13 research outputs found
The PAF Complex and Prf1/Rtf1 Delineate Distinct Cdk9-Dependent Pathways Regulating Transcription Elongation in Fission Yeast
<div><p>Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast <i>Schizosaccharomyces pombe</i> that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF) complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways.</p></div
Prf1 does not stably associate with the PAF complex.
<p>(<b>A</b>) TAP purification was carried out using whole cell extracts from the indicated strains and purified material was analyzed by SDS-PAGE and silver staining. The proteins contained in the labeled bands were identified by mass spectrometry (as indicated on the right). âCBPâ denotes the residual fusion to the calmodulin binding peptide resulting from the TAP procedure. Leo1 was detected in two bands that also contained either Paf1-CBP (in the <i>paf1-TAP</i> lane) or Paf1 (in the <i>tpr1-TAP</i> lane). Molecular weight standards (in kD) are denoted on the left. (<b>B</b>) Single-step TAP purifications were performed using extracts from the indicated strains. 5% of input fractions (âinputâ) and 50% of bead-bound fractions (âbeadsâ) were analyzed by SDS-PAGE and western blotting.</p
Prf1 and PAF pathways have opposing biological effects.
<p>(<b>A</b>) Quantification of the abnormal septation patterns in the indicated strains. Strains carrying the <i>cdk9<sup>as</sup></i> allele were cultured in DMSO (â) or 2 ”M 3-MB-PP1 (+) for 15 hours prior to fixation for microscopy. Error bars denote standard deviations from 3 independent experiments. (<b>B</b>) ChIP of RNAPII was carried out in the indicated strains and quantified by qPCR using primers specific to the <i>nup189</i><sup>+</sup> gene. Values were normalized to that for primer pair 1. Error bars denote standard deviations from three independent experiments. Significant differences from wild-type values (unpaired t-test) are indicated.</p
Prf1 and PAF have shared roles in histone H2B ubiquitylation but are functionally distinct.
<p>(<b>A</b>) Whole-cell extracts from strains of the indicated genotypes were analyzed by SDS-PAGE and western blotting with the indicated antibodies. (<b>B</b>) Cells of the indicated genotypes were fixed, stained with DAPI/calcofluor, and visualized by microscopy. For each strain fluorescent images (denoting DAPI/calcofluor staining) are shown on the left; bright-field images are shown on the right. (<b>C</b>) Quantification of the abnormal septation patterns in the indicated strains. âSeptaâ refers to cells with at least one visible division septum, âmulti-sepâ refers to cells with twinned septa or multiple septa between two nuclei, and âchainsâ refers to unseparated chains of cells. Error bars denote standard deviations from 3 independent experiments.</p
Prf1 and PAF are recruited to chromatin via alternate mechanisms.
<p>(<b>A</b>) Strains used for ChIP with IgG resin (recognizing the TAP tag) are indicated at the bottom. All strains also harbored the <i>cdk9<sup>as</sup></i> allele and were treated with either DMSO (â) or 20 ”M 3-MB-PP1 (+) for two hours before crosslinking for ChIP (also indicated by â3MBâ at the far right). Assays were quantified by qPCR using primers specific for the <i>act1</i><sup>+</sup> (left) or <i>adh1</i><sup>+</sup> (right) genes. Lengths of the gene coding regions (in base pairs) and positions of PCR amplicons are indicated at the top. Numbering of the datasets for the corresponding PCR amplicons shown for <i>act1</i><sup>+</sup> (left) is used throughout the figure. Error bars denote standard deviations from 2â3 independent experiments. (<b>B and C</b>) Strains of genotypes indicated at the bottom were used for ChIP of either Prf1-TAP (left) or Tpr1-TAP (right). Cultures were treated with DMSO or 3-MB-PP1 as in (A).</p
Model depicting the roles of the Prf1/Rtf1 and PAF pathways in RNAPII elongation.
<p>The Prf1 pathway, involving direct association of Prf1 with phosphorylated Spt5 CTD, is labeled â1.â The PAF pathway, involving multiple Cdk9 targets, is labeled â2.â Potential crosstalk between the two pathways is indicated by the broken double arrow. See text for details.</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 (â) or presence (+) of 20 ”M 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. âT212Aâ and âT212Eâ 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
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<sup>as</sup> htb1-K119R</i> (LV193) cells grown in the absence (top) or presence (middle) of 10 ”M 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>brl2Î</i> (JTB331), <i>brl2Î cdk9-T212A</i> (JTB335), <i>ubp8Î</i> (JTB297), <i>ubp8Î 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
Opposing effects of H2Bub1 and Cdk9 activity on RNAPII distribution revealed by ChIPâchip.
<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; ÎŒ<sub>0</sub>â=â0). Note that there is only light shading for the last row (corresponding to the blue curve).</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 (â) or presence (+) of 20 ”M 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