Gcn5 facilitates Pol II progression, rather than recruitment to nucleosome-depleted stress promoters, in Schizosaccharomyces pombe

In the fission yeast, the MAP kinase Sty1 and the transcription factor Atf1 regulate up to 400 genes in response to environmental signals, and both proteins have been shown to bind to their promoters in a stress-dependent manner. In a genetic search, we have isolated the histone H3 acetyltransferase Gcn5, a component of the SAGA complex, as being essential for oxidative stress survival and activation of those genes. Upon stress, Gcn5 is recruited to promoters and coding sequences of stress genes in a Sty1- and Atf1-dependent manner, causing both an enhanced acetylation of histone H3 and nucleosome eviction. Unexpectedly, recruitment of RNA polymerase II (Pol II) is not impaired in Δgcn5 cells. We show here that stress genes display a 400-bp long nucleosome depleted region upstream of the transcription start site even prior to activation. Stress treatment does not alter promoter nucleosome architecture, but induces eviction of the downstream nucleosomes at stress genes, which is not observed in Δgcn5 cells. We conclude that, while Pol II is recruited to nucleosome-free stress promoters in a transcription factor dependent manner, Gcn5 mediates eviction of nucleosomes positioned downstream of promoters, allowing efficient Pol II progression along the genes

Cuf2 Is a Novel Meiosis-Specific Regulatory Factor of Meiosis Maturation

Meiosis is the specialized form of the cell cycle by which diploid cells produce the haploid gametes required for sexual reproduction. Initiation and progression through meiosis requires that the expression of the meiotic genes is precisely controlled so as to provide the correct gene products at the correct times. During meiosis, four temporal gene clusters are either induced or repressed by a cascade of transcription factors

A Gene Cluster Involved in Metal Homeostasis in the Cyanobacterium Synechocystis sp. Strain PCC 6803

A gene cluster composed of nine open reading frames (ORFs) involved in Ni(2+), Co(2+), and Zn(2+) sensing and tolerance in the cyanobacterium Synechocystis sp. strain PCC 6803 has been identified. The cluster includes an Ni(2+) response operon and a Co(2+) response system, as well as a Zn(2+) response system previously described. Expression of the Ni(2+) response operon (nrs) was induced in the presence of Ni(2+) and Co(2+). Reduced Ni(2+) tolerance was observed following disruption of two ORFs of the operon (nrsA and nrsD). We also show that the nrsD gene encodes a putative Ni(2+) permease whose carboxy-terminal region is a metal binding domain. The Co(2+) response system is composed of two divergently transcribed genes, corR and corT, mutants of which showed decreased Co(2+) tolerance. Additionally, corR mutants showed an absence of Co(2+)-dependent induction of corT, indicating that CorR is a transcriptional activator of corT. To our knowledge, CorR is the first Co(2+)-sensing transcription factor described. Our data suggest that this region of the Synechocystis sp. strain PCC 6803 genome is involved in sensing and homeostasis of Ni(2+), Co(2+), and Zn(2+)

The <i>cuf2</i>Δ/<i>cuf2</i>Δ mutant is defective in FSM formation.

<p><i>A</i>, Both wild-type diploid (<i>cuf2⁺/cuf2⁺</i>) and <i>cuf2Δ/cuf2Δ</i> mutant cells expressing GFP-Psy1 were synchronously induced to undergo azygotic meiosis. At the 9 h time point, FSM formation was monitored by detecting the GFP-Psy1 signal by fluorescence microscopy. When GFP-Psy1 localized to 4 circular FSM structures, these FSMs were classified as normal (i). When GFP-Psy1 localized to either lesser or greater than 4 circular FSMs or shmoo-like structures, FSMs were classified as abnormal (ii, iii and iv). <i>B</i>, Histograms showing the percentages of each normal (i) and abnormal (ii, iii, iv) FSM structure in both wild-type (<i>cuf2⁺/cuf2⁺</i>) and <i>cuf2Δ/cuf2Δ</i> mutant cells, as well as in a diploid <i>cuf2Δ/cuf2Δ</i> disruption strain in which wild-type copies of the <i>cuf2⁺-GFP</i> fusion gene were reintegrated. <i>C</i>, Typical images of FSM structures 9 h after meiotic induction in both wild-type (<i>cuf2⁺/cuf2⁺</i>) and <i>cuf2Δ/cuf2Δ</i> mutant cells (top panels). Each strain had previously been transformed with pJK210GFP-Psy1, which encodes GFP-Psy1 that is used as an FSM-resident marker. Hoechst 33342 staining was used to visualize the chromosomal DNA (middle panels). The merged images of the GFP-Psy1 and the Hoechst 33342 dye are shown in the bottom panels. Anucleated FSM structures, or unpackaged nuclei, are indicated by the white arrows.</p

Riboprobes used to detect steady-state levels of transcripts.

<p>Riboprobes used to detect steady-state levels of transcripts.</p

Comparison of Cuf2 with the <i>S. pombe</i> copper metalloregulatory transcription factor Cuf1.

<p><i>A</i>, Schematic representations of Cuf1 and Cuf2. The amino acid sequences of both proteins are numbered relative to their initiator codons. The locations of the domains required for Cuf1 function are indicated, including the N-terminal nuclear localization signal (NLS) (11–53) that is located within the DNA-binding module (1–174), the C-terminal Cu-sensing module (C-rich) (328–342) and the C-terminal nuclear export signal (NES) (349–358). The positions of some of the Cys (C) and His (H) residues within both Cuf1 and Cuf2 are also indicated. <i>B</i>, Amino acid alignment of the N-terminal 61 amino acid residues of Cuf1 with the N-terminal 60-residue segment of Cuf2. The black boxes indicate identical amino acids, and the gray boxes indicate amino acids that are similar between Cuf2 and Cuf1. The asterisks highlight the 7 Cys residues that are conserved between Cuf2 and Cuf1. The indicated N-terminal 1–60 amino acid region of Cuf2 exhibits 42% sequence identity and 62% sequence similarity with the N-terminal 61 amino acids of Cuf1 that are part of its DNA-binding domain.</p

PCR primers for amplification of the modules in Figure 3.

1<p>The forward primer is identical for all modules described here and is the same as for previously described modules containing <i>nmt1-</i>derived promoters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001428#pone.0001428-Bhler1" target="_blank">[26]</a>. The gene-specific portion of the primer is typically chosen to correspond to sequences 100–200 bp upstream of the start codon. A web-based tool for automated primer design is available for these primers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001428#pone.0001428-Penkett1" target="_blank">[35]</a>.</p>2<p>A 25-mer universal sequence is used to anneal to P<i>urg1</i> due to the AT-rich nature of this sequence; the gene-specific portion is therefore reduced to 75 bp for 100-mer primers, which does not seem to affect targeting efficiency. The complement start codon is indicated in <i>italic</i>. For regulated expression of full length proteins, the gene-specific portion of the primer corresponds to the complement of the N-terminal codons of the target gene (<i>without</i> start codon).</p>3<p>The reading frames of the tag sequences are indicated. These primers are the same as for the corresponding modules containing <i>nmt1-</i>derived promoters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001428#pone.0001428-Bhler1" target="_blank">[26]</a>. For N-terminal tagging of full-length proteins, the gene-specific portion of the primer corresponds to the complement of the N-terminal codons of the target gene (<i>including</i> start codon). Note that the 3′ portions of these primers are specific to the tags and correspond to the complement of the C-terminal tag codons (without stop codon). A web-based tool for automated primer design is available for these primers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001428#pone.0001428-Penkett1" target="_blank">[35]</a>.</p

Assessment of the mRNA and protein steady-state levels of Cuf2 during meiosis.

<p><i>A</i>, Representative expression profiles of the <i>cuf2</i>⁺ and <i>ctr4</i>⁺ mRNAs in <i>h</i>⁺ haploid cells that were either left untreated (−) or were treated with either 50 µM TTM or 50 µM CuSO₄ during mitosis. <i>B</i>, Cultures of <i>pat1-114/pat1-114</i> diploid cells were either maintained in vegetative growth at 25°C, or were induced to initiate and proceed through meiosis at 34°C. <i>pat1-114/pat1-114</i> diploid cells were either left untreated, or incubated in the presence of 50 µM TTM or 50 µM CuSO₄. Total RNA was isolated at the indicated time points after the induction of meiosis. Shown are representative RNase protection assays of both the <i>cuf2⁺</i> and the <i>act1⁺</i> (internal control) mRNA steady-state levels during meiosis. <i>C</i>, Cuf2-TAP protein expression during meiosis. <i>pat1-114/pat1-114 cuf2Δ/cuf2Δ</i> diploid cells expressing Cuf2-TAP were either left uninduced (25°C), or were induced (34°C) under basal conditions or in the presence of 50 µM TTM or 50 µM CuSO₄. Shown are Western blots of both Cuf2-TAP and α-tubulin (control loading) levels at different time points after meiotic induction. <i>D</i>, Meiotic progression of cells under either basal (untreated) conditions, or in the presence of TTM (50 µM) or CuSO₄ (50 µM). The values shown for each condition (TTM, basal or Cu) correspond to the percentage of cells with 1, 2, or 3–4 nuclei, and the percentage of cells with horse tails. The graphed values represent the averages of triplicate determinations +/− the standard deviations.</p

<i>S pombe</i> strains used in this study.

<p><i>S pombe</i> strains used in this study.</p