Centromeric localization of dispersed Pol III genes in fission yeast

Abstract

The eukaryotic genome is a complex three-dimensional entity residing in the nucleus. We present evidence that Pol III-transcribed genes such as tRNA and 5S rRNA genes can localize to centromeres and contribute to a global genome organization. Furthermore, we find that ectopic insertion of Pol III genes into a non-Pol III gene locus results in the centromeric localization of the locus. We show that the centromeric localization of Pol III genes is mediated by condensin, which interacts with the Pol III transcription machinery, and that transcription levels of the Pol III genes are negatively correlated with the centromeric localization of Pol III genes. This centromeric localization of Pol III genes initially observed in interphase becomes prominent during mitosis, when chromosomes are condensed. Remarkably, defective mitotic chromosome condensation by a condensin mutation, cut3-477, which reduces the centromeric localization of Pol III genes, is suppressed by a mutation in the sfc3 gene encoding the Pol III transcription factor TFIIIC subunit, sfc3-1. The sfc3-1 mutation promotes the centromeric localization of Pol III genes. Our study suggests there are functional links between the process of the centromeric localization of dispersed Pol III genes, their transcription, and the assembly of condensed mitotic chromosomes.

Figure 1.

Localization of Pol III factors within the nucleus. (A) Deconvolved immunofluorescent images of cells stained for Sfc6-Myc (TFIIIC component), Brf1-Myc (TFIIIB), Rpc25-Myc (Pol III), and Sfc2-Myc (TFIIIA) proteins (red) were merged with DAPI signals (blue). Arrowheads indicate focal localization of Brf1 and Rpc25 proteins. (B) Localization of Brf1 and Rpc25 near the nucleolus. Schematic representation of fission yeast nucleus is shown on top. Localizations of Brf1-Myc and Rpc25-Myc (red) are shown in the middle and bottom panels, respectively. Localization of Nop1, a fibrillarin protein, is shown as nucleolar marker (green). Nucleoplasm was visualized by DAPI staining (blue). The percentage with which Brf1 or Rpc25 foci associate (left) and do not associate (right) with the nucleolus is noted in each image. (C) Colocalization of Pol III factors. Pairs of Pol III factors, selected from Sfc6, Brf1, Rpc25, and Sfc2 carrying either Myc (green) or Flag (red) epitope, were immunostained and merged with DAPI signals (blue). Arrowheads indicate the positions at which foci derived from two different Pol III factors overlap in the percentage of cells noted in each image. (D) Binding of Pol III factors to distant genomic loci. ChIP was performed using strains expressing Sfc6-TAP, Brf1-TAP, and Rpc25-TAP. DNA isolated from ChIP or whole-cell extract (WCE) fractions was subjected to multiplex PCR to amplify DNA fragments from COC loci (including the IR boundary at the mating-type region) and tRNA^phe genes (top bands), as well as an act1 fragment (bottom bands) as an internal amplification control. The ratios of top and bottom bands present in WCE were used to calculate the relative enrichment of precipitated samples. ChIP experiments were repeated at least twice for each factor, and representative results are shown.

Figure 2.

The centromeric localization of Pol III factors and genes. (A) Sfc6 (TFIIIC), Brf1 (TFIIIB), and Rpc25 (Pol III) foci colocalizing with heterochromatin protein Swi6. Immunofluorescent images of cells stained for Sfc6-Myc, Brf1-Myc, and Rpc25-Myc (red) were merged with Swi6 localization (green) and DAPI signals (blue). Arrowheads indicate the positions at which the foci derived from Sfc6, Brf1, or Rpc25 overlap with Swi6 spots in the percentage of cells noted in the merged image. (B) Focal localization of Sfc6, Brf1, and Rpc25 at centromeres. Immunofluorescent images of cells stained for Sfc6-Myc, Brf1-Myc, and Rpc25-Myc (green) were merged with FISH signals visualizing centromeres (red) and DAPI signals (blue). Arrowheads indicate the positions at which the foci derived from Sfc6, Brf1, or Rpc25 localize at centromeres in the percentage of cells noted in the merged image. (C) tRNA and 5S rRNA genes clustering at centromeres. Three tRNA gene families (tRNA^ala, tRNA^gly, and tRNA^pro) and 5S rRNA genes were separately visualized (green) and merged with centromeres (red) and DAPI signals (blue). The 7 tRNA^ala genes of a total of 12 members are encoded at centromeric regions, whereas only 2 tRNA^gly genes of 12 members and 0 tRNA^pro gene of 9 members are encoded at centromeres. Arrowheads show the positions at which the FISH spots indicating either tRNA or 5S rRNA genes overlap with centromeres in the percentage of cells noted in the merged image. (D) Frequent localization of genomic locus containing tRNA and 5S rRNA genes in the vicinity of centromeres. The two genomic loci (green) were visualized by FISH using the cosmid clones (c417 and c162), and merged with centromeres (red) and DAPI signals (blue). The chromosomal positions of the cosmids are shown in Supplemental Figure S1. Several typical images are shown on top. In each image, measurement of the distance between the genomic locus and centromeres is indicated by linking the image with the histogram. The distance was measured between two focal centers. A histogram of distributions of observed distances between the genomic loci and centromeres is shown at the bottom. The percent populations of the observed distances between the genomic loci and centromeres were binned into 0.1 μm. (E) The insertion of Pol III genes into the non-Pol III gene locus results in frequent localization of the locus near centromeres. The non-Pol III gene locus (green) was visualized by FISH using the cosmid clone (c162) and merged with centromeres (red) and DAPI signals (blue). Schematic representations of the c162 locus with (left) and without (right) the insertion of the Pol III genes are shown on top. The Pol III genes (tRNA^asn and 5S rRNA) are derived from the c417 locus (Supplemental Figure S4A). The microscopic images typified intranuclear positioning of the c162 locus and centromeres. The plotting in the histogram was carried out as described in D.

Figure 3.

Condensin, but not cohesin, mutations disrupt the centromeric localization of Pol III genes. (A) The centromeric localization of Pol III genes was defective in the condensin mutants. The Pol III gene locus (green) was visualized using a FISH probe specific to the cosmid clones (c417) and merged with centromeres (red) and DAPI signals (blue). The cut3-477, cut14-208, and wild-type (wt) cells grown at 26°C were subsequently cultured at the restricted temperature (36°C) for 2 h for cut3-477 and wt cells and 1 h for cut14-208 cells and then subjected to FISH analysis. Most cells used for FISH analysis were in interphase. The microscopic images on top typified intranuclear positioning of the c417 locus and centromeres in the respective strains. The quantitative measurements of distances between the c417 locus and centromeres, and their plotting in the histogram, were carried out as described in Figure 2D. (B) Positioning of the non-Pol III gene locus relative to centromeres was not affected by the condensin mutations. The non-Pol III gene locus (green) was visualized using a FISH probe specific to the cosmid clones (c162) and merged with centromeres (red) and DAPI signals (blue). The experiment was performed as described in A. (C) The centromeric localization of the Pol III gene locus was not visibly impaired in the cohesin mutant. The wt and cohesin mutant (rad21-K1) cells grown at 26°C were subsequently cultured at the restricted temperature (36°C) for 4 h and subjected to FISH analysis. The cohesin mutant typically contains two foci for the c417 locus (green) and centromeres (red), respectively (top).

Figure 4.

Association of Cut3-Myc with Rpc25-Flag. Top, extracts prepared from strains expressing tagged proteins were incubated with anti-Flag antibody, and immunoprecipitated (IP) fractions were analyzed by Western blotting using anti-Myc and anti-Flag antibodies. Bottom, the control experiment showing the interaction between Brf1-Myc (TFIIIB) and Rpc25-Flag (Pol III). IP experiments were repeated at least twice, and representative results are shown.

Figure 5.

The centromeric localization of Pol III genes is linked to mitotic chromosome condensation. (A) The temperature sensitivity phenotype of the condensin mutant, cut3-477, is suppressed by the sfc3-1 mutation. Logarithmically growing cells (OD ∼ 0.5) in YEA liquid medium at 27°C were serially diluted by 10-fold and spotted onto nonselective YEA plates that were incubated at indicated temperatures for 2–3 d. (B) The sfc3-1 mutation promotes the centromeric localization of Pol III genes. Wild-type (wt), cut3-477, cut3-477 sfc3-1, and sfc3-1 cells grown at 26°C were subsequently cultured at the restricted temperature (36°C) for 2 h and then subjected to FISH analysis. Most cells used for FISH analysis were in interphase. The Pol III gene locus was visualized using a FISH probe specific to the cosmid clone (c417), whose signal was merged with that of centromeres. The quantitative measurements of distances between the c417 locus and centromeres, and their plotting in the histogram, were carried out as described in Figure 2D. (C) The ϕ-shaped chromosomes phenotype of the cut3-477 mutant is suppressed by the sfc3-1 mutation. The indicated strains were cultured at 36°C for 2 h. Immunofluorescent images of cells stained for tubulin (red) were merged with DAPI signals (blue). The percentage of the ϕ-shaped chromosomes phenotype in each strain is shown on the right. More than 300 cells were counted for each strain. (D) The sfc3-1 mutation facilitates mitotic chromosome condensation. The indicated strains were cultured at 36°C for 2 h. The Pol III genes locus (c417) and non-Pol III gene locus (c162) visualized by FISH were merged with IF images of cells stained for tubulin, and distances between the two loci were measured in interphase cells (n > 100) and in mitotic cells with short spindles (n > 20). (E) The centromeric localization of the Pol III gene locus becomes prominent during prometaphase compared with interphase. For cell-cycle synchronization, exponentially growing cells were arrested in S phase by culturing in YEA medium containing 11 mM hydroxyurea (HU) at 30°C for 4 h, released by further culturing without HU for 1.5 h, and then subjected to IF-FISH experiments. Immunofluorescent images of cells stained for tubulin (cyan) were merged with FISH signals visualizing the c417 locus (green) and centromeres (red). During prometaphase, a few centromeric signals attached with short spindles were observed. Arrowheads indicate the positions at which foci derived from centromeres completely overlap with the c417 locus. Measurements of the distance between the c417 locus and centromeres in the respective images were indicated by linking the image with the histogram.

Figure 6.

Binding of condensin and Pol III factors to the Pol III gene region in the sfc3-1 mutant. (A) The sfc3-1 mutation results in the increased level of Cut3 (condensin) binding to the Pol III gene region. The wt and sfc3-1 cells were cultured at 36°C for 2 h and subjected to ChIP analysis. Cut3-Myc levels at the Pol III gene region (c417) and the cnt1 region of centromere 1 were determined by ChIP. Quantitative measurements of ChIP results were carried out as described in Figure 1D. ChIP analyses were repeated three times. The difference in the ChIP relative enrichment levels between the wt and sfc3-1 mutant is significant; **p < 0.05, two-tailed t test. (B) Binding of Sfc6 (TFIIIC) and Rpc25 (Pol III) to the Pol III gene region (c417) in the sfc3-1 mutant. ChIP was performed using strains expressing Sfc6-Myc and Rpc25-Myc in the wt and sfc3-1 mutant.

Figure 7.

Transcription levels of Pol III genes are negatively correlated with the centromeric localization of Pol III genes. (A) Pol III transcription is affected by the cut3-477 and sfc3-1 mutations. Transcription of tRNA gene is monitored by the assay as described (Huang et al., 2005). The typical colony phenotypes (top) and percent frequencies of white, pink, and red colonies derived from the same genotypes after genetic cross (bottom) are shown. (B) Pol III transcription is decreased and increased in the sfc3-1 and cut3-477 mutants, respectively. The sfc3-1, cut3-477 sfc3-1 mutants and their control wt cells were cultured at 36°C for 10 h. The cut3-477 mutant and its control wt cells were cultured at 36°C for 2 h. Harvested cells were lysed to extract nucleic acids containing total RNA and genomic DNA. These mutations might affect the total RNA amount in a cell. Thus, RNA samples were first normalized based on the genomic DNA concentration in nucleic acid fractions before being subjected to RT-PCR analysis. Genomic DNA concentration in the nucleic acid fraction was quantified by PCR and normalized against act1 gene. Normalized nucleic acid fractions were treated with RNase-free DNaseI, and subjected to RT-PCR. The transcript levels from six distinct tRNA genes in the mutants and wt cells were measured, and the relative transcript levels (mutants vs. wt) are shown. The RNA preparation and RT-PCR were repeated three times, and average transcript levels are shown. The tRNA gene for ser+met indicates that these two tRNA genes are known to be transcribed as a dimeric transcript (Johnson et al., 1989). (C) Treatment with a Pol III inhibitor reduces transcript levels from tRNA genes in fission yeast. The Pol III inhibitor treatment was performed as described previously (Wu et al., 2003). The cells were cultured in YEA liquid medium containing 0, 50, and 200 μM of the Pol III inhibitor (ML-60218) for 4 h. RNA samples were prepared and subjected to RT-PCR analysis. The transcript levels from four tRNA genes and act1 gene were measured. The tRNA levels were normalized against the expression levels from act1 gene. The relative transcript levels of tRNAs are shown below each lane. (D) Treatment with the Pol III inhibitor promotes the centromeric localization of Pol III genes. Cells were cultured in YEA liquid medium containing 0, 50, and 200 μM of the Pol III inhibitor (ML-60218) for 4 h and then subjected to FISH analysis. The Pol III gene locus was visualized by FISH using the cosmid clone (c417) and merged with the centromeric signal. The quantitative measurements of distances between the c417 locus and centromeres, and their plotting in the histogram, were carried out as described in Figure 2D.