Sin3/Elp3 is required for modification of uridine-34 (U₃₄) at the anticodon of some cytoplasmic tRNAs.

<p>(A) Sin3/Elp3 and the Ctu1-Ctu2 pathways are required for the mcm⁵ and s² modifications, respectively, at U₃₄ of some anticodons. (B) Northern blot analysis of bulk tRNA isolated from WT (972), IV16 (<i>Δsin3/elp3</i>), YDH 644 (<i>Δctu1</i>), IV86 (<i>Δctu2</i>), and YDH 254 (<i>Δctu1 Δctu2</i>) using specific probes against tRNA^Lys_UUU, tRNA^Gln_UUG, tRNA^Glu_UUC and tRNA^Arg_UCU (negative control) by the APM-gel retardation method. The position of the unmodified (<i>tRNA</i>) or modified (<i>mcm⁵s² tRNA</i>) tRNAs are indicated with arrows. (C) <i>Δctu1</i> and <i>Δctu2</i> strains are sensitive to oxidative stress. Serial dilutions from cultures of strains 972 (WT), AV18 (<i>Δsty1</i>), IV16 (<i>Δsin3/elp3</i>), IV86 (<i>Δctu2</i>), YDH 644 (<i>Δctu1</i>), YDH 254 (<i>Δctu1 Δctu2</i>) were spotted onto rich plates without (Untreated) or with 1 mM H₂O₂. (D) Double mutant <i>Δsin3</i>/<i>elp3 Δctu2</i> colonies are similar in size to single mutant <i>Δsin3</i>/<i>elp3</i>. Tetrad analysis of a cross between IV16 (<i>Δsin3/elp3</i>) and IV86 (<i>Δctu2</i>) strains. Each vertical box corresponds to the four spores of a tetrad. The genotypes of each colony of the top panel are indicated in the corresponding positions of the bottom panel: WT, <i>Δctu2 (Δc), Δsin3/elp3 (Δe) or Δsin3/elp3 Δctu2 (ΔΔ)</i>. (E) Single mutant <i>Δsin3</i>/<i>elp3</i> and double mutant <i>Δsin3</i>/<i>elp3 Δctu2</i> show similar sensitivity to oxidative stress. Serial dilutions from cultures of strains 972 (WT), IV16 (<i>Δsin3/elp3</i>) and JF73 (<i>Δsin3/elp3 Δctu2</i>) were spotted onto rich plates without (Untreated) or with 1 mM H₂O₂.</p

Protein levels of the stress transcription factors Atf1 and Pcr1 depend on the U₃₄ modifying activities Sin3/Elp3 and Ctu2.

<p>(A) Scheme illustrating the activation of the stress gene expression program by Atf1 and Pcr1 (see text for details). (B and C) Absence of Sin3/Elp3 or Ctu2 barely affects transcription of the <i>atf1</i> and <i>pcr1</i> genes but largely affects Atf1 and Pcr1 protein levels. Rich media cultures of strains 972 (WT), IV16 (Δ<i>sin3/elp3</i>) and IV86 (Δ<i>ctu2</i>) treated with 1 mM H₂O₂ at the indicated time points were analyzed to determine transcription levels of <i>atf1</i> and <i>pcr1</i> genes by Northern blot (B), or Atf1 and Pcr1 protein levels by Western blot using polyclonal antibodies (C). <i>act1</i> mRNA or tubulin were used as loading controls for B and C, respectively. (D) Quantification of the relative mRNA and protein levels for <i>atf1</i>, <i>pcr1</i>, Atf1 and Pcr1 in wild-type and mutant strains. The Northern or Western blot panels of experiments as in B and C, respectively, were quantified and represented here relative to untreated wild-type levels (with an assigned value of 1). The <i>atf1</i> and <i>pcr1</i> mRNA levels normalized to <i>act1</i> are shown in the left two panels, <i>w</i>hereas the Atf1 and Pcr1 protein levels normalized to tubulin are shown in the two right panels. Error bars (SEM) were calculated from biological duplicates.</p

Expression of a synthetic AAA-to-AAG <i>atf1</i> gene rendered wild-type Atf1 protein levels in Elongator mutants.

<p>(A and B) Vectors carrying a constitutively expressed wild-type (p<i>HA-atf1</i>′) or mutated <i>atf1</i> gene (p<i>HA-atf1</i>_AAG′) were integrated in the chromosomes of wild-type or <i>Δsin3/elp3</i> strains. Rich media cultures of strains JF91 (WT+p<i>HA-atf1</i>′), JF92 (<i>Δsin3/elp3</i>+p<i>HA-atf1</i>′), JF94 (WT+p<i>HA-atf1</i>_AAG′) and JF95 (<i>Δsin3/elp3</i>+p<i>HA-atf1</i>_AAG′), either untreated (0) or treated with 1 mM H₂O₂ for the indicated times, were analyzed to determine <i>HA-atf1</i> mRNA levels by Northern blot using an anti-HA probe (A) or HA-Atf1 protein levels by Western blot using monoclonal antibody against HA (B). The numbers below the Northern or Western blot panels indicate the relative levels of <i>HA-atf1/act1</i> mRNAs (panel A) or HA-Atf1/tubulin protein levels (panel B). (C) Expression of the mutant AAA-to-AAG Atf1 protein does not suppress the growth defects of <i>Δsin3/elp3</i> cells upon oxidative stress. Empty vector or plasmids carrying a wild-type (p<i>HA-atf1</i>′) or a mutated <i>atf1</i> gene (p<i>HA-atf1</i>_AAG′) were integrated in the chromosomes of wild-type or <i>Δsin3/elp3</i> strains. Cultures from the resulting strains JF88 (WT+empty vector), JF89 (<i>Δsin3/elp3</i>+empty vector), JF91 (WT+p<i>HA-atf1</i>′), JF92 (<i>Δsin3/elp3</i>+p<i>HA-atf1</i>′), JF94 (WT+p<i>HA-atf1</i>_AAG′) and JF95 (<i>Δsin3/elp3</i>+p<i>HA-atf1</i>_AAG′) were serially diluted and spotted onto rich media plates without (Untreated) or with 1 mM H₂O₂.</p

Codon usage and <i>tRNA</i> gene copy number for Lys, Gln and Glu.

<p>N.P: Not provided.</p>a<p>4932 <i>S. pombe</i> ORFs are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003647#pgen.1003647-Hiraoka1" target="_blank">[42]</a>.</p>b<p>30 ORFs with highest expression levels, obtained from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003647#pgen.1003647-Hiraoka1" target="_blank">[42]</a>.</p>c<p>50 genes most up-regulated after treatment with 0.5 mM of H₂O₂ during 30 minutes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003647#pgen.1003647-Chen2" target="_blank">[4]</a>.</p>d<p>The number of mRNA molecules per cell at basal levels was obtained from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003647#pgen.1003647-Hiraoka1" target="_blank">[42]</a>.</p><p>The number of mRNA molecules per cell at induced levels was calculated using the fold induction ratios upon 0.5 mM of H₂O₂ during 30 minutes from microarray experiments reported at <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003647#pgen.1003647-Chen2" target="_blank">[4]</a>.</p>e<p>Codon usage values of ‘Total <i>S. pombe</i> ORFs’ and ‘Highly expressed ORFs’ were obtained from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003647#pgen.1003647-Hiraoka1" target="_blank">[42]</a>.</p><p>The other codon usage values were calculated using free software ACUA v1.0 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003647#pgen.1003647-Vetrivel1" target="_blank">[43]</a>.</p>f<p>tRNA gene copy number was extracted from PomBase <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003647#pgen.1003647-Wood1" target="_blank">[61]</a>.</p

Over-expression of tRNA^Lys_UUU supresses the growth defects of <i>Δsin3/elp3</i> upon oxidative stress.

<p>(A) Relative levels of tRNA over-expression by Northern blot. Total RNA from strains 972 (WT), IV16 (<i>Δsin3/elp3</i>), or JF77 (<i>Δsin3/elp3</i>) transformed with episomal plasmids p465 (ptRNA^Lys_UUU), p466 (ptRNA^Lys_CUU), p467 (ptRNA^Gln_UUG), p468 (ptRNA^Glu_UUC) or the empty vector pREP.42x, was analyzed by Northern blot with probes of dsDNA of the indicated tRNAs labeled at their antisense strand. a<i>ct1</i> is shown as a loading control. The numbers below last panel indicate the relative levels of the corresponding over-expressed tRNA normalized to basal levels in wild-type strain. (B) Serial dilutions from cultures of strains as in A were spotted onto synthetic rich media plates without (Untreated) or with 2 mM H₂O₂.</p

Mutual phenotypic suppression in <i>cdk9 htb1-K119R</i> double mutants.

<p>(A) For indicated strains (JS78, LV7, JTB62-1, JTB67-1, MS249, LV193), growth in increasing [3-MB-PP1] is plotted as a percentage of growth in the absence of 3-MB-PP1. Error bars denote standard deviations from 3 independent experiments. (B) Images of DAPI- and calcofluor-stained <i>htb1-K119R</i> (JTB67-1) and <i>cdk9^as htb1-K119R</i> (LV193) cells grown in the absence (top) or presence (middle) of 10 µM 3-MB-PP1 for 7 hr, or after inhibitor washout and return to growth (bottom). (C) Fluorescent images of DAPI/calcofluor-stained wild-type (JS78), <i>cdk9-T212A</i> (HD7-24), <i>brl2Δ</i> (JTB331), <i>brl2Δ cdk9-T212A</i> (JTB335), <i>ubp8Δ</i> (JTB297), <i>ubp8Δ cdk9-T212A</i> (JTB336), <i>htb1-K119R</i> (JTB67-1), <i>cdk9-T212A htb1-K119R</i> (LV252) <i>cdk9-T212E htb1-K119R</i> (LV256), and <i>mcs6-S165A htb1-K119R</i> (LV254) cells. (D) Quantification of abnormal septation patterns in strains of indicated genotypes (JTB62-1, JTB67-1, JTB325, JTB326, JTB331, JTB335, JTB377, JTB333 respectively). Error bars represent standard deviations from 2 independent experiments; at least 200 cells were counted in each. (E) Flocculation of indicated <i>htb1-FLAG</i> strains (JTB62-1, JTB67-1, JTB325, JTB326) was quantified as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002822#s4" target="_blank">Materials and Methods</a>. Error bars denote standard deviations from 2 independent experiments.</p

H2Bub1 enhances Cdk9 recruitment and Spt5 phosphorylation at transcribed genes.

<p>(A,B) Spt5 phosphorylation was measured by ChIP using anti-Spt5-P in <i>spt5-myc</i> (MS265; black bars) and <i>spt5-myc htb1-K119R</i> (LV239; gray bars) strains and quantified at the indicated genes by qPCR. Enrichment is plotted as a percentage of input signal for each primer pair. Positions of PCR primer pairs within coding regions are indicated schematically at top. (C,D) Spt5-myc occupancy was measured by ChIP as in A and B. (E,F) Spt5-P enrichment normalized to total Spt5-myc occupancy. (G,H) Cdk9-myc occupancy was measured by ChIP in <i>cdk9-myc</i> (MS264; black bars) and <i>cdk9-myc htb1-K119R</i> (KL259; gray bars) strains. (I,J) H2Bub1 enrichment was measured by ChIP and normalized to H2B-FLAG occupancy. Error bars denote standard deviations from 3 independent experiments. Asterisks denote a significant difference between wild-type and mutant (“*” p<0.04, “**” p<0.02; unpaired t-test).</p

H2Bub1 independently stimulates Cdk9-mediated Spt5 phosphorylation and Set1-dependent H3K4 methylation.

<p>(A) Immunoblots of extracts from strains carrying <i>spt5</i>-<i>myc</i> with or without an <i>as</i> kinase allele as indicated (CS111, CS112, CS155, CS159, LV125 and LV167, respectively). Cultures were grown in the absence (−) or presence (+) of 20 µM 3-MB-PP1, added 20 min prior to harvest. Antibody reactivities are indicated at right. (B) Immunoblots of extracts from indicated strains (JS78, JTB67-1, JTB62-1, JTB331, HD7-24 and JTB297, respectively), probed for phosphorylated (Spt5-P) or total Spt5. (C) Fluorescent images of DAPI/calcofluor-stained cells from indicated strains (<i>spt5-WT</i>, JTB350, <i>spt5-T1A</i>, JTB352, <i>spt5-T1E</i>, JTB354, respectively). (D) Quantification of abnormal septation in strains of indicated genotypes (JTB67-1, <i>spt5-WT</i>, JTB350, <i>spt5-T1A</i>, JTB352, <i>spt5-T1E</i>, JTB354, JTB418 and JTB428, respectively). Error bars represent standard deviations from 2 independent experiments; at least 200 cells were counted in each. (E) Immunoblots of extracts from indicated strains (JTB204, JTB80-2, and JTB331, respectively), probed for phosphorylated (Spt5-P) or total Spt5. (F) Immunoblots of extracts from indicated strains (JTB204, JTB80-2, JTB67-1, <i>spt5-WT, spt5-T1A</i> and <i>spt5-T1E</i>, respectively), probed for H3K4me3 or total H3.</p

H2Bub1 depends on Cdk9 activity and Spt5 phosphorylation.

<p>(A) Immunoblots of whole-cell extracts from wild-type (wt) (JS78) or AS mutant strains (LV7, LV77, LV42), as indicated, grown in the absence (−) or presence (+) of 20 µM 3-MB-PP1, added 20 min prior to harvest. Antibodies are indicated at right. (B) Immunoblots of extracts from indicated strains probed for total histone H2B and H2Bub1, as indicated at right. “T212A” and “T212E” denote strains <i>cdk9-T212A</i> (HD7-24) and <i>cdk9-T212E</i> (HG127). (C) Immunoblots of extracts from wild-type (JS78) or indicated <i>spt5</i> mutant strains.</p

Opposing effects of H2Bub1 and Cdk9 activity on RNAPII distribution revealed by ChIP–chip.

<p>(A) Average distribution of RNAPII at 540 <i>S. pombe</i> genes, as determined by ChIP-chip in a <i>cdk9-T212A</i> strain (JTB325). Genes were grouped according to total levels of RNAPII enrichment. (B) As in (A) for <i>cdk9-T212A htb1-K119R</i> (JTB326). (C) Average distributions of differences between <i>cdk9-T212A</i> (JTB325) and wild-type (JTB62-1) RNAPII enrichment grouped according to RNAPII enrichment in wild-type cells. (D) As in (C) for differences between <i>cdk9-T212A htb1-K119R</i> and wild-type RNAPII enrichment. (E) As in (C) for differences between <i>cdk9-T212A htb1-K119R</i> and <i>cdk9-T212A</i> RNAPII enrichment. (F) As in (C) for differences between <i>cdk9-T212A htb1-K119R</i> and <i>htb1-K119R</i> RNAPII enrichment. The keys below C-F illustrate the statistical significance of the differences for each group at 50 positions along the average gene. The rows of the key are color-coded according to the graph. Open squares denote p>0.01; light shading denotes 0.01>p>10exp-5; dark shading denotes p<10exp-5 (one-sample t-tests; μ₀ = 0). Note that there is only light shading for the last row (corresponding to the blue curve).</p