CENP-A^Cse4 peak information.

<p>CENP-A^Cse4 peak information.</p

CENP-A^Cse4 mislocalization does not depend on H2A.Z^Htz1 incorporation.

<p>(A) ChIP was performed with anti-Cse4 antibody on negative control cells (SBY15924), as well as <i>pTET-CSE4</i> (SBY15903), <i>psh1Δ pTET-CSE4</i> (SBY15904) and <i>psh1Δ htz1Δ pTET-CSE4</i> (SBY15906) cells overexpressing CENP-A^Cse4 for six hours. As a control, we also performed a ChIP experiment with no antibody in <i>psh1Δ pTET-CSE4</i> cells. Input dilutions are 1:100, 1:300, 1:100 and ChIP dilutions are 1x, 1:3, 1:9. (B) ChIP-PCR of 3HA-Htz1 at the <i>RDS1</i> promoter. Strains used: negative (neg.) control (SBY3), <i>psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12833), <i>psh1Δ pGAL-3Flag-CSE4</i>, <i>3HA-HTZ1 swr1Δ</i> (SBY12924). Input dilutions: 1:100, 1:300, 1:900. ChIP dilutions: 1:3, 1:9, 1:21. (C) Relative CENP-A^Cse4 levels were measured by quantifying the mean chromatin CENP-A^Cse4:H2B fold change vs. <i>pGAL-3Flag-CSE4 HA-HTZ1</i> +/- 1 SEM using quantitative immunoblots of chromatin fraction (n = 3), p = 0.0204 (paired t-test comparing the Cse4:H2B relative ratio in <i>psh1Δ pGAL-3Flag-CSE4</i> to <i>swr1Δ psh1Δ pGAL-3Flag-CSE4</i>). Strains used were <i>pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12832), <i>swr1Δ pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12956), <i>psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12833) and <i>swr1Δ psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12924).</p

INO80-C contributes to CENP-A^Cse4 misincorporation.

<p>(A) Mean chromatin CENP-A^Cse4:H2B fold change vs. <i>pGAL-3Flag-CSE4 HA-HTZ1</i> strain +/- 1 SEM from quantitative immunoblots of the chromatin fraction. Strains used: <i>pGAL-3Flag-CSE4 HA-HTZ1</i> (SBY12832), <i>nhp10Δ pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12930), <i>psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12833), and <i>nhp10Δ psh1Δ pGAL-3Flag-CSE4 3HA-HTZ1</i> (SBY12959). (n = 3). p = 0.0593 (paired t-test comparing the Cse4:H2B relative ratio in <i>psh1Δ pGAL-3Flag-CSE4</i> to <i>nhp10Δ psh1Δ pGAL-3Flag-CSE4</i>). (B) Co-Immunoprecipitation (co-IP) of overexpressed 3Flag-Cse4 with either Psh1-13Myc or Ino80-13Myc from the following strains: <i>PSH1-13Myc pGAL-3Flag-CSE4</i> (SBY14482), <i>pGAL-3Flag-CSE4</i> (SBY9540), <i>INO80-13Myc</i> (SBY14527), <i>INO80-13Myc pGAL-3Flag-CSE4</i> (SBY14526), <i>INO80-13Myc psh1Δ pGAL-3Flag-CSE4</i> (SBY14515). (C) Five-fold serial dilutions of strains <i>3Flag-CSE4</i> (SBY10419), <i>nhp10Δ 3Flag-CSE4</i> (SBY12958), <i>psh1Δ 3Flag-CSE4</i> (SBY10484), <i>nhp10Δ psh1Δ 3Flag-CSE4</i> (SBY12928), <i>pGAL-3Flag-CSE4</i> (SBY10425), <i>nhp10Δ pGAL-3Flag-CSE4</i> (SBY12930), <i>psh1Δ pGAL-3Flag-CSE4</i> (SBY10484), and <i>nhp10Δ psh1Δ pGAL-3Flag-CSE4</i> (SBY12959) were plated on indicated media.</p

Intergenic regions are the major sites of overexpressed CENP-A^Cse4 mislocalization.

<p>(A) Quantification of CENP-A^Cse4 levels in MNase-digested chromatin in untagged (SBY3, black), <i>3Flag-CSE4</i> (SBY10419, blue), <i>psh1Δ 3Flag-CSE4</i> (SBY10484, pink), <i>pGAL-3Flag-CSE4</i> (SBY10425, green) and <i>psh1Δ pGAL-3Flag-Cse4</i> (SBY10483, orange) strains. The ratio of CENP-A^Cse4:H2B in the chromatin in each strain was quantified relative to the CENP-A^Cse4:H2B ratio from the <i>3Flag-CSE4</i> (SBY10419) strain. Quantification is based on two biological replicates. Error bars are +/- 1 standard error of the mean (SEM) of the two biological replicates. (B) The total number of CENP-A^Cse4 peaks called in the indicated strains: <i>3Flag-CSE4</i> (SBY10419, blue), <i>psh1Δ 3Flag-CSE4</i> (SBY10484, pink), <i>pGAL-3Flag-CSE4</i> (SBY10425, green) and <i>psh1Δ pGAL-3Flag-CSE4</i> (SBY10483, orange). (C) A representative region of the CENP-A^Cse4 ChIP-seq coverage on Chromosome 4 between 429,000 base pairs (bp) and 470,000 bp is shown. The CENP-A^Cse4 ChIP-seq coverage for the strains in (B) is normalized to the coverage at the centromeres after subtracting the input. Peaks are shown as lines below each coverage signal (the cutoff is the average minimum coverage at the centromere in the <i>3Flag-CSE4</i> strain). The scale of the normalized coverage is from 0–20,000 for all strains. (D) The percentage of CENP-A^Cse4 peak centers in each type of genomic region is graphed for each strain, as in (B). The percentage of each feature in the genome is: genes (68.23%), intergenic (27.04%), pericentromeres (2.62%), telomeres (1.16%), origins (0.92%) and centromeres (0.02%).</p

CENP-A^Cse4 mislocalization to promoters alters transcription.

<p>(A) Graph of the number of transcripts significantly increased or decreased compared to the WT strain by RNA-seq of <i>psh1Δ</i> and <i>htz1Δ</i> cells at t = 2 hours. Differential expression analysis statistics were performed using edgeR [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005930#pgen.1005930.ref078" target="_blank">78</a>]. (B) Graph of the number of transcripts significantly increased or decreased at t = 2 hours compared to t = 0 in <i>pGAL-3Flag-CSE4</i>, <i>psh1Δ pGAL-3Flag-CSE4</i>, and <i>pGAL-H3</i> that are not also changed at t = 2 hours compared to t = 0 in the WT strain. (C) TSS and TTS profiles of CENP-A^Cse4 enrichment based on the measured transcriptional changes by RNA-seq in the <i>psh1Δ pGAL-3Flag-CSE4</i> strain. (D) Proportional Venn diagram of genes up-regulated in <i>psh1Δ pGAL-3Flag-CSE4</i> at t = 2 hours and genes up-regulated in <i>htz1Δ</i> at t = 2 hours. p = 2.699x10^-24 (p-value from a cumulative hypergeometric distribution test, which represents the probability of the number of genes overlapped or greater between the two strains.) (E) Model: At chromosome arms, INO80-C-mediated full nucleosome turnover may lead to CENP-A^Cse4 deposition and H2A.Z^Htz1 removal. Psh1 blocks stable CENP-A^Cse4 promoter incorporation by ubiquitylating mislocalized CENP-A^Cse4 and targeting it for degradation. Mislocalization of CENP-A^Cse4 to promoters leads to misregulation of a subset of downstream genes, which could affect survival of these cells.</p