Antisense Transcription Controls Cell Fate in Saccharomyces cerevisiae

SummaryEntry into meiosis is a key developmental decision. We show here that meiotic entry in Saccharomyces cerevisiae is controlled by antisense-mediated regulation of IME4, a gene required for initiating meiosis. In MAT a/α diploids the antisense IME4 transcript is repressed by binding of the a1/α2 heterodimer at a conserved site located downstream of the IME4 coding sequence. MAT a/α diploids that produce IME4 antisense transcript have diminished sense transcription and fail to initiate meiosis. Haploids that produce the sense transcript have diminished antisense transcription and manifest several diploid phenotypes. Our data are consistent with transcription interference as a regulatory mechanism at the IME4 locus that determines cell fate

A Toggle Involving Cis-Interfering Noncoding RNAs Controls Variegated Gene Expression in Yeast

The identification of specific functional roles for the numerous long noncoding (nc)RNAs found in eukaryotic transcriptomes is currently a matter of intense study amid speculation that these ncRNAs have key regulatory roles. We have identified a pair of cis-interfering ncRNAs in yeast that contribute to the control of variegated gene expression at the FLO11 locus by implementing a regulatory circuit that toggles between two stable states. These capped, polyadenylated ncRNAs are transcribed across the large intergenic region upstream of the FLO11 ORF. As with mammalian long intervening (li)ncRNAs, these yeast ncRNAs (ICR1 and PWR1) are themselves regulated by transcription factors (Sfl1 and Flo8) and chromatin remodelers (Rpd3L) that are key elements in phenotypic transitions in yeast. The mechanism that we describe explains the unanticipated role of a histone deacetylase complex in activating gene expression, because Rpd3L mutants force the ncRNA circuit into a state that silences the expression of the adjacent variegating gene

Neurospora Importin α Is Required for Normal Heterochromatic Formation and DNA Methylation

<div><p>Heterochromatin and associated gene silencing processes play roles in development, genome defense, and chromosome function. In many species, constitutive heterochromatin is decorated with histone H3 tri-methylated at lysine 9 (H3K9me3) and cytosine methylation. In <i>Neurospora crassa</i>, a five-protein complex, DCDC, catalyzes H3K9 methylation, which then directs DNA methylation. Here, we identify and characterize a gene important for DCDC function, <i>dim-3</i> (<i>d</i>efective <i>i</i>n <i>m</i>ethylation-3), which encodes the nuclear import chaperone NUP-6 (Importin α). The critical mutation in <i>dim-3</i> results in a substitution in an ARM repeat of NUP-6 and causes a substantial loss of H3K9me3 and DNA methylation. Surprisingly, nuclear transport of all known proteins involved in histone and DNA methylation, as well as a canonical transport substrate, appear normal in <i>dim-3</i> strains. Interactions between DCDC members also appear normal, but the <i>nup-6dim-3</i> allele causes the DCDC members DIM-5 and DIM-7 to mislocalize from heterochromatin and NUP-6^<i>dim-3</i> itself is mislocalized from the nuclear envelope, at least in conidia. GCN-5, a member of the SAGA histone acetyltransferase complex, also shows altered localization in <i>dim-3</i>, raising the possibility that NUP-6 is necessary to localize multiple chromatin complexes following nucleocytoplasmic transport.</p></div

Interactions between DCDC members are not compromised by the mutations in <i>dim-3</i>.

<p>[A] Western blots detecting DIM-5 interaction with DIM-7-3X-FLAG or DIM-9-3XFLAG from <i>dim-3</i>⁺ (WT) or <i>dim-3</i> nuclei. For quantification of purified DIM-5, levels of DIM-7/9-3xFLAG IP from WT and <i>dim-3</i> nuclei were equalized and the level of purified DIM-5 from <i>dim-3</i> was normalized to the adjusted DIM-7/9-3xFLAG IP level from <i>dim-3</i>. The experiment performed in triplicate, and average percent and standard deviation of wild type DIM-5 is noted below. [B] Western blots detecting DIM-9-3xHA interaction with purified DIM-8-3X-FLAG from <i>dim-3</i>⁺ (WT) or <i>dim-3</i> nuclei. Experiment performed in duplicate and normalized as in [A], with average noted. Control experiment with DIM-9-3xHA co-IP from WT and <i>dim-3</i> nuclei without FLAG-tagged protein demonstrates α-FLAG IP specificity. The star indicates a non-specific band routinely detected in experiments with Neurospora nuclear extracts probed with the rabbit-derived FLAG antibody. [C] Western blots detecting DIM-9-3xHA interaction with purified DIM-7-3X-FLAG from <i>dim-3</i>⁺ (WT) or <i>dim-3</i> nuclei. The experiment was performed in triplicate and normalized as in [A].</p

Histidine supplementation exacerbates loss of DNA methylation in <i>dim-3</i> strains by displacing DIM-2 from heterochromatin.

<p>[A] Southern blot assay (as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005083#pgen.1005083.g001" target="_blank">Fig. 1A</a>) of DNA methylation in <i>dim-3</i>⁺ (WT) or <i>dim-3</i> prototrophic strains grown in minimal or histidine supplemented medium. [B] (left) α-FLAG western blots showing DIM-2-3xFLAG levels in WT and <i>dim-3</i> nuclei grown in medium with or without histidine compared with a loading control (histone H3, [hH3]); (right) Quantification of DIM-2-3xFLAG levels from three independent experiments, normalized to hH3 levels. [C-D] Southern blot assay of DamID experiments with WT and <i>dim-3</i> strains containing [C] DIM-2-DAM or [D] HP1-DAM, grown with or without histidine, and probed for the heterochromatic region 2:B3. Genomic DNA was digested with DpnI (DI), specifically cutting GA^mTC sequences or with the A^m-insensitive isoschizomer DpnII (DII) to monitor complete digestion.</p

Abnormal localization of NUP-6^<i>dim-3</i> and increased euchromatic localization of SAGA.

<p>[A-B] Representative differential interference contrast (DIC), GFP fluorescent, Hoechst33342 stained DNA, and merged images of [A] <i>dim-3</i>^<i>+</i> cells expressing NUP-6-GFP or [B] <i>dim-3</i> cells expressing NUP-6^<i>dim-3</i>-GFP. Each panel displays one conidium, with one to three nuclei visualized. Top panels show examples of localization at nuclear periphery and bottom panels show examples of dispersed nuclear localization. Percentages of conidia exhibiting each pattern are listed on the right; the P value for loss of membrane localization is <1x10⁻¹²⁴ (X²-test). Densely staining DNA foci are thought to represent centromeric heterochromatin [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005083#pgen.1005083.ref011" target="_blank">11</a>]. Scale bar indicates 5 μm. [C-D] Differential interference contrast (DIC), GFP fluorescence, mCherry fluorescence, and merged images of strains expressing the nuclear pore complex member NUP-84-mCherry and [C] NUP-6-GFP or [D] NUP-6^<i>dim-3</i>-GFP. Scale bar indicates 5 μm. For C, one conidium with three nuclei (one out of the focal plane) is displayed. For D, a conidium with one nucleus is displayed. [E-F] DamID experiment, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005083#pgen.1005083.g003" target="_blank">Fig. 3</a>, of SAGA complex members GCN-5-DAM and TAF-5-DAM in <i>dim-3</i>^<i>+</i> (WT) and <i>dim-3</i> strains, probed for [E] euchromatic regions (<i>am</i>, <i>hpo</i>) or [F] a heterochromatic region (8:A6). [G] Illustrations depicting how heterochromatin and DNA methylation depend, at least in part, on a nuclear transport-independent role of NUP-6. In wild type strains (left), DCDC components are hypothetically transported into the nucleus by the NUP-6/Importin β dimer through the Nuclear Pore Complex (NPC), and NUP-6 facilitates formation of active DCDC, which then catalyzes H3K9 methylation. HP1 binds H3K9me3 and directly recruits DIM-2, catalyzing DNA methylation (mC). In the <i>dim-3</i> mutant (right), decreased H3K9me3 and DNA methylation at heterochromatin results from diminished localization of DIM-7 and DIM-5 to A-T-rich DNA, despite appropriate nuclear transport. Remaining H3K9me3 is bound by HP1, which efficiently recruits DIM-2 if histidine is absent. <i>Dim-3</i> strains also exhibit an increase in nuclear DIM-9 and an increase in GCN-5 targeting to euchromatin.</p

Nuclear transport of DCDC components is unaffected by the <i>dim-3</i> mutation.

<p>[A] α-FLAG and α-histone H3 (hH3; loading control) western blots of <i>dim-3</i>⁺ (WT) or <i>dim-3</i> nuclei expressing individually FLAG-tagged DCDC components. All strains were grown in the presence of histidine. Average and standard deviation of nuclear FLAG-tagged protein levels of three experiments are indicated below the representative images shown. [B] α-FLAG western blots of the total (T) proteins, cytoplasmic (C) fraction, and nuclear (N) fraction of nuclei preparations from <i>dim-3</i>⁺ (WT) and <i>dim-3</i> strains expressing DIM-5-3xFLAG, DIM-7-3xFLAG, or DIM-9-3xFLAG. Representative α-histone H3 (hH3) and α-PGK blots from the DIM-5-3xFLAG nuclei are shown as nuclear and cytoplasmic fraction controls, respectively; equivalent results were obtained for all nuclei preparations. [C] Representative differential interference contrast (DIC) and GFP fluorescent images of (left) <i>dim-3</i>^<i>+</i> or (right) <i>dim-3</i> strains expressing an overexpressed NLS^SV40-GFP reporter construct (pCCG::NLS^SV40::LexADBD::GFP). While just one representative, multinucleate cell is displayed in the figures here and elsewhere in the paper, we visualized numerous vegetative cells, including both conidia and hyphal cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005083#pgen.1005083.ref062" target="_blank">62</a>]. Percentages of conidia exhibiting each pattern are listed on the right (P value = 0.43, X²-test). Scale bar indicates 5μm.</p

Global reduction of H3K9me3 in <i>dim-3</i>compromises telomeric silencing.

<p>[A] Western blots of histones isolated from indicated strains were probed for H3K9me3, or histone H3 (hH3) as a loading control. [B] (Left) Western blots of histones isolated from the indicated strains grown with or without histidine probed for H3K9me3 or hH3. (Right) Quantification of H3K9me3 levels from three independent experiments, normalized to hH3 levels. [C] Chromatin Immunoprecipitation sequencing (ChIP-seq) of H3K9me3 from <i>dim-3</i>⁺ (WT) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005083#pgen.1005083.ref056" target="_blank">56</a>] and <i>dim-3</i> strains displayed on IGV, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005083#pgen.1005083.g001" target="_blank">Fig. 1B</a>. Due to the vastly reduced trimethylation of H3K9 in the <i>dim-3</i> strain, this strain shows a reduced signal-to-noise ratio, rendering background more prominent. [D] Growth of strains containing the <i>telVR</i>::<i>hph</i> or <i>cenVIR</i>::<i>bar</i> reporter cassettes on minimum medium (MIN) plates or plates supplemented with hygromycin (200 μg/mL, +HYG, left) or phosphinothricin (8 mg/mL, +BASTA, right). Approximate numbers of spotted conidia indicated below the pictures.</p