcr1Δ strain level in the chromosome I centromere region. A schematic figure of the centromere is shown aligned to the x axis. Black bars represent previously identified siRNA regions (10). Dots represent siRNA probe locations for the Northern blot analyses shown in Fig. 1F. (F) Quantification by qPCR of dh and imr transcripts. Samples are normalized to act1⁺ levels. Bars show standard deviations of at least three independent cultures. (G) RT-PCR showing transcript levels in the centromere regions (dh, imr, and cnt) in the wt, med20Δ, rrp6Δ, and med20Δ rrp6Δ strains. Actin and −RT are shown as controls.

Fig 4

Deletion of med20⁺ leads to loss of CENP-A^Cnp1. (A) Localization of CENP-A^Cnp1 fused to CFP in wt, med20Δ, and dcr1Δ cells. Scale bar, 5 μm. (B) ChIP analysis of CENP-A^Cnp1 occupancy over the cnt region. Bars show standard deviations. (C) The CENP-A^Cnp1 protein was detected by Western blotting using a GFP antibody. Tubulin was used as the loading control. (D) Quantification by qPCR of CENP-A^Cnp1 transcript levels. Bars indicate standard deviations. (E) Diagram showing the effect of actinomycin D on the percentage of cells with an identifiable CENP-A^Cnp1 signal in the indicated strains (n > 160 per strain). CENP-A^Cnp1 localization was blindly scored, and the statistical significance (**, P < 0.01; ***, P < 0.001) was calculated as described in Materials and Methods.

Fig 5

A suggested model for the function of Mediator at centromeres. The Mediator complex binds to the core centromere region and regulates Pol II binding and activity. Med20 represses Pol II binding to the cnt region, which promotes incorporation of CENP-A^Cnp1 at the core centromere, forming a silenced chromatin structure. Disruption of med20⁺ (indicated in yellow) causes unregulated binding of Pol II to the centromere. The continued transcription impairs the incorporation of CENP-A^Cnp1, which leads to a dysfunctional kinetochore and chromosome segregation defects.