59 research outputs found
Transcriptional Memory: Staying in the Loop
No full textSummaryActively transcribed genes are organized into loops in which the 5′ and 3′ ends of the gene physically associate. Two new papers show that gene looping can persist after genes are repressed, promoting rapid reactivation of transcription, a phenomenon known as transcriptional memory
Nuclear Architecture: The Cell Biology of a Laminopathy
No full textSummaryLamin mutations cause muscular dystrophies, but the mechanism is unclear. A new study shows that lamin mutant worms display muscle-specific defects linked to altered subnuclear localization of heterochromatin, leading to altered gene expression
Gene recruitment of the activated INO1 locus to the nuclear membrane.
No full textThe spatial arrangement of chromatin within the nucleus can affect reactions that occur on the DNA and is likely to be regulated. Here we show that activation of INO1 occurs at the nuclear membrane and requires the integral membrane protein Scs2. Scs2 antagonizes the action of the transcriptional repressor Opi1 under conditions that induce the unfolded protein response (UPR) and, in turn, activate INO1. Whereas repressed INO1 localizes throughout the nucleoplasm, the gene is recruited to the nuclear periphery upon transcriptional activation. Recruitment requires the transcriptional activator Hac1, which is produced upon induction of the UPR, and is constitutive in a strain lacking Opi1. Artificial recruitment of INO1 to the nuclear membrane permits activation in the absence of Scs2, indicating that the intranuclear localization of a gene can profoundly influence its mechanism of activation. Gene recruitment to the nuclear periphery, therefore, is a dynamic process and appears to play an important regulatory role
Cdk Phosphorylation of a Nucleoporin Controls Localization of Active Genes through the Cell Cycle
No full textMany inducible genes in yeast are targeted to the nuclear pore complex when active. We find that the peripheral localization of the INO1 and GAL1 genes is regulated through the cell cycle. Active INO1 and GAL1 localized at the nuclear periphery during G1, became nucleoplasmic during S-phase, and then returned to the nuclear periphery during G2/M. Loss of peripheral targeting followed the initiation of DNA replication and was lost in cells lacking a cyclin-dependent kinase (Cdk) inhibitor. Furthermore, the Cdk1 kinase and two Cdk phosphorylation sites in the nucleoporin Nup1 were required for peripheral targeting of INO1 and GAL1. Introduction of aspartic acid residues in place of either of these two sites in Nup1 bypassed the requirement for Cdk1 and resulted in targeting of INO1 and GAL1 to the nuclear periphery during S-phase. Thus, phosphorylation of a nuclear pore component by cyclin dependent kinase controls the localization of active genes to the nuclear periphery through the cell cycle.</jats:p
UPR-Dependent Dissociation of Opi1 from Chromatin
No full text<div><p>(A) Chromatin-associated Opi1 dissociates upon activation of the UPR. Cells of the indicated genotypes were harvested after growth for 4.5 h with or without <i>myo</i>-inositol, fixed, and processed as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020342#pbio-0020342-g002" target="_blank">Figure 2</a>. The <i>scs2</i>Δ mutant was transformed with pRS315-Opi1-<i>myc</i>, a <i>CEN ARS</i> plasmid that expresses Opi1-<i>myc</i> at endogenous levels. Input DNA (In) and immunoprecipitated DNA (IP) were analyzed by PCR using primers to amplify the <i>INO1</i> promoter and the <i>URA3</i> gene. Amplified DNA was separated by electrophoresis on ethidium bromide–stained agarose gels.</p>
<p>(B) Quantitative PCR analysis. Input and IP fractions were analyzed by real-time quantitative PCR. The ratio of <i>INO1</i> promoter to <i>URA3</i> template in the reaction is shown. Error bars represent the SEM between experiments.</p></div
Ino2/Ino4 Bind to the <i>INO1</i> Promoter Constitutively
No full text<div><p>(A) Untagged control cells (upper images), or cells in which the endogenous copies of <i>INO2</i> and <i>INO4</i> were replaced with HA-tagged Ino2 (center images) or HA-tagged Ino4 (lower images) were harvested in mid-logarithmic phase and washed into medium with or without <i>myo</i>-inositol. After 4.5 h, about 1.5 × 10<sup>8</sup> cells were harvested and processed for Northern blot analysis (light images with dark bands, right). Northern blots were probed against both <i>INO1</i> and <i>ACT1</i> (loading control) mRNA. The remaining cells were fixed with formaldehyde and lysed. Chromatin was sheared by sonication and then subjected to immunoprecipitation with anti-HA agarose. Input DNA (In) and immunoprecipitated DNA (IP) were analyzed by PCR using primers to amplify the <i>INO1</i> promoter and the <i>URA3</i> gene. Amplified DNA was size-fractionated by electrophoresis on ethidium bromide-stained agarose gels (dark images with light bands, left).</p>
<p>(B) Quantitative PCR analysis. Input and IP fractions were analyzed by real-time quantitative PCR. The ratio of <i>INO1</i> promoter to <i>URA3</i> template in the reaction is shown. Error bars represent the standard error of the mean (SEM) between experiments.</p></div
Artificial Relocalization of <i>INO1</i> Bypasses the Requirement for Scs2
No full text<div><p>(A) Northern blot analysis of membrane-targeted <i>INO1</i>. Strains of the indicated genotypes having the Lac operator array integrated at <i>INO1</i> and expressing either the wild-type GFP-Lac repressor or GFP-FFAT-Lac repressor were grown in the presence or absence of 1 μg/ml tunicamycin (Tm) for 4.5 h, harvested, and analyzed by Northern blot. Blots were probed for either <i>INO1</i> or <i>ACT1</i> (as a loading control) mRNA. The wild-type strain CRY1, lacking both the Lac operator array and the Lac repressor, was included in the first two lanes for comparison.</p>
<p>(B) Wild-type or <i>scs2</i>Δ mutant strains in which the Lac operator had been integrated at <i>INO1</i> were transformed with either GFP-Lac repressor or GFP-FFAT-Lac repressor. The resulting transformants were serially diluted (tenfold between wells) and spotted onto medium lacking inositol, uracil, and histidine, and incubated for 2 d at 37 °C.</p>
<p>(C) Wild-type and <i>scs2</i>Δ mutant strains transformed with either GFP-Lac repressor or GFP-FFAT-Lac repressor, but lacking the Lac operator, were streaked onto medium lacking inositol and histidine and incubated for 2 d at 37 °C.</p></div
Genetic and Epigenetic Strategies Potentiate Gal4 Activation to Enhance Fitness in Recently Diverged Yeast Species
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