Suppression of heat-sensitive phenotype of htbK123R cells. (A) Dilutions (1:9) of yeast strains WT (FGY8.2), htbK123R (FGY8.3), or Δset1 (FGY8.6) were spotted onto either YPAD or YPAD containing 1 M sorbitol and grown for 3 d at either 30 or 37°C. (B) Spotting assay was performed on either WT yeast (FGY8.2) or on htbK123R cells (FGY8.3) that were untransformed, carried an empty pVV215 vector (vector), or carried a pVV215-based vector expressing the indicated open reading frames (ORFs). Wild-type HTB1 and HTB2 are not included here. (C) Reverse-transcriptase quantitative PCR was used to measure transcript levels from the normally silent YFR057W locus in the same yeast strains described in B. YFR057W cDNA levels were normalized to those of ACT1. Error bars show standard errors from three independent experiments.

Comparative transcriptomics reveals a set of euchromatic genes suppressed by Sir4 in the absence of H2B ubiquitylation. RNA sequencing was performed on yeast strains WT (FGY8.2), htbK123R (FGY8.3), Δsir4 (FGY8.10), and htbK123R Δsir4 (FGY8.11) and transcript levels expressed as RPKM values. (A) Venn diagram, comparing the 1361 genes differentially expressed between WT and htbK123R cells, and the 1347 genes differentially expressed between htbK123R and htbK123R Δsir4 cells. A total 576 of these genes overlap. (B). Scatterplot comparing the relative expression of the 576 genes highlighted in A. Category 4 genes (top left) are those whose expression is decreased in htbK123R cells but increased upon subsequent deletion of SIR4. (C) Histogram of the fraction of category 4 genes as a function of their distance to the nearest telomere. The red line shows the cumulative fraction of category 4 genes as a function from distance to the nearest telomere. The green line indicates that 25% of category 4 genes lie within 100 kb of a telomere.

Model for ectopic silencing of euchromatic genes in the absence of H2B–H3 cross-talk. In WT cells, H3K4 methylation at subtelomeric and genome-wide euchromatin keeps Sir proteins localized to telomeric heterochromatin and prevents them from associating from transcribed genes. In the absence of H3K4 methylation (as occurs in htbK123R and Δset1 cells), Sir proteins dissociate from the telomeres, leading to activation of subtelomeric genes (e.g., gene A) and their ectopic association with select euchromatic genes (e.g., gene D), silencing their expression. In this model, gene D would correspond to a category 4 gene.

Novel phenotypes associated with loss of H2B–H3 cross-talk. (A) PCR was used to determine VI-R telomere length in WT (FGY8.2), htbK123R (FGY8.3), Δrad6 (FGY8.4), Δbre1 (FGY8.5), Δset1 (FGY8.6), Δset1 Δdot1 (FGY8.7), Δdot1 (FGY8.8), hht2K4R (FGY8.17), htbK123R Δsir4 (FGY8.11), and Δsir4 (FGY8.10) cells. Note that the congenic WT strain for hht2K4R (FGY8.18) is different from FGY8.2, which is congenic with all other strains. Representative image of three independent experiments. (B) Fluorescence microscopy analysis, comparing localization of telomere-proximal GFP-LacI (green) vs. Nsp1 (red). Left, representative image of cells, showing those that were scored as GFP-LacI being at the nuclear periphery or the nucleoplasm. Right, quantification of GFP-LacI signals localized to the nuclear periphery for each of the three telomeres in three yeast backgrounds WT (ICY119, ICY123, ICY127), htbK123R (ICY120, ICY124, ICY128), and Δset1 (ICY131, ICY132, ICY133). Error bars indicate standard errors from at least two independent experiments. (C) Fluorescence microscopy analysis, comparing localization of LacO sites integrated proximal to the GAL1 locus in WT (ICY74) or Δrad6 (ICY31) cells. Left, representative image of cells, showing those that were scored as GFP-LacI being at the nuclear periphery or the nucleoplasm. Right, quantification of GFP-LacI signals localized to the nuclear periphery in media with either glucose (off) or galactose (on) as the carbon source. Error bars indicate standard errors from at least two biological replicates. (D) ChIP analysis, examining association of NUPs with sequences proximal to the VI-R telomere. ChIP DNA was sampled with two sets of primers: 4040 and 160 base pairs from the TG repeats of VI-R. Error bars indicate standard errors from four independent experiments. (E) Dilutions (1:9) of yeast strains WT (FGY8.2), htbK123R (FGY8.3), Δbre1 (FGY8.5), Δset1 (FGY8.6), and Δdot1 (FGY8.8) were spotted on YPAD plates and grown for 3 d at either 30 or 37°C.

Loss of SIR function rescues the heat-sensitive phenotype of htbK123R and Δset1 cells. Dilutions 1:9 of yeast were plated on YPAD and grown for 3 d at either 30 or 37°C. Strains used here are WT (FGY8.2), htbK123R (FGY8.3), htbK123R Δsir4 (FGY8.11), Δsir4 (FGY8.10), Δsir2 (FGY8.12), htbK123R Δsir2 (FGY8.13), Δsir3 (FGY8.14), htbK123R Δsir3 (FGY8.15), Δset1 Δsir4 (FGY8.16), and Δset1 (FGY8.6). To analyze the effects of expressing a dominant-negative version of Sir4 (SIR4–CTERM), htbK123R cells (FGY8.3) carried a pVV214-based plasmid expressing the carboxy-terminal 167 residues of Sir4.

Ectopic association of Sir3 and Sir4 with category 4 genes in the absence of H3K4 methylation. DamID chromatin profiling was performed on cells expressing Sir3-Dam or Sir4-Dam fusion proteins in an otherwise WT (black bars) or Δset1 (open bars) background. The percentage of methylated DNA was determined by using real-time PCR—with primers that flank candidate methylation sites—to compare relative signals for each amplicon in digested vs. undigested DNA. The percentage of methylation by Sir3-Dam or Sir4-Dam in each strain was normalized to their respective strain expressing the unfused Dam protein. Strains YM2302, YM2312, YM2304, YM2313, YM2306, and YM2314 were used in these experiments. (A) DamID comparison of Sir3 and Sir4 association with the promoter regions of the indicated genes. (B) DamID comparison of Sir3-Dam and Sir4-Dam association with candidate methylation sites [amplicons A (−964 to −836), B (−344 to −266), and C (+541 to +642) across the CTT1 gene; CTT1 is a category 4 gene involved in the cellular response to heat. Error bars show standard errors from three independent experiments.