Figure 6.

Effect of htb1 mutations on silencing of the inserted ura4⁺ marker gene and histone modification patterns in the centromere region. The ura4⁺ insertion sites within cen1: otr1R∷ura4⁺ and cnt1∷ura4⁺ are indicated by the short bar in the top panel of Figure 5B. (A) Cells were serially diluted (1:5) and spotted onto the YPD plates: non-selective (N/S) or containing 5-FOA. They were incubated at 30°C for 3 days, or they were spotted onto the EMM2 plates: nonselective (N/S), lacking uracil (−URA) or containing 5-FOA. The highest-density spots contained 1 × 10⁴ cells. (B) Lagging chromosomes observed in anaphase of htb1-72 cells at 18°C. (C) Histone acetylation and methylation patterns in the centromere region were altered in htb1-223 mutant. The levels of histone H3 and H4 acetylation and methylation in the centromeric regions were examined by CHIP using antibodies against acetylated H3 and H4. Anti-AcH4 is against acetylated K5, K8, K12, K16 in H4, whereas anti-AcH3 is against acetylated K9 and K14 in H3. Antibodies against dimethylated K4 in H3 were also used. The centromere probes used were central cnt1, although pericentric probe lys1 was also used. Indicated strains were cultured at the restrictive temperature (37°C) for 0 or 8 h. (D) The levels of histone H2B and (di- or tri-) methylated histone H3 (Lys4) in htb1 mutant cells were examined by immunoblotting. The levels of Lys4 (di- or tri-) methylated histone H3 were diminished in htb1 mutants, particularly in htb1-223 cells.