(A) Barplots indicating gene expression ratios of mutant/wt in log2 format for 106 genes within 20kb of the chromosome ends. The genes are ordered left to right in terms of increasing distance from the end of the chromosome. (B) Heatmap of gene expression within 20 kb of the end of the 16 yeast chromosomes in swi4Δ, ard1Δ, and dot1Δ strains, which are specifically defective in di- and trimethylation of H3K79 respectively, are compared to a wild-type strain. Gene expression ratios are indicated by colored rectangles with red indicating a higher expression in the mutant, and green indicating a higher expression in the wild-type cells. Gray indicates equal expression in both strains. The color scale is plus or minus 3-fold. The numbered rectangles indicate the ends of each yeast chromosome. The genes within 20 kb of the end of the left arm of each chromosome are indicated to the left of the gray line in each rectangle, whereas genes within 20 kb of the end of the right arm of the chromosome are indicated to the right of the gray line. “L” and “R” indicate the left and right arm of each chromosome.

(A) The log2 ratio of gene expression for mutant/wt is shown for genes within 5kb of chromosomal ends. The dashed grey line indicates two-fold enrichment in the mutant over wt. (B) Table of gene expression values. The log2 ratio of gene expression for mutant/wt is shown for genes within 5kb of chromosomal ends for the mutants indicated in the table, along with the chromosomal position of the gene, the gene midpoint, and the distance between the midpoint and the end of the chromosome. (C) H3K79 methylation status in cells expressing dot1-1, dot1-2, and dot1-3 alleles were examined by Western blotting using anti-H3K79 mono-, di- and trimethylation specific antibodies. Dot1 alleles were cloned in pRS315 vector as described under Materials and Methods. Dot1 (G401R) is a catalytically dead mutant (van Leeuwen et al., 2002). dot1-3 exhibits slightly less accumulation of H3K79 trimethylation, but comparable levels of dimethylation than wild-type; while dot1-2 and -1 lost most of their H3K79 tri- and dimethylation with an observed slight loss in the H3K79 monomethylation signal in dot1-1. (C) Gene expression analysis using dot1-1, -2, and -3 strains. The ratio of gene expression in log2 format for various Dot1 mutants relative to wt is shown for four genes within 5 kb of the chromosomal ends.

A) 20 kb of chromosome ends I to III were plotted along the x axis against the relative occupancy (R/O) of Sir2, H3K79 di- and trimethylation in WT cells. Superimposed ChIP-on-chip profiles indicated the distinct distribution of Sir2 and H3K79 methylation. Each chromosome end was unique for its Sir2, H3K79me2 and H3K79me3 occupancy. B) ORFs bound by Sir2 separated from H3K79 di- and trimethylated ORFs. Heatmap visualizing the average enrichment of Sir2 in WT and dot1Δ cells as well as H3K79me2 and H3K79me3 for all known yeast ORFs (left panel) and for all ORFs within 20kB from the chromosome ends (right panel). Each row color-codes the average enrichment score for a particular ORF on the spectrum from red indicating enrichment to blue for depletion. The distribution patterns were hierarchically clustered and plotted.

A) The right arms of chromosome III and XIVR were plotted along the x axis against the relative occupancy (R/O) of Sir3 in a wild-type and dot1 deletion strain. Increased Sir3 occupancy coincided with a lower expression of AAD3 and COS10. B) The right arm of chromosome XIII was plotted along the x axis against the relative occupancy of Sir2 in a wild-type and dot1 deletion strain. Decreased Sir2 occupancy coincided with a higher expression of YMR323W. C) The left arms of chromosome VII and chromosome I were plotted along the x axis against the relative occupancy of Sir2 in a wild-type and dot1 deletion strain as well as H3K79 di- and trimethylation. Only minor changes in Sir2 occupancy were observed at the subtelomere of ChrVIIL and ChrIL in the absence of H3K79 methylation. The superimposed profiles with H3K79me2 and H3K79me3 indicate that COS12 and YAL067C were moderately enriched for H3K79 methylation and not Sir2.

Wild-type, dot1Δ, sir3Δ, or H3K79A strains were plated on YPD and tested for growth inhibition in the presence of the indicated concentration of alpha factor in a halo assay test. Both sir3Δ and H3K79A mutants were resistant to alpha factor arrest. However, strains lacking H3K79 methylation due to deletion of DOT1 are inhibited to a similar extent as the wild-type strain. This indicates that H3K79 methylation in itself is not required for proper HML silencing.