Genes exhibiting aberrant expression in cell lacking CHZ1 map to distinct chromosome regions. Chromosome position analysis was done on genes with significant differential expression in Δchz1 that were repressed (A) or induced (B) more than 2-fold. For each gene, the distance to the nearest telomere was determined and these distances were grouped into 5-kb bins and plotted as a function of telomeric distance. The shaded histograms indicate the distribution of telomeric distances for all ORFs plotted at one-third scale on the y-axis.

Deletion of Chz1p leads to increased binding of Sir3p and Sir4p to subtelomeric chromatin. The association of Sir2p (A), Sir3p (B), Sir4p (C) and Rap1p (D) with subtelomeric chromatin regions was determined by ChIP using anti-myc antibodies, followed by qPCR. The relative enrichment ratio for each is plotted. ACT1 was used as an internal control to normalize signals of enrichment. Error bars show the standard deviation from three independent biological replicates with two technical replicates of each.

Deletion of CHZ1 leads to enhanced association of Ubp10p near telomeric regions. (A) Double chromatin immunoprecipitation (ChDIP) of ubH2B. Formaldehyde cross-linked chromatin was obtained from wild-type, Δchz1 and htb1-KR mutant cells bearing Flag-tagged H2B and HA-tagged ubiquitin. Relative enrichment values (y axes) are the averages of the results from three independent ChIPs with qPCR determination performed twice per biological replicate. Non-promoter IGRi YMR325W was used as an internal control to normalize signals of enrichment. (B) ChIP assays of Ubp10p were performed using anti-GFP antibody specific for GFP tag fused to the C terminal of Ubp10p. All values are the averages of the at least three independent experiments. Errors bars represent the standard deviation. (C) Chz1p negatively regulates histone H2B ubiquitination. Global steady-state H2B ubiquitination levels from whole-cell lysates were assayed using anti-FLAG antibodies that recognize the FLAG epitope placed on the N terminus of H2B expressed from a plasmid and replacing the native HTB1 gene. Δubp10 and htb1-KR serve as positive and negative control strains for H2B ubiquitination, respectively. The loading equivalents (one and one-fourth) are indicated above each lane.

Comparison of genes with altered transcriptional profiles in Δchz1, Δubp10 and Δchz1, Δubp10 mutants. Venn diagram depicting the numbers of genes affected in their transcriptional levels (downregulated ≥ 2-fold) within 50 kb from telomeres as detected by microarray in Δchz1, Δubp10 and Δchz1, Δubp10 mutant strains.

Chz1p is not involved in Htz1p incorporation near telomeres. Anti-HA antibodies were used to pull down HA-tagged Htz1p to determine its enrichment by ChIP assays. Each primer set is ∼2.5 kb apart, interspersed along the 20-kb region from the right telomere of chromosome VI. Relative enrichment values (y axes) are the averages of the results from three independent ChIPs with qPCR performed twice per biological replicate. Non-promoter IGRi YMR325W was used as an internal control to normalize signals of enrichment.

Di-methylation of H3K79 near telomeres is dramatically reduced in the absence of Chz1p. ChIP was performed on wild-type and Δchz1 mutant strains. Formaldehyde cross-linked protein-DNA complexes were immunoprecipitated by using antibodies against individual di-methylated H3 lysine residue 4 (A) and 79 (B). Relative enrichment values (y axes) are the averages of the results from three independent ChIPs with qPCR determination performed twice per biological replicate. Non-promoter IGRi YMR325W was used as an internal control to normalize signals of enrichment.

Genetic interaction between CHZ1 and factors involved in H2B ubiquitination. Five-fold serial dilutions of each indicated haploid strain were spotted on complete medium containing 2% glucose for 3 days at 25°C, and for 2 days at 30, 33 and 37°C and photographed.