Volume 42, Issue 1, Pages 127-136 (April 2011) A Common Telomeric Gene Silencing Assay Is Affected by Nucleotide Metabolism  Marlies P. Rossmann, Weijun Luo, Olga Tsaponina, Andrei Chabes, Bruce Stillman  Molecular Cell  Volume 42, Issue 1, Pages 127-136 (April 2011) DOI: 10.1016/j.molcel.2011.03.007 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 42, 127-136DOI: (10.1016/j.molcel.2011.03.007) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 CDC21 Is a High-Copy Suppressor of the pol30-8 URA3-VII-L TPEV Defect (A) Schematic representation of (1) the endogenous telomere on chromosome VII, left arm (VII-L), (2) telomere VII-L with URA3 inserted adjacent to a truncated ADH4. Arrows above genes depict the direction of transcription. (B) Ten-fold serial dilution of a pol30-8 hmr::ADE2 URA3-VII-L strain (MRY0828) transformed with indicated plasmids on indicated selective plates. (C) Western blot analysis of whole-cell extracts from an experiment as in (B). Short and long refer to different exposure times. (D) Ten-fold serial dilution of wild-type (MRY1655, MRY1657), pol30-8 (MRY1653, MRY1652), cdc21-216 (MRY1656, MRY1651), and pol30-8 cdc21-216 (MRY1654, MRY1650) hmr::ADE2 URA3-VII-L strains. Plates were incubated at room temperature. (E) Ten-fold serial dilution of wild-type (MRY1237), dot1Δ (MRY1242), dot1Δ asf1Δ (MRY1288), or pol30-8 dot1Δ asf1Δ (MRY1226) hmr::ADE2 URA3-VII-L strains transformed with pRS425 or CDC21. Molecular Cell 2011 42, 127-136DOI: (10.1016/j.molcel.2011.03.007) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 pol30-8 and dot1Δ Are Not Defective in TPEV at HIS3-VII-L (A) Schematic representation of telomere VII-L carrying HIS3 distal to URA3. Arrows above genes depict the direction of transcription. (B) Ten-fold serial dilution of HIS3-VII-L strains of wild-type (MRY1525), dot1Δ (MRY1530), pol30-8 (MRY1521), sir3Δ (MRY1519), and double (MRY1529, MRY1528, MRY1523) and triple (MRY1533) mutants. 3-AT: see text. (C) hmr::ADE2 and URA3-VII-L expression ratios measured by RT-qPCR of pol30-8 (MRY1098, MRY1092), sir3Δ (MRY1084, MRY1080) and pol30-8 sir3Δ (MRY1088, MRY1102) compared to wild-type (MRY1081, MRY1097) ade2Δ ura3Δ strains. The average result for three experiments with two primer pairs per gene is shown. (D) Expression levels of URA3 measured as in (C) of wild-type (MRY1097) or pol30-8 (MRY1092) ura3Δ URA3-VII-L strains transformed with indicated plasmids. ACT1: reference. Error bars denote the standard error of the mean (SEM) for two transformants and three primer pairs tested. (E) Expression levels of HIS3 measured as in (C) of wild-type (MRY1418) or pol30-8 (MRY1414) his3Δ HIS3-VII-L strains transformed with indicated plasmids. Analysis was performed as in (D). (F) ChIP analysis for IgG and Sir2 followed by qPCR of wild-type (MRY1073) and dot1Δ (MRY1072) ura3Δ URA3-VII-L strains transformed with indicated plasmids. Error bars denote the SEM for two experiments. Molecular Cell 2011 42, 127-136DOI: (10.1016/j.molcel.2011.03.007) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 dot1Δ and pol30-8 Cells Do Not Have a General Telomere-Specific Silencing Defect (A) A log2-based gene expression ratio in the regions 20 kb from each telomere (from top to bottom: chromosome I-L to XVI-R; 125 genes in total) for three biological replicates of dot1Δ (MRY1627) compared to wild-type (MRY1629) ade2Δ ura3Δ hmr::ADE2 URA3-VII-L strains. Genes at the engineered telomere VII-L are labeled. (B) Gene expression level changes in three biological replicates of pol30-8 (MRY1071), dot1Δ (MRY1627), and pol30-8 dot1Δ (MRY1069) compared to wild-type (MRY1629) strains from all 32 pooled telomeres to the pooled centers of the chromosomes. Differential gene expression was determined by GAGE test statistics (Luo et al., 2009). Each data point spans a 10 kb window of the corresponding regions in all 32 chromosome halves. (C) Gene expression level changes of two asf1Δ and cac1Δ compared to two wild-type strains. The raw data files were obtained from J. Tyler (Zabaronick and Tyler, 2005) and processed as in (B). (D) Average log2-based expression ratio of all genes in pol30-8 compared to wild-type strains as in (B) versus baseline gene expression level in wild-type strains. Data points are for all three replicates of each strain. A local weighted polynomial smoothing (Loess) curve (in red) was fitted to the data. (E) A log2-based expression ratio of all genes in wild-type replicate 1 versus baseline expression level in wild-type replicate 2. Analysis was performed as in (D). (F) ChIP analysis for IgG and histone H3 of wild-type (MRY1551) and pol30-8 (MRY1550) strains. VIII ig: intergenic region on VIII-R. Error bars denote the SEM for two experiments. (G) Ten-fold serial dilution of wild-type (MRY1081), 2 bas1Δ pho2Δ (MRY1866, MRY1867), dot1Δ (MRY1063), and 2 dot1Δ bas1Δ pho2Δ (MRY1871, MRY1872) strains (upper panel) as well as wild-type (MRY1081), 2 bas1Δ pho2Δ (MRY1866, MRY1867), pol30-8 (MRY1098), and 2 pol30-8 bas1Δ pho2Δ (MRY1868, MRY1869) strains (lower panel). All strains are hmr::ADE2 URA3-VII-L. Molecular Cell 2011 42, 127-136DOI: (10.1016/j.molcel.2011.03.007) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 RNR Is Induced by 5-FOA and RNR Inhibition Rescues 5-FOA Sensitivity of pol30-8 and dot1Δ URA3-VII-L Cells (A) Expression levels of RNR1, RNR2, RNR3, and RNR4 measured by RT-qPCR in wild-type (MRY1629) or pol30-8 (MRY1071) strains. ACT1: reference. Error bars denote the SEM for six strains per genotype. (B) Western blot analysis of whole-cell extracts from two wild-type (MRY1767, MRY1773) and two pol30-8 (MRY1768, MRY1772) strains. Lanes 7 and 8 show a wild-type strain (MRY1638), either left untreated or treated with 4-NQO (see text) for 2 hr. (C) Schematic overview of 5-fluorouracil (5-FU) metabolism in the cell (modified from Hardman et al., 2001); added on is the metabolism of 5-FOA (Jones and Fink, 1982). For abbreviations see text. ↑ or ↑↑: magnitude of increased expression of the gene(s) encoding the enzyme (in orange) for indicated genotypes. Ura3: protein encoded by URA3-VII-L. (D) Ten-fold serial dilution of wild-type (MRY0656), pol30-8 (MRY0041), cac1Δ (MRY0462), pol30-8 asf1Δ (MRY0658), pol30-8 hir1Δ (MRY0661), and pol30-8 asf1Δ hir1Δ (MRY0655) hmr::ADE2 URA3-VII-L strains. (E) Expression levels of RNR4 measured by RT-qPCR for wild-type (MRY1082, MRY1090) and pol30-8 (MRY1086, MRY1101) ura3Δ URA3-VII-L strains before or at indicated time points after treatment with either DMSO or 5-FOA. PGK1: reference. Error bars denote the SEM for two experiments. (F) Western blot analysis of whole-cell extracts from a pol30-8 (MRY0828) strain transformed with indicated plasmids (two transformants each). Short and long refer to different exposure times. Asterisk indicates a cross-reacting band. (G) Western blot analysis of whole-cell extracts from wild-type (MRY1807), asf1Δ (MRY1811), dot1Δ (MRY1802), and asf1Δ dot1Δ (MRY1797) strains, either left untreated or treated with 4-NQO (see text) for 2 hr. Molecular Cell 2011 42, 127-136DOI: (10.1016/j.molcel.2011.03.007) Copyright © 2011 Elsevier Inc. Terms and Conditions