PMC

The hrp1Δ mutant alleviates silencing at the mating-type region, outer repeats and central core of the centromere. Cell suspensions of wt and hrp1Δ strains were 5-fold serially diluted and spotted on selective (−ura or −ade) or counter-selective plates (FOA) as indicated. The plates were incubated at 30°C for 3 days. (A) Silencing in the mating-type region (mat3-M::ade6⁺) was assayed in the Hu15 (wt) and Hu33 (hrp1Δ) strains. (B) Silencing at the telomeres (tel::ade6⁺) was assayed in the FY375 (wt) and Hu26 (hrp1Δ) strains. (C) Silencing at the rDNA (rDNA::ura4⁺) was assayed in the Hu393 (wt) and Hu466 (hrp1Δ) strains. (D) Left: schematic diagram of the otr1R::ade6⁺ insertion in centromere 1; right: silencing at the dg-dh repeats (otr1R::ade6⁺) was assayed in the FY1180 (wt) and Hu22 (hrp1Δ) strains. (E) Top: schematic diagram of the cen2::ura4⁺ insertion in centromere 2; bottom: silencing at the central core of the centromere (cen2::ura4⁺) was assayed in the FY412 (wt) and Hu584 (hrp1Δ) strains.

Hrp1 directly binds to the centromere in a periodic, cell cycle-dependent manner. (A) An EtBr-stained gel with PCR products from ChIP with Hrp1-HA. The α-HA antibody was used for precipitating epitope-tagged Hrp1-HA in an asynchronous cell culture. cnt1, dg1 and tRNA synthetase (control) primers were used in the duplex PCR for quantification of Hrp1-HA binding. The strain was Hu615. (B) Left: an IF image with Hrp1-myc (red), combined with FISH using pRS140 centromere probe (green). Overlapping signals, indicating co-localization between Hrp1-myc and the cen-FISH signal, is visualized by a yellow signal. Right: a bar diagram showing the percentages of mononucleated and binucleated cells, in which Hrp1-myc signals co-localized with the cen-FISH signal. Size bar = 5 µm. The strain was Hu764. (C) An EtBr-stained gel of PCR quantified products from ChIP of synchronized cells. Half of the cells were collected immediately after HU arrest, the other half were washed and transferred to fresh media without HU and grown 1 h before harvest, and both cell batches were subjected to ChIP. The strain was Hu615 (Hrp1-HA).

The hrp1Δ and hrp3Δ mutants are TBZ and TSA sensitive and display different chromosome segregation defects. (A) A spotting assay showing TBZ sensitivity of the hrp1Δ and hrp3Δ single and double mutants. Cells were 5-fold serially diluted and spotted on YEA plates with and without 15 μg/ml TBZ. The plates were incubated at 30°C for 3 days. Strains were FY1180 (wt) Hu61 (hrp1Δ), Hu602 (hrp3Δ) and Hu603 (hrp1Δ hrp3Δ). (B) Growth curves showing TSA sensitivity of wt (blue line), hrp1Δ (green line), hrp3Δ (black line) and hrp1Δ hrp3Δ double mutants (red line). Log phase cells grown in YEA were inoculated to a final concentration of 5 × 10⁵ cells/ml in YES media without TSA (left panel) and with media containing 200 nM TSA (right panel). Growth was monitored measuring the optical density at 595 nm (OD₅₉₅) for 64 h. Strains were Fy367 (wt) Hu61 (hrp1Δ), Hu602 (hrp3Δ) and Hu603 (hrp1Δ hrp3Δ). (C) Left, IF micrographs showing a representative cells in anaphase. The wt cell shows normal segregation and the hrp1Δ cell shows a lagging chromosome (arrow). Right: a bar diagram showing the percentages of wt and hrp mutant cells that displayed lagging chromosomes. Cells were grown at 18°C without TSA (black bars) and at 30°C in media containing 100 nM TSA (grey bars) prior to fixation. Strains were Fy367 (wt) Hu61 (hrp1Δ), Hu602 (hrp3Δ) and Hu603 (hrp1Δ hrp3Δ). (D) The hrp1Δ mutant and the hrp1Δ hrp3Δ double mutant display asymmetric segregation of nuclei. Left, an IF micrograph of an hrp1Δ cell with asymmetric nuclei. Right: a bar diagram that shows the percentages of wt and hrp mutant cells that display asymmetric segregation of nuclei. (C and D) The TAT1 antibody (red) was used for tubulin staining and DAPI (green) for staining DNA. Size bar = 5 µm.

The hrp1Δ mutant has elevated histone H4 acetylation levels, reduced levels of Cnp1 (SpCENP-A) in chromatin of the central core region, and it shows synthetic growth defects with mis6-302. (A) Quantitative PCR (³²P images) analyses of ChIP samples. For each experiment, the normalized competitive PCR input ratios (IN), the mean ratio values and the SD is indicated below the lanes. The experiments were performed in duplicates or triplicates and both results were statistically significant (P < 0.05, independent sample t-test). α-H4K8ac and α-H4K12ac antibodies were used to measure H4K8 and H4K12 acetylation levels at cnt1 in wt and hrp1Δ cells with the glutamyl tRNA synthetase gene as a control. Strains were FY412 (wt) and Hu584 (hrp1Δ). (B) Quantitative PCR of ChIP samples. α-Cnp1 and α-H3 antibodies were used to determine Cnp1 and H3 levels, respectively, at cnt1 in wt and hrp1Δ cells. Strains were FY412 (wt) and Hu61 (hrp1Δ). (C) A spotting assay showing hrp1 genetically interacts with other genes important for Cnp1 loading. hrp1Δ, mis6-302, ams2Δ single and double mutant cells were serially diluted, spotted onto YES plates and incubated for 3 days at 25 or 30°C as indicated. Strains were Hu721 (mis6-302), Sp45 (ams2Δ), Hu465 (hrp1Δ), Hu1181 (hrp1Δ mis6-302) and Hu1176 (hrp1Δ ams2Δ). (D) Central core structure is not altered in the hrp1Δ mutant. MNase assay of wt and hrp1Δ chromatin extracts. Top: ethidium bromide (EtBr) stained gel with partially digested chromatin samples 0, 25, 50, 100, 200, 300 and 500 U MNase. Bottom: a Southern blot of the same gel hybridized with a [³²P]labeled cnt2 probe. (E) Picture of RT–PCR products from cDNA samples to investigate a putative termination defect of hrp1Δ in dg-dh transcription. Top: schematic diagram of centromere 3. The position of the dhIII and imrIII primers used RT–PCR are indicated. Bottom: dhIII and imrIII RT–PCR analysis of dcr1Δ, hrp1Δ and hrp1Δ dcr1Δ mutant cells (as indicated). Primers for the tRNA synthetase gene were used as a control. Strains were Fy367 (wt), Hu61 (hrp1Δ), Hu659 (dcr1Δ) and Hu1121 (hrp1Δ dcr1Δ).