Epistasis analysis with homologous recombination genes.

<p>(A) Survival after irradiation with UV or IR of the strains: (A) wild type (JS78), <i>csk1Δ</i> (JS155), <i>rhp51Δ</i> (YP6), <i>csk1Δ rhp51Δ</i> (HD4-6); (B) wild type (JS78), <i>csk1Δ</i> (JS155), <i>rhp54Δ</i> (YP25), <i>csk1Δ rhp54Δ</i> (HD2-55). (C) Cells were streaked onto YES plates and incubated for 4 days at 30°C before being photographed. Strains: wild type (JS78), <i>csk1Δ</i> (JS155), <i>rhp57Δ</i> (YP27), <i>csk1Δ rhp57Δ</i> (HG123). (D) Survival after UV irradiation of the strains: wild type (JS78), <i>csk1Δ</i> (JS155), <i>sfr1Δ</i> (HG24), <i>csk1Δ sfr1Δ</i> (HG31).</p

A <i>csk1Δ</i> strain is hypersensitive to DNA damaging agents.

<p>Survival was measured after irradiation with UV (A) or IR (B) of the following strains: wild type (JS78), <i>csk1Δ</i> (JS155). (C) 10-fold serial dilutions of the wild-type and <i>csk1Δ</i> strains [as in (A)] in mid-log phase were plated on fresh media containing no drug (top), 0.005% MMS (middle) or 6 mM hydroxyurea (bottom), and incubated 3–5 days before photographing.</p

Strains used in this study

<p>Strains used in this study</p

Csk1 is not required for activation of the DNA damage checkpoint or for NER.

<p>(A) WT (JS78), <i>csk1Δ</i> (JS155), <i>rad3Δ</i> (YP46) and <i>csk1Δ rad3Δ</i> (YP68) cells were synchronized in G2 by fractionation in lactose gradients and irradiated with 40 J/m² UV light. Samples were taken every 30 min and the percent of cells passing through mitosis was measured by counting binucleated cells, septated cells and doublets. (B) Survival after UV irradiation of the following strains: wild type (JS78), <i>csk1Δ</i> (JS155), <i>rad13Δ</i> (YP1), <i>csk1Δ rad13Δ</i> (YP85).</p

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 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^as</i> allele were cultured in DMSO (−) or 2 µM 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>⁺ 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 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). “CBP” 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 (“input”) and 50% of bead-bound fractions (“beads”) were analyzed by SDS-PAGE and western blotting.</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^as</i> allele and were treated with either DMSO (−) or 20 µM 3-MB-PP1 (+) for two hours before crosslinking for ChIP (also indicated by “3MB” at the far right). Assays were quantified by qPCR using primers specific for the <i>act1</i>⁺ (left) or <i>adh1</i>⁺ (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>⁺ (left) is used throughout the figure. Error bars denote standard deviations from 2–3 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 “1.” The PAF pathway, involving multiple Cdk9 targets, is labeled “2.” Potential crosstalk between the two pathways is indicated by the broken double arrow. See text for details.</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. “Septa” refers to cells with at least one visible division septum, “multi-sep” refers to cells with twinned septa or multiple septa between two nuclei, and “chains” refers to unseparated chains of cells. Error bars denote standard deviations from 3 independent experiments.</p