Figure 3.

Sem1p and Ubp6p regulate histone H2B ubiquitination. (A) The sem1Δ ubp6Δ mutant shows increased level of histone H2B ubiquitination. (B) Deletion of UBP10 in the ubp6Δ background restores the level of ub-H2B back to that in the wild-type cells. A plasmid copy of HTA1-FLAG-HTB1 was introduced into cells with both HTA1-HTB1 and HTA2-HTB2 deleted from the genome. (C) Sem1p and Ubp6p are involved in the regulation of H2B ubiquitination at telomere. Whole cell extracts were prepared from 25-ml cultures in mid-log phase and 25 µg protein were analyzed by western blotting. To calculate the fold-change, the density of wild-type strain was designated as 1.0, which was reset from the value obtained by dividing the density of each band with the density of the corresponding input band (H2B). SeqChIP was performed with anti-FLAG M5 and anti-HA (Covance) antibodies. Relative IP represents the IP signal normalized to the respective input signal and then to the respective background signal obtained from the yeast strain with K123R mutation. Densities of protein and DNA bands were quantitated as described in ‘Materials and methods’ section. Standard deviations were calculated from three replicates. Genotypes of yeast strains are indicated on the top. NT, non-tagged.