elife03635.xml

<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" article-type="research-article" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">03635</article-id><article-id pub-id-type="doi">10.7554/eLife.03635</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Computational and systems biology</subject></subj-group><subj-group subj-group-type="heading"><subject>Genes and chromosomes</subject></subj-group></article-categories><title-group><article-title>Transcription mediated insulation and interference direct gene cluster expression switches</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-15469"><name><surname>Nguyen</surname><given-names>Tania</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="other" rid="par-6"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15470" equal-contrib="yes"><name><surname>Fischl</surname><given-names>Harry</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="equal-contrib1">&#x2020;</xref><xref ref-type="fn" rid="equal-contrib2">&#x2021;</xref><xref ref-type="other" rid="par-8"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15473" equal-contrib="yes"><name><surname>Howe</surname><given-names>Fran&#xe7;oise S</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="equal-contrib1">&#x2020;</xref><xref ref-type="fn" rid="equal-contrib2">&#x2021;</xref><xref ref-type="other" rid="par-8"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15474" equal-contrib="yes"><name><surname>Woloszczuk</surname><given-names>Ronja</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="equal-contrib1">&#x2020;</xref><xref ref-type="fn" rid="equal-contrib2">&#x2021;</xref><xref ref-type="other" rid="par-9"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15471" equal-contrib="yes"><name><surname>Serra Barros</surname><given-names>Ana</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="equal-contrib3">&#xa7;</xref><xref ref-type="other" rid="par-7"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15472" equal-contrib="yes"><name><surname>Xu</surname><given-names>Zhenyu</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="fn" rid="equal-contrib3">&#xa7;</xref><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15475"><name><surname>Brown</surname><given-names>David</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="other" rid="par-8"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15476"><name><surname>Murray</surname><given-names>Struan C</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="other" rid="par-9"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15501"><name><surname>Haenni</surname><given-names>Simon</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-9493"><name><surname>Halstead</surname><given-names>James M</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15479"><name><surname>O'Connor</surname><given-names>Leigh</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="con11"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-20317"><name><surname>Shipkovenska</surname><given-names>Gergana</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="con12"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-15481"><name><surname>Steinmetz</surname><given-names>Lars M</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-5"/><xref ref-type="fn" rid="con13"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" corresp="yes" id="author-8056"><name><surname>Mellor</surname><given-names>Jane</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1">&#x2a;</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con14"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><label>1</label><institution content-type="dept">Department of Biochemistry</institution>, <institution>University of Oxford</institution>, <addr-line><named-content content-type="city">Oxford</named-content></addr-line>, <country>United Kingdom</country></aff><aff id="aff2"><label>2</label><institution content-type="dept">Genome Biology Unit</institution>, <institution>European Molecular Biology Laboratory</institution>, <addr-line><named-content content-type="city">Heidelberg</named-content></addr-line>, <country>Germany</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Espinosa</surname><given-names>Joaquin M</given-names></name><role>Reviewing editor</role><aff><institution>Howard Hughes Medical Institute, University of Colorado</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>&#x2a;</label>For correspondence: <email>jane.mellor@bioch.ox.ac.uk</email></corresp><fn fn-type="con" id="equal-contrib1"><label>&#x2020;</label><p>These authors contributed equally to this work</p></fn><fn fn-type="con" id="equal-contrib2"><label>&#x2021;</label><p>These authors are joint second authors to this work</p></fn><fn fn-type="con" id="equal-contrib3"><label>&#xa7;</label><p>These authors are joint third authors to this work</p></fn></author-notes><pub-date publication-format="electronic" date-type="pub"><day>19</day><month>11</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e03635</elocation-id><history><date date-type="received"><day>08</day><month>06</month><year>2014</year></date><date date-type="accepted"><day>17</day><month>11</month><year>2014</year></date></history><permissions><copyright-statement>&#xa9; 2014, Nguyen et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Nguyen et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife03635.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.03635.001</object-id><p>In yeast, many tandemly arranged genes show peak expression in different phases of the metabolic cycle (YMC) or in different carbon sources, indicative of regulation by a bi-modal switch, but it is not clear how these switches are controlled. Using native elongating transcript analysis (NET-seq), we show that transcription itself is a component of bi-modal switches, facilitating reciprocal expression in gene clusters. <italic>HMS2</italic>, encoding a growth-regulated transcription factor, switches between sense- or antisense-dominant states that also coordinate up- and down-regulation of transcription at neighbouring genes. Engineering <italic>HMS2</italic> reveals alternative mono-, di- or tri-cistronic and antisense transcription units (TUs), using different promoter and terminator combinations, that underlie state-switching. Promoters or terminators are excluded from functional TUs by read-through transcriptional interference, while antisense TUs insulate downstream genes from interference. We propose that the balance of transcriptional insulation and interference at gene clusters facilitates gene expression switches during intracellular and extracellular environmental change.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.001">http://dx.doi.org/10.7554/eLife.03635.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.03635.002</object-id><title>eLife digest</title><p>A DNA double helix is made up of two DNA strands, which in turn are made of molecules that are each known by a single letter&#x2014;A, T, C, or G. The sequence of these &#x2018;letters&#x2019; in each DNA strand contains biological information.</p><p>Genes are sections of DNA that can be &#x2018;expressed&#x2019; to produce proteins and RNA molecules. To express a gene, the DNA strands in the double helix must first be partially separated so that one of them can be used as a template to build an RNA molecule in a process called transcription. Either of the DNA strands in a helix can be used as an RNA template, but contain different genes and are read in opposite directions. One of the two strands is called the &#x2018;sense&#x2019; strand, the other the &#x2018;antisense&#x2019; strand.</p><p>The RNA molecule does not transcribe a whole DNA strand; instead, it transcribes a section of DNA, known as a transcription unit, which contains at least one gene. The end of a transcription unit is marked by certain signals that stop transcription. However, some transcription units in a DNA strand overlap, so there must be some way that the transcription machinery can sometimes ignore these stop signals.</p><p>The activity of some genes is linked to the activity of their immediate neighbours. Furthermore, some genes are expressed in different amounts in response to changes in environmental conditions. Researchers have previously suggested that there must be some form of switch that controls when these genes are expressed.</p><p>Nguyen et al. now engineer start and stop signals at a neighbouring pair of genes, called <italic>HMS2</italic> and <italic>BAT2</italic>, in yeast. When one gene is switched on, the other is switched off and which gene is active depends on the diet of the yeast cells.</p><p>On the antisense DNA strand opposite to <italic>HMS2</italic> is another gene, <italic>SUT650</italic>. Nguyen et al. show that when this gene is transcribed, the transcription of <italic>HMS2</italic> on the other DNA strand is blocked. This has the knock-on effect of turning on <italic>BAT2</italic>. Conversely, transcribing <italic>HMS2</italic> switches off <italic>SUT650</italic> and <italic>BAT2</italic> because the end of <italic>HMS2</italic> overlaps with the beginning of both <italic>SUT650</italic> and <italic>BAT2</italic>. Switching between different genes relies on loops that physically link the start and stop signals of the gene to be transcribed while ignoring the start and stop signals for neighbouring genes.</p><p>Proteins called transcription factors can bind to DNA and affect whether a gene is transcribed. Nguyen et al. found that a transcription factor that binds near the start of the <italic>HMS2</italic> gene helps to control which DNA strand is transcribed. When transcription factors do not bind to the start of <italic>HMS2</italic>, antisense transcription&#x2014;and the expression of <italic>SUT650</italic>&#x2014;occurs instead.</p><p>Overall, Nguyen et al. show that the transcription process itself makes up part of a switch that can control the expression of several genes on both the sense and antisense strands of a DNA double helix. This may also explain how many other, more complex, gene networks are activated in response to changes in the environment.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.002">http://dx.doi.org/10.7554/eLife.03635.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>transcription interference</kwd><kwd>yeast metabolic cycle</kwd><kwd>HMS2:BAT2</kwd><kwd>transcription insulation</kwd><kwd>cycling transcripts</kwd><kwd>gene clusters</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>S. cerevisiae</italic></kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100004440</institution-id><institution>Wellcome Trust</institution></institution-wrap></funding-source><award-id>WT089156MA</award-id><principal-award-recipient><name><surname>Mellor</surname><given-names>Jane</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution content-type="university">National Institutes of Health</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Steinmetz</surname><given-names>Lars M</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>Oxford Biodynamics</institution></institution-wrap></funding-source><award-id>ALRNEI1</award-id><principal-award-recipient><name><surname>Mellor</surname><given-names>Jane</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100000780</institution-id><institution>European Commission</institution></institution-wrap></funding-source><award-id>Epigenesys FP7 Network</award-id><principal-award-recipient><name><surname>Mellor</surname><given-names>Jane</given-names></name></principal-award-recipient></award-group><award-group id="par-5"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001659</institution-id><institution>Deutsche Forschungsgemeinschaft</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Steinmetz</surname><given-names>Lars M</given-names></name></principal-award-recipient></award-group><award-group id="par-6"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100000038</institution-id><institution>Natural Sciences and Engineering Research Council of Canada</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Nguyen</surname><given-names>Tania</given-names></name></principal-award-recipient></award-group><award-group id="par-7"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001871</institution-id><institution>Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Serra Barros</surname><given-names>Ana</given-names></name></principal-award-recipient></award-group><award-group id="par-8"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100004440</institution-id><institution>Wellcome Trust</institution></institution-wrap></funding-source><award-id>Graduate Studentships</award-id><principal-award-recipient><name><surname>Fischl</surname><given-names>Harry</given-names></name><name><surname>Howe</surname><given-names>Fran&#xe7;oise S</given-names></name><name><surname>Brown</surname><given-names>David</given-names></name></principal-award-recipient></award-group><award-group id="par-9"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100000266</institution-id><institution>Engineering and Physical Sciences Research Council</institution></institution-wrap></funding-source><award-id>Graduate Studentships</award-id><principal-award-recipient><name><surname>Woloszczuk</surname><given-names>Ronja</given-names></name><name><surname>Murray</surname><given-names>Struan C</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>The formation of mutually exclusive coding and non-coding transcription units contributes to transcriptional interference and insulation at gene clusters and manages state-switching in response to environmental change.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type="intro" id="s1"><title>Introduction</title><p>Genome-wide mapping of RNA transcripts in the budding yeast <italic>Saccharomyces cerevisiae</italic> has revealed an extensive array of coding and non-coding transcripts, giving rise to a genome that is heavily interleaved. Individual genes can possess multiple, overlapping transcripts, in the sense and the antisense orientations with respect to the pre-mRNA, as well as poly-cistronic transcripts, which span neighbouring genes (<xref ref-type="bibr" rid="bib16">Kapranov et al., 2007</xref>; <xref ref-type="bibr" rid="bib2">Berretta et al., 2008</xref>; <xref ref-type="bibr" rid="bib35">Pelechano et al., 2013</xref>). Genome-wide mapping of nascent transcription, using techniques such as NET-seq, shows that transcription commonly extends into and over the intergenic regions of both convergent and tandemly arranged genes (<xref ref-type="bibr" rid="bib7">Churchman and Weissman, 2011</xref>). For tandemly arranged genes, transcription into the promoter of the downstream gene would be expected to interfere with its expression by a variety of mechanisms, including modifying the local chromatin environment and interference by removal of transcription factors (<xref ref-type="bibr" rid="bib25">Martens et al., 2004</xref>; <xref ref-type="bibr" rid="bib26">Martianov et al., 2007</xref>; <xref ref-type="bibr" rid="bib12">Hainer et al., 2011</xref>). Furthermore, extensive transcription antisense to (<xref ref-type="bibr" rid="bib54">Venters and Pugh, 2009</xref>; <xref ref-type="bibr" rid="bib33">Murray et al., 2012</xref>), and into the promoter of (<xref ref-type="bibr" rid="bib36">Perocchi et al., 2007</xref>; <xref ref-type="bibr" rid="bib58">Xu et al., 2009</xref>), the canonical coding transcript is also implicated in modulating gene expression by similar mechanisms (<xref ref-type="bibr" rid="bib14">Hongay et al., 2006</xref>; <xref ref-type="bibr" rid="bib5">Camblong et al., 2007</xref>; <xref ref-type="bibr" rid="bib52">Uhler et al., 2007</xref>; <xref ref-type="bibr" rid="bib15">Houseley et al., 2008</xref>; <xref ref-type="bibr" rid="bib37">Pinskaya et al., 2009</xref>; <xref ref-type="bibr" rid="bib57">Xu et al., 2011</xref>; <xref ref-type="bibr" rid="bib53">van Werven et al., 2012</xref>; <xref ref-type="bibr" rid="bib6">Castelnuovo et al., 2013</xref>). An interleaved genome with overlapping transcription units requires that polyadenylation and transcription termination signals in the sense and antisense orientations are by-passed but it is not clear how this is achieved. In addition, questions are commonly raised about whether transcription of these overlapping transcription units is contemporaneous.</p><p>It is now clear that much transcription is organised into biologically relevant temporal windows within phenomena such as the metabolic cycle (<xref ref-type="bibr" rid="bib50">Tu and McKnight, 2006</xref>). Indeed, periodic or cycling expression of genes can be detected using fluorescent reporters or dual-labelled RNA FISH in cultures of asynchronous cells (<xref ref-type="bibr" rid="bib21">Laxman et al., 2010</xref>; <xref ref-type="bibr" rid="bib40">Silverman et al., 2010</xref>), or in the absence of cell division (<xref ref-type="bibr" rid="bib41">Slavov et al., 2011</xref>). This periodic expression is a result of synchronization of respiratory and glycolytic activities into robust oscillations in oxygen consumption, characterized by phase-specific transcript signatures involving over 3000 genes, known as the Yeast Metabolic Cycle (YMC) (<xref ref-type="bibr" rid="bib18">Klevecz et al., 2004</xref>; <xref ref-type="bibr" rid="bib49">Tu et al., 2005</xref>, <xref ref-type="bibr" rid="bib51">2007</xref>; <xref ref-type="bibr" rid="bib42">Soranzo et al., 2009</xref>; <xref ref-type="bibr" rid="bib41">Slavov et al., 2011</xref>; <xref ref-type="bibr" rid="bib4">Cai and Tu, 2012</xref>). In the long-period YMC, a single cell alternates between periods of high (oxidative (OX) phase) or low oxygen consumption (reductive building (RB) and charging (RC) phases), the residence time in each phase being nutrient-dependent. For exponentially growing cells in batch culture, the majority of cells in the population will be in the OX phase of the YMC (<xref ref-type="bibr" rid="bib41">Slavov et al., 2011</xref>). Transcript levels for genes that cycle in the YMC will change as the cell moves through these phases; at any time, some cells in an asynchronous population will contain a transcript and some will not. These shifts through transcriptional states are robust but not invariable, as YMC-regulated genes switch on and off in response to cues prompted by both regulated and erratic changes in the intracellular and extracellular environment.</p><p>In addition to the partitioned gene expression patterns during the YMC, genomic spatial arrangements also contribute to regulatory relationships between genes, albeit on a smaller scale (<xref ref-type="bibr" rid="bib8">Cohen et al., 2000</xref>; <xref ref-type="bibr" rid="bib22">Lee and Sonnhammer, 2003</xref>; <xref ref-type="bibr" rid="bib1">Batada et al., 2007</xref>). While genes displaying functional relatedness, such as the <italic>GAL</italic> genes, are regulated similarly by possessing the same <italic>cis</italic>-acting sequences (i.e. the UAS<sub><italic>GAL</italic></sub>), co-expression of clustered genes in <italic>S. cerevisiae</italic> is largely independent of similarly controlled transcription, gene orientation, and/or shared regulatory sequences. However, members of adjacent gene pairs or clusters are more likely to belong to the same functional pathway than expected by chance (<xref ref-type="bibr" rid="bib8">Cohen et al., 2000</xref>; <xref ref-type="bibr" rid="bib22">Lee and Sonnhammer, 2003</xref>; <xref ref-type="bibr" rid="bib1">Batada et al., 2007</xref>). The mechanism by which these clustered genes are regulated remains largely elusive.</p><p>Here we show that overlapping transcription, in both the sense and antisense orientations, constitutes an additional layer of regulation at clustered genes by managing state-switching in response to environmental change. We use a simple carbon source shift coupled with NET-seq to define genes whose transcription increases or decreases &#x3e;threefold, after transfer from glucose (GLU)- to galactose (GAL)-containing media and show a remarkable enrichment for genes whose transcripts cycle during the YMC. The majority of these genes have no functional associations with transcription factors that might mediate repression or induction in GLU or GAL, but are organised in clusters and subject to overlapping sense and antisense transcription. We exemplify this mode of gene regulation, state-switching by transcriptional interference and insulation, at the <italic>HMS2:BAT2</italic> tandem gene cluster. By engineering promoters and terminators at <italic>HMS2:BAT2</italic>, we demonstrate the formation of alternative transcription units underlies state-switching. We suggest that overlapping transcription and the formation of alternative transcription units, associated with temporally segregated gene expression, will be a general feature of gene regulation.</p></sec><sec sec-type="results" id="s2"><title>Results</title><sec id="s2-1"><title>Genome-wide response to a change in carbon source reveals genes whose transcripts cycle in the YMC</title><p>The question we address in this work is whether transcriptional interference can explain the switching on and off of genes and thus altered gene expression in response to environmental change. We used a change in carbon source by shifting exponentially growing cells from glucose- (GLU) to galactose-containing media (GAL) for 3 h and analysed the native transcripts associated with elongating RNA polymerase II (NET-seq) (<xref ref-type="bibr" rid="bib7">Churchman and Weissman, 2011</xref>) to identify genes whose transcription is altered during this environmental change (<xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1A</xref>). 10.45% (551) of ORF-Ts (open reading frame&#x2014;transcripts) showed a &#x3e;threefold increase and 9.99% (527) showed a &#x3e;threefold decrease in transcription in GAL relative to GLU (<xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1B</xref>). By comparing the NET-seq output with a microarray of Poly(A)<sup>&#x2b;</sup> RNA, we show that for the majority (&#x2248;88%) of genes, the change in transcript levels on the GLU to GAL shift reflects altered transcription rather than altered transcript stability (<xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1A</xref>). Gene ontology (GO) analysis revealed highly significant associations to growth, quiescence, and transcripts that cycle in the YMC, particularly during the oxidative (OX) and the reductive charging (RC) phases of the YMC (<xref ref-type="fig" rid="fig1">Figure 1A</xref>; <xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1C,D</xref>). The genes whose transcription decreases in GAL are enriched (467; 88.6% p &#x3c; 1x10<sup>&#x2212;5</sup>) for OX phase genes that are mainly involved in ribosome biosynthesis and growth (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). By contrast, for genes whose transcription increases in GAL, there is significant enrichment (392; 71.1%, p &#x3c; 1 &#xd7; 10<sup>&#x2212;5</sup>) for genes whose expression peaks in the reductive charging phase (RC) of the YMC (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). These genes are associated with stress resistance, metabolism, and quiescence. 33.1% (891) of the 2691 annotated OX- and RC-regulated genes also show &#x3e;threefold change on the GLU to GAL shift, supporting a shared regulatory mechanism between the YMC and the GLU to GAL shift (<xref ref-type="fig" rid="fig1">Figure 1D</xref>; <xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1C</xref>). In addition to ORF-Ts, many of the non-coding transcription events are regulated by the GLU to GAL shift. These include antisense transcription reads (to ORF-Ts), which increase in GAL compared to GLU (<xref ref-type="fig" rid="fig1">Figure 1E</xref>) and 25.7% of the 1772 annotated non-coding transcripts, the stable unannotated transcripts (SUTs), or cryptic unstable transcripts (CUTs), which show &#x3e;threefold change in transcription on the GLU to GAL shift (<xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1E</xref>).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03635.003</object-id><label>Figure 1.</label><caption><title>Reciprocal switching of expression by carbon source reveals links to the YMC.</title><p>(<bold>A</bold>) The distribution of all ORF-Ts into different YMC phases. OX, oxidative phase; RB, reductive building phase; RC, reductive charging phase of the YMC. (<bold>B</bold>, <bold>C</bold>, <bold>F</bold>) YMC profiles (SCEPTRANS; <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://moment.utmb.edu/cgi-bin/main_cc.cgi">http://moment.utmb.edu/cgi-bin/main_cc.cgi</ext-link>) (<xref ref-type="bibr" rid="bib20">Kudlicki et al., 2007</xref>) showing cycling expression at 124 genes with the (<bold>B</bold>) most reduced and (<bold>C</bold>) most increased transcription (NET-seq) after a switch from glucose (GLU) to galactose (GAL) for 3 h and (<bold>F</bold>) at the <italic>HMS2:BAT2</italic> locus. (<bold>D</bold>) Overlap between all YMC classes and all ORF-Ts showing &#x3e;threefold change in transcription on the GLU to GAL shift. (<bold>E</bold>) NET-seq reads on sense or antisense strands genome-wide in GLU (red) or GAL (blue) for ORF-Ts with peak expression in phases of the YMC indicated. (<bold>G</bold>) Strand-specific TIF-seq (<xref ref-type="bibr" rid="bib35">Pelechano et al., 2013</xref>), microarray and NET-seq data at the <italic>HMS2:BAT2</italic> locus. Profiles from cells cultured in GLU or GAL on the Watson strand (top) or Crick strand (bottom) are shown. The TIF-seq data indicate each different transcript isoform, microarray data indicate levels of steady-state poly(A)<sup>&#x2b;</sup> RNA (blue&#x2014;darker colour for more RNA) and the NET-seq data (normalized, unique, and clipped to 3&#x2032;OH) display transcript reads (scale 0&#x2013;30) associated with elongating RNAPII. Screen shots are displayed using IGV (<xref ref-type="bibr" rid="bib39">Robinson et al., 2011</xref>; <xref ref-type="bibr" rid="bib45">Thorvaldsdottir et al., 2013</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.003">http://dx.doi.org/10.7554/eLife.03635.003</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03635.004</object-id><label>Figure 1&#x2014;figure supplement 1.</label><caption><title>Exemplary tandem gene clusters and their regulation by carbon source and the YMC.</title><p>Supplements 1A&#x2013;D, 2A&#x2013;D, 3A&#x2013;C show examples of tandem gene clusters with reciprocal switching of transcription by carbon source, di-cistrons, and antisense transcripts. Supplements 3D and 4A&#x2013;D show examples of tandem gene clusters that do not show reciprocal switching of transcription by carbon source. Gene clusters were selected from the analyses in <xref ref-type="supplementary-material" rid="SD6-data">Supplementary files 1F&#x2013;J</xref>. Each panel shows the poly(A)<sup>&#x2b;</sup> RNA profile by microarray, NET-seq, TIF-seq, and YMC profile screenshots for the Watson strand (top) or the Crick strand (bottom). Transcription over intergenic regions and di-cistonic transcripts is common to both groups of clusters.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.004">http://dx.doi.org/10.7554/eLife.03635.004</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03635.005</object-id><label>Figure 1&#x2014;figure supplement 2.</label><caption><title>Exemplary tandem gene clusters and their regulation by carbon source and the YMC.</title><p>Supplements 1A&#x2013;D, 2A&#x2013;D, 3A&#x2013;C show examples of tandem gene clusters with reciprocal switching of transcription by carbon source, di-cistrons, and antisense transcripts. Supplements 3D and 4A&#x2013;D show examples of tandem gene clusters that do not show reciprocal switching of transcription by carbon source. Gene clusters were selected from the analyses in <xref ref-type="supplementary-material" rid="SD6-data">Supplementary files 1F&#x2013;J</xref>. Each panel shows the poly(A)<sup>&#x2b;</sup> RNA profile by microarray, NET-seq, TIF-seq, and YMC profile screenshots for the Watson strand (top) or the Crick strand (bottom). Transcription over intergenic regions and di-cistonic transcripts is common to both groups of clusters.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.005">http://dx.doi.org/10.7554/eLife.03635.005</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635fs002"/></fig><fig id="fig1s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03635.006</object-id><label>Figure 1&#x2014;figure supplement 3.</label><caption><title>Exemplary tandem gene clusters and their regulation by carbon source and the YMC.</title><p>Supplements 1A&#x2013;D, 2A&#x2013;D, 3A&#x2013;C show examples of tandem gene clusters with reciprocal switching of transcription by carbon source, di-cistrons, and antisense transcripts. Supplements 3D and 4A&#x2013;D show examples of tandem gene clusters that do not show reciprocal switching of transcription by carbon source. Gene clusters were selected from the analyses in <xref ref-type="supplementary-material" rid="SD6-data">Supplementary files 1F&#x2013;J</xref>. Each panel shows the poly(A)<sup>&#x2b;</sup> RNA profile by microarray, NET-seq, TIF-seq, and YMC profile screenshots for the Watson strand (top) or the Crick strand (bottom). Transcription over intergenic regions and di-cistonic transcripts is common to both groups of clusters.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.006">http://dx.doi.org/10.7554/eLife.03635.006</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635fs003"/></fig><fig id="fig1s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03635.007</object-id><label>Figure 1&#x2014;figure supplement 4.</label><caption><title>Exemplary tandem gene clusters and their regulation by carbon source and the YMC.</title><p>Supplements 1A&#x2013;D, 2A&#x2013;D, 3A&#x2013;C show examples of tandem gene clusters with reciprocal switching of transcription by carbon source, di-cistrons, and antisense transcripts. Supplements 3D and 4A&#x2013;D show examples of tandem gene clusters that do not show reciprocal switching of transcription by carbon source. Gene clusters were selected from the analyses in <xref ref-type="supplementary-material" rid="SD6-data">Supplementary files 1F&#x2013;J</xref>. Each panel shows the poly(A)<sup>&#x2b;</sup> RNA profile by microarray, NET-seq, TIF-seq, and YMC profile screenshots for the Watson strand (top) or the Crick strand (bottom). Transcription over intergenic regions and di-cistonic transcripts is common to both groups of clusters.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.007">http://dx.doi.org/10.7554/eLife.03635.007</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635fs004"/></fig></fig-group></p></sec><sec id="s2-2"><title>Global analysis reveals distinct organisation and regulation</title><p>We used a computer simulation to obtain the expected number of occurrences of genome-wide features, by randomly shuffling genes, their orientation, YMC status, and expression levels (data from <xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1A</xref>, codes in <xref ref-type="supplementary-material" rid="SD7-data">Source code 1</xref>). Selected data from this analysis are shown in <xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1F</xref>. In summary, pairs of ORF-Ts in the convergent orientation occur more often than expected, while tandemly arranged ORF-Ts occur less often than expected. YMC genes are clustered, with 503 (16.9%) YMC genes flanked by non-cycling genes, 1431 (48.4%) with a single adjacent YMC partner either upstream or downstream and 1023 (34.5%) with a neighbouring cycling gene both upstream and downstream. Compared to more isolated genes, ORF-T clusters are more likely to show increased transcription in GLU or GAL than expected, suggesting that clustering is associated with some aspect of their regulation. In addition, ORF-Ts flanked by annotated SUTs or CUTs (in any orientation) occur more often than expected and show a twofold to fourfold higher median expression upon change from GLU to GAL than the average median expression obtained through the simulations. This is consistent with CUTs and SUTs playing a role in modulating ORF-T transcription, particularly on environmental change (<xref ref-type="bibr" rid="bib57">Xu et al., 2011</xref>). Convergent overlapping arrangements occur three times more frequently than expected, whether the feature is a non-coding transcript (NC) such as a SUT or a CUT, or an ORF-T, with a median overlap of 92 bp for convergent ORF-Ts and 462 bp for ORF-Ts with non-coding transcripts. Thus clustering with overlapping transcription and transcriptional regulation are common features of the yeast genome.</p><p>As the genes whose transcription changed during the GLU/GAL shift are enriched for OX and RC YMC genes, we examined whether the nature of the flanking gene influences transcription, focusing on the tandem, and divergent combinations of these genes (<xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1F</xref>). OX genes, regardless of the nature (OX or RC) or orientation (divergent or tandem) of the upstream feature showed higher than expected transcriptional rates in GLU, but not in GAL, suggesting they are activated in GLU but not repressed in GAL. Similarly, RC genes are more likely to show higher than expected transcriptional rates in GAL. The behaviour of RC genes in GLU, however, does appear to be dependent on the orientation of the upstream OX gene. With a divergent upstream OX gene, the transcription of the downstream RC pair is not enriched or depleted in GLU. However, if the OX gene is in tandem, the RC gene is repressed in GLU. This raises the possibility that at the OX.RC tandem pair, transcription of the OX gene is mediating transcriptional interference and repressing the transcription of the RC gene in GLU. We note that the downstream gene in a tandem RC.RC pair also shows significant repression in glucose. To investigate further, we examined three different data sets: (i) three groups of tandemly arranged genes; OX.RC, RC.OX, and non-cycling (<xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1G</xref>); (ii) three sets of 20 consecutive ORF-Ts from <xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1A</xref> showing a sevenfold increase, no change or a sevenfold decrease on the GLU/GAL shift (<xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1H</xref>); and (iii) GLU/GAL-regulated genes with an annotated antisense CUT or SUT that is also GLU/GAL-regulated (<xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1I</xref>). From this analysis, we picked tandem gene clusters where the transcription of the target gene is regulated by GAL/GAL shift or not, and examined how neighbouring genes behave (<xref ref-type="fig" rid="fig1s1 fig1s2 fig1s3 fig1s4">Figure 1&#x2014;figure supplements 1&#x2013;4</xref>; <xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1H</xref>; <xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1J</xref>). The tandem partner of a GLU/GAL-regulated gene is often also regulated by the GLU/GAL shift and the YMC, while non-regulated genes are less likely to be in tandem and if they are, are surrounded by genes that are also not regulated by the GLU/GAL shift or the YMC. Common features of the regulated tandem clusters include reciprocal transcription in GLU and GAL, reciprocally cycling transcripts in the YMC, reciprocal AS transcription to OX genes, di-cistronic transcripts, and/or antisense transcripts spanning promoters that could mediate temporal transcriptional interference and thus facilitate cycling transcription. We chose the OX.RC pair <italic>HMS2:BAT2</italic> for further study, as all the above characteristics are present and because the relatively abundant <italic>HMS2</italic> antisense transcript, <italic>SUT650</italic>, can be detected experimentally in both GLU and GAL (<xref ref-type="fig" rid="fig1">Figure 1F,G</xref>).</p></sec><sec id="s2-3"><title>Characterisation of transcripts and transcription at the <italic>HMS2:BAT2</italic> locus</title><p>We examined the relationship between <italic>HMS2</italic> and <italic>BAT2</italic> (transcripts A and D respectively). <italic>BAT2</italic> transcripts peak in the RC phase of the YMC and in GAL, reciprocal to <italic>HMS2</italic> sense transcripts, which peak in the OX phase of the YMC and in GLU (<xref ref-type="fig" rid="fig1">Figure 1F,G</xref> and <xref ref-type="fig" rid="fig2s1">Figure 2&#x2014;figure supplement 1A&#x2013;C</xref>). Interestingly, there is also a reciprocal relationship between <italic>HMS2</italic> and its antisense <italic>SUT650</italic> (transcripts A and B respectively). Dual-labelled RNA FISH revealed a degree of temporal separation in the production of the <italic>HMS2</italic> and <italic>SUT650</italic> transcripts. In multiple analyses, no examples of FISH signals for both <italic>HMS2</italic> and <italic>SUT650</italic> at the site of transcription in the nucleus are observed in either GLU or GAL. Cells with nascent transcripts (arrows, <xref ref-type="fig" rid="fig2">Figure 2A</xref>) often have the opposite transcript type or both <italic>SUT650</italic> and <italic>HMS2</italic> transcripts in the cytoplasm, suggesting they are changing state. 35.5 &#xb1; 7.9% of the cells contained both transcripts in GLU, 22.8 &#xb1; 10.5% of all cells lacked both the <italic>SUT650</italic> and <italic>HMS2</italic> sense transcripts. The remaining cells contained either <italic>HMS2</italic> or <italic>SUT650</italic> transcripts but not both (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). For cells containing a single type of transcript, more cells contained antisense transcripts in GAL than GLU while more cells contained sense transcripts in GLU than GAL (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). Thus, the increase in levels of <italic>SUT650</italic> transcripts in GAL reflects an increase in the number of cells expressing <italic>SUT650</italic> and a concomitant decrease in the number of cells expressing <italic>HMS2</italic>, rather than a change in the levels of transcripts within a fixed number of cells in the population. We conclude that the expression of <italic>HMS2</italic> and <italic>SUT650</italic> is temporally separated, similar to the <italic>PHO84</italic> sense and antisense transcripts (<xref ref-type="bibr" rid="bib6">Castelnuovo et al., 2013</xref>). This suggests potential co-regulation between <italic>BAT2</italic> and <italic>SUT650</italic> and reciprocal regulation of <italic>HMS2</italic> and <italic>BAT2</italic>.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03635.008</object-id><label>Figure 2.</label><caption><title>Characterization of transcripts around the <italic>HMS2:BAT2</italic> locus (<bold>A</bold>).</title><p>Visualizing <italic>HMS2</italic> transcripts using RNA fluorescence in situ hybridization (RNA FISH) in single cells using a combination of four, 50 nt DNA probes labelled with four fluorophores, either Cy5 (sense) or Cy3 (antisense), hybridised to paraformaldehyde-fixed yeast cells. DAPI (blue) marks the nucleus. Smaller boxes are zoomed images of selected cells in the field of view. Cells with nascent nuclear transcription event are marked with arrows. The images presented here are part of larger data sets. The graph (<bold>B</bold>) represents the proportion of sense- and antisense-expressing cells between growth conditions from different experiments. A total of &#x2248;250 cells were assessed in each growth condition. The mean (MAX) signal from an <italic>hms2&#x394;</italic> strain prepared at the same time was used to threshold the signal intensity. Error bars are SEM, n &#x3d; 2, <xref ref-type="supplementary-material" rid="SD1-data">Figure 2&#x2014;source data 1A</xref>. (<bold>C</bold>) Northern blots of total and poly(A)<sup>&#x2b;</sup> selected RNA from WT cells probed with <italic>HMS2</italic> (<bold>H</bold>) and <italic>BAT2</italic> (<bold>B</bold>) sense (S)- and <italic>HMS2</italic> antisense (AS)-specific probes (positions indicated in <xref ref-type="fig" rid="fig2">Figure 2E</xref>). Note the <italic>HMS2</italic> antisense probe shows cross-hybridization with the 25S rRNA and is marked &#x2a; in this and subsequent figures. Ethidium bromide-stained rRNA is used as a loading control. (<bold>D</bold>) Northern blot of total RNA showing <italic>HMS2</italic> sense transcripts in strains indicated. (<bold>E</bold>) Map of transcripts (<bold>A</bold> to <bold>D</bold>) around the <italic>HMS2</italic> locus showing approximate length, initiation sites, and termination sites relative to the ATG for each gene (see also <xref ref-type="fig" rid="fig2s1">Figure 2&#x2014;figure supplement 1</xref>). The Cbf1 transcription factor binding site is shown (grey boxes). Different transcripts are coloured: blue for <italic>HMS2</italic>, orange for <italic>SUT650</italic>, black for <italic>BAT2</italic>, and grey for <italic>RPS4A</italic> and <italic>YJR149W</italic>. Transcripts are shown on the top or bottom of the schematic to reflect linked expression. Thick black lines indicate probe position for Northern blots, with the name of the probe above.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.008">http://dx.doi.org/10.7554/eLife.03635.008</ext-link></p><p><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.03635.009</object-id><label>Figure 2&#x2014;Source data 1.</label><caption><p>(<bold>A</bold>) Source data for <xref ref-type="fig" rid="fig2">Figure 2B</xref>, (<bold>B</bold>) Source data for <xref ref-type="fig" rid="fig2s1">Figure 2&#x2014;Supplement 1B</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.009">http://dx.doi.org/10.7554/eLife.03635.009</ext-link></p></caption><media xlink:href="elife03635s001.xlsx" mimetype="application" mime-subtype="xlsx"/></supplementary-material></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03635.010</object-id><label>Figure 2&#x2014;figure supplement 1.</label><caption><title>Characterization of transcripts around the <italic>HMS2:BAT2</italic> locus.</title><p>(<bold>A</bold>) Map of transcripts. (<bold>B</bold>) Histogram showing quantitation of the Northern blot in (<bold>C</bold>) <xref ref-type="supplementary-material" rid="SD1-data">Figure 2&#x2014;source data 1B</xref>. (<bold>C</bold>) Northern blot showing carbon source regulation of <italic>HMS2</italic>, <italic>SUT650,</italic> and <italic>BAT2</italic> transcripts. Total RNA was prepared from cells growing exponentially in glucose, after 5, 30, or 60 min in galactose and then after transfer back to glucose for 1 or 2 hr. (<bold>D</bold>) Chromatograms showing sequence from 3&#x2032;RACE mapping of transcripts at <italic>HMS2</italic>, <italic>BAT2,</italic> and <italic>SUT650</italic> (<italic>HMS2</italic> antisense). The positions of the poly(A) tails are indicated. (<bold>E</bold>) Screen shot of poly(A) site mapping at the <italic>HMS2</italic> sense transcript (red, top panel) and <italic>SUT650</italic> antisense (blue, bottom panel). Data in (<bold>E</bold>) taken from <xref ref-type="bibr" rid="bib35">Pelechano et al. (2013)</xref>; <xref ref-type="bibr" rid="bib56">Wilkening et al. (2013)</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.010">http://dx.doi.org/10.7554/eLife.03635.010</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635fs005"/></fig></fig-group></p><p>A number of different methods revealed heterogeneity at both the 5&#x2032; and 3&#x2032; ends of the <italic>HMS2</italic> and <italic>SUT650</italic> transcripts and longer RNA species (C<sub>1</sub> and C<sub>2</sub>) on the <italic>HMS2</italic> sense strand extending through the coding region of <italic>BAT2</italic> and beyond, consistent with low-level read-through transcripts initiating at the <italic>HMS2</italic> promoter (<xref ref-type="fig" rid="fig2">Figure 2C&#x2013;E</xref> and <xref ref-type="fig" rid="fig2s1">Figure 2&#x2014;figure supplement 1D,E</xref>). These read-through transcripts increase upon loss of nuclear exosome (<italic>rrp6</italic>&#x394;) (<xref ref-type="fig" rid="fig2">Figure 2D</xref>). Thus <italic>HMS2</italic> is at the head of a contiguous gene cluster including the downstream genes <italic>BAT2</italic> and <italic>YJR149W</italic> (<xref ref-type="fig" rid="fig2">Figure 2E</xref>). The 3&#x2032; UTR of the <italic>HMS2</italic> transcript is of variable length. Polyadenylation (pA) occurs either 85 nt (proximal) or 426 nt (distal) downstream from the <italic>HMS2</italic> stop codon. The distal pA site is 45 nt upstream of the <italic>BAT2</italic> initiation codon, such that this longer transcription event is likely to interfere with initiation of <italic>BAT2</italic> transcription. Additional transcriptional interference might be mediated through the production of the longer <italic>HMS2-BAT2</italic> di-cistronic RNA species.</p><p>In summary, analysis of transcripts around <italic>HMS2</italic> and <italic>BAT2</italic> supports (i) an overlapping, reciprocally related and temporally segregated, sense&#x2013;antisense pair (SAP) at <italic>HMS2</italic>, (ii) reciprocally expressed sense transcripts at <italic>HMS2</italic> and <italic>BAT2</italic>, and (iii) low-level read-through di-cistronic transcripts (<italic>HMS2:BAT2</italic> and <italic>SUT650:RPS4A</italic>). We ask if overlapping transcription in the sense and antisense orientations at <italic>HMS2</italic> contributes to the reciprocal regulation of <italic>HMS2</italic> and <italic>BAT2</italic>, by dissociating one transcription unit from the other.</p></sec><sec id="s2-4"><title><italic>HMS2</italic> transcription interferes with <italic>BAT2</italic> transcription</title><p>Insertion of the <italic>ADH1</italic> transcription terminator (<italic>ADH1</italic>t) into the middle to <italic>HMS2</italic>, to separate <italic>HMS2</italic> transcription from <italic>BAT2</italic>, allows us to test directly whether the <italic>HMS2</italic> sense transcription over the <italic>HMS2:BAT2</italic> intergenic region represses <italic>BAT2</italic> transcription (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). We confirmed that <italic>ADH1</italic>t efficiently terminates the <italic>HMS2</italic> transcript (<xref ref-type="fig" rid="fig3">Figure 3B,C</xref>) and that there are no detectable transcripts over the <italic>HMS2</italic> 3&#x2032; region (<xref ref-type="fig" rid="fig3">Figure 3D</xref>). The strain with <italic>ADH1</italic>t inserted showed a &#x2248;twofold increase in <italic>BAT2</italic> transcript levels compared to WT in both GLU and GAL (<xref ref-type="fig" rid="fig3">Figure 3B,C</xref>) supporting a role for <italic>HMS2</italic> sense transcription in repressing the <italic>BAT2</italic> promoter. We conclude that <italic>HMS2</italic> sense transcription interferes with <italic>BAT2</italic> transcription.<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.03635.011</object-id><label>Figure 3.</label><caption><title><italic>HMS2</italic> mediates transcriptional interference of <italic>BAT2</italic>.</title><p>(<bold>A</bold>) Schematic showing constructs and transcripts at the WT <italic>HMS2:BAT2</italic> locus and after insertion of the <italic>ADH1</italic> terminator (T). (<bold>B</bold>) Exemplarily autoradiographs of Northern blots of total RNA prepared from the constructs in (<bold>A</bold>) cultured in glucose or after 60 min in galactose probed for the <italic>HMS2</italic> sense, the <italic>SUT650</italic> antisense, and the <italic>BAT2</italic> sense transcripts. (<bold>C</bold>) Quantitation of autoradiographs showing average signal normalized to rRNA for the transcripts indicated. n &#x3d; 2, errors are SEM, <xref ref-type="supplementary-material" rid="SD2-data">Figure 3&#x2014;source data 1A</xref>. (<bold>D</bold>, <bold>E</bold>) Exemplarily autoradiographs of Northern blots of total RNA prepared from the strains indicated containing the constructs in (<bold>A</bold>) cultured in glucose probed for the regions indicated. (<bold>F</bold>) Visualizing <italic>HMS2</italic> sense transcripts using fluorescence in situ hybridization (FISH) in single cells using a combination of four, 50 nt DNA probes labelled with four Cy5 fluorophores (green, sense), hybridised to paraformaldehyde-fixed yeast cells. The nucleus is shown in blue (DAPI). Smaller boxes are zoomed images of select cells in the field of view. The images presented here are part of a larger data set. (<bold>G</bold>) The graphs represent the proportion of sense-expressing cells in the WT compared to <italic>HMS2:ADH1t</italic> and the mean signal per cell. A total of &#x2248;500 cells were assessed for each strain. Error bars are SEM; n &#x3d; 4, <xref ref-type="supplementary-material" rid="SD2-data">Figure 3&#x2014;source data 1A,C</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.011">http://dx.doi.org/10.7554/eLife.03635.011</ext-link></p><p><supplementary-material id="SD2-data"><object-id pub-id-type="doi">10.7554/eLife.03635.012</object-id><label>Figure 3&#x2014;source data 1.</label><caption><p>(<bold>A</bold>) Source data for <xref ref-type="fig" rid="fig3">Figure 3C</xref>, (<bold>B</bold>) Source data for <xref ref-type="fig" rid="fig3">Figure 3F</xref>, (<bold>C</bold>) Source data for <xref ref-type="fig" rid="fig3">Figure 3F</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.012">http://dx.doi.org/10.7554/eLife.03635.012</ext-link></p></caption><media xlink:href="elife03635s002.xlsx" mimetype="application" mime-subtype="xlsx"/></supplementary-material></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635f003"/></fig></p></sec><sec id="s2-5"><title><italic>SUT650</italic> transcription limits the number of cells expressing <italic>HMS2</italic></title><p>Insertion of the <italic>ADH1</italic>t into the <italic>HMS2</italic> coding region resulted in a &#x2248;fourfold increase in levels of a truncated <italic>HMS2</italic> sense transcript (A<sup>A</sup>) (<xref ref-type="fig" rid="fig3">Figure 3A,B,D</xref>) and blocks <italic>SUT650</italic> antisense transcription, resulting in the truncated antisense transcript (B<sup>A</sup>) (<xref ref-type="fig" rid="fig3">Figure 3A,D</xref>). We confirm there are no antisense transcripts over the 5&#x2032; region of <italic>HMS2</italic> (<xref ref-type="fig" rid="fig3">Figure 3B,E</xref>). In the absence of <italic>SUT650</italic>, expression of the <italic>HMS2:ADH1t</italic> transcript remains sensitive to the change in carbon source, showing a similar decrease to full-length <italic>HMS2</italic> 1 h after transfer from GLU to GAL (<xref ref-type="fig" rid="fig3">Figure 3B,C</xref>). This suggests that the carbon source responsive signal is received at the <italic>HMS2</italic> promoter and that reciprocal switching of <italic>HMS2</italic> and <italic>SUT650</italic> is a function of sense transcription. However, as the levels of <italic>HMS2</italic> sense transcripts are higher without the antisense transcript, we asked if antisense transcription plays a role in limiting the number of cells containing sense transcript using RNA FISH (<xref ref-type="fig" rid="fig3">Figure 3F</xref>). When grown in glucose, optimal conditions for <italic>HMS2</italic> sense transcription, we observed an increase in the number of <italic>HMS2:ADH1t</italic> cells containing sense transcript compared to WT (<xref ref-type="fig" rid="fig3">Figure 3G</xref>), consistent with a role for antisense transcription in reducing the number of sense transcription initiation events, leading to a decrease in the number of cells in the population that contain the sense transcript. We conclude that <italic>SUT650</italic> antisense transcription does not influence the environmental responsiveness of <italic>HMS2</italic> transcription but acts to limit the number of cells in the population that respond.</p></sec><sec id="s2-6"><title><italic>SUT650</italic> antisense transcription insulates <italic>BAT2</italic> from interference by <italic>HMS2</italic></title><p>Next, we asked whether transcription of <italic>SUT650</italic> antisense might prevent <italic>HMS2</italic> sense transcription from interfering with <italic>BAT2</italic>, thus insulating <italic>BAT2</italic>. To ablate <italic>SUT650</italic> whilst ensuring that the regulatory elements in the flanking sequences between <italic>HMS2</italic> and <italic>BAT2</italic> remained intact, we removed the transcription start sites (TSS) for <italic>SUT650</italic> located between &#x2b;953 and &#x2b;1038 relative to the <italic>HMS2</italic> ATG, near the end of the <italic>HMS2</italic> coding region. We chose to do this by substituting the complete coding region of <italic>HMS2</italic> with that of <italic>URA3</italic>, to ensure a natural ORF between the <italic>HMS2</italic> promoter and terminator, rather than mutating the regions containing multiple TSSs for <italic>SUT650</italic> within the ORF (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). We note that insertion of the <italic>URA3</italic> ORF could affect transcript stability, although the nature of the promoter may be the primary determinant of transcript stability (<xref ref-type="bibr" rid="bib3">Bregman et al., 2011</xref>; <xref ref-type="bibr" rid="bib47">Trcek et al., 2011</xref>), so we focused on transcript size and relative abundance. We observed two clear effects. First, loss of the antisense transcript (B), which could be explained either by introduction of a unidirectional terminator in the <italic>URA3</italic> ORF coding region or loss of TSSs for <italic>SUT650</italic> (<xref ref-type="fig" rid="fig4">Figure 4B&#x2013;D</xref> and <xref ref-type="fig" rid="fig4s1">Figure 4&#x2014;figure supplement 1</xref>). Second, an increase in the read-through transcripts (C<sub>1</sub><sup>U</sup>) relative to the <italic>HMS2</italic>:<italic>URA3</italic> sense transcript (A<sup>U</sup>) (<xref ref-type="fig" rid="fig4">Figure 4B&#x2013;D</xref>). We confirmed these were read-through transcripts by inserting a terminator (T) at the 3&#x2032; region of the <italic>HMS2:URA3</italic> hybrid gene (<xref ref-type="fig" rid="fig4">Figure 4A</xref>) and observing loss of the read-through transcripts and increased levels of a shorter transcript (A<sup>U</sup>T) (<xref ref-type="fig" rid="fig4">Figure 4E,F</xref>). We note that transcripts initiated at the <italic>HMS2</italic> promoter remain sensitive to a change in carbon source in the absence of antisense transcription (<xref ref-type="fig" rid="fig4">Figure 4D</xref>), as we observed using <italic>HMS2:ADH1t</italic> (see <xref ref-type="fig" rid="fig3">Figure 3</xref>) and consistent with a regulatory input at the promoter. The increase in the read-through transcript (C<sub>1</sub><sup>U</sup>) relative to the <italic>HMS2</italic>:<italic>URA3</italic> sense transcript (A<sup>U</sup>) in the absence of antisense transcription suggests a role for antisense transcription in attenuating sense transcription and preventing interference at the <italic>BAT2</italic> promoter. Consistent with this, a twofold decrease in <italic>BAT2</italic> sense transcript is observed in the antisense-less <italic>HMS2:URA3</italic> strain relative to WT in GLU, concomitant with a threefold increase in A<sup>U</sup> and C<sub>1</sub><sup>U</sup> (<xref ref-type="fig" rid="fig4">Figure 4B,C</xref>). Taken together, the effect of blocking (<xref ref-type="fig" rid="fig3">Figure 3</xref>) or increasing (<xref ref-type="fig" rid="fig4">Figure 4</xref>) <italic>HMS2</italic> transcription on levels of <italic>BAT2</italic> transcript supports transcriptional interference by <italic>HMS2</italic> at the <italic>BAT2</italic> promoter and a reciprocal relationship between <italic>HMS2</italic> and <italic>BAT2</italic>. We sought evidence for transcriptional interference resulting from increased levels of A<sup>U</sup> and C<sub>1,2</sub><sup>U</sup>. Chromatin immunoprecipitation (ChIP) analysis at the <italic>HMS2:BAT2</italic> intergenic region reveals reduced initiation-related modifications (H3K4me3 and H3K56ac) and increased elongation-related modifications (H3K79me3 and H3K36me3) in the <italic>HMS2</italic>:<italic>URA3</italic> hybrid gene, consistent with transcriptional interference leading to repression of <italic>BAT2</italic> transcription initiation (<xref ref-type="fig" rid="fig4">Figure 4G</xref>). We conclude that <italic>SUT650</italic> antisense transcription (i) changes the proportion of cells expressing <italic>HMS2</italic> and (ii) controls the amount of overlapping and read-through transcription from <italic>HMS2</italic> and thus levels of <italic>BAT2</italic> transcript. We envisage that at any one time in a single cell, formation of the <italic>HMS2</italic> and <italic>SUT650</italic> transcription units (TUs) is mutually exclusive, but the formation of the <italic>SUT650</italic> TU and the <italic>BAT2</italic> TU is not. Thus formation of the <italic>SUT650</italic> transcription unit insulates <italic>BAT2</italic> from interference by <italic>HMS2</italic> transcription, as the <italic>HMS2</italic> TU cannot form when <italic>SUT650</italic> is expressed.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.03635.013</object-id><label>Figure 4.</label><caption><title><italic>SUT650</italic> antisense transcription insulates <italic>BAT2</italic> from interference by <italic>HMS2.</italic></title><p>(<bold>A</bold>) Schematic showing transcripts after replacement of the <italic>HMS2</italic> coding region (top) with the <italic>URA3</italic> coding region (middle) or <italic>URA3</italic> plus a transcription terminator (T) (bottom). Transcripts resulting from the <italic>URA3</italic> insertions are identified with a superscript U or UT. (<bold>B</bold>, <bold>D</bold>, <bold>E</bold>) Northern blots of total RNA in strains with the <italic>HMS2</italic> coding region replaced with the <italic>URA3</italic> coding region in glucose (<bold>B</bold>), after 1 h in galactose (<bold>D</bold>) or in glucose with a terminator (T) inserted after <italic>URA3</italic> (<bold>E</bold>). In (<bold>E</bold>), samples were run on the same gel but intervening tracks removed. (<bold>C</bold>) Quantitation of transcripts for WT and <italic>HMS2:URA3</italic> in glucose, n &#x3d; 2, errors are SEM, <xref ref-type="supplementary-material" rid="SD3-data">Figure 4&#x2014;source data 1A</xref>. (<bold>F</bold>) Quantitation of transcripts in (<bold>E</bold>) for <italic>HMS2:URA3</italic> and <italic>HMS:URA3:T</italic> in glucose, n &#x3d; 2, errors are SEM, <xref ref-type="supplementary-material" rid="SD3-data">Figure 4&#x2014;source data 1B</xref>. (<bold>G</bold>) Chromatin immunoprecipitation (ChIP-qPCR) at the <italic>HMS2:BAT2</italic> intergenic region (IG) in strains indicated using antibodies with the specificities indicated. Signals were normalized to Histone H3 and then the signal in the coding region of <italic>TUB2</italic>. Error bars are SEM for n &#x3d; 2; <xref ref-type="supplementary-material" rid="SD4-data">Figure 4&#x2014;source data 2</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.013">http://dx.doi.org/10.7554/eLife.03635.013</ext-link></p><p><supplementary-material id="SD3-data"><object-id pub-id-type="doi">10.7554/eLife.03635.014</object-id><label>Figure 4&#x2014;source data 1.</label><caption><p>(<bold>A</bold>) Source data for <xref ref-type="fig" rid="fig4">Figure 4C</xref>, (<bold>B</bold>) Source data for <xref ref-type="fig" rid="fig4">Figure 4F</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.014">http://dx.doi.org/10.7554/eLife.03635.014</ext-link></p></caption><media xlink:href="elife03635s003.xlsx" mimetype="application" mime-subtype="xlsx"/></supplementary-material></p><p><supplementary-material id="SD4-data"><object-id pub-id-type="doi">10.7554/eLife.03635.015</object-id><label>Figure 4&#x2014;source data 2.</label><caption><p>Source data for <xref ref-type="fig" rid="fig4">Figure 4G</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.015">http://dx.doi.org/10.7554/eLife.03635.015</ext-link></p></caption><media xlink:href="elife03635s004.xlsx" mimetype="application" mime-subtype="xlsx"/></supplementary-material></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03635.016</object-id><label>Figure 4&#x2014;figure supplement 1.</label><caption><title>No <italic>SUT650</italic> antisense transcription in either GLU or GAL in the <italic>HMS2:URA3</italic> strain.</title><p>(<bold>A</bold>) Schematic showing transcripts after replacement of the <italic>HMS2</italic> coding region (top) with the <italic>URA3</italic> coding region (bottom). (<bold>B</bold>) Northern blots of total RNA in WT strain or with the <italic>HMS2</italic> coding region replaced with the <italic>URA3</italic> coding region in glucose or after 5, 30, and 60 min in galactose.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.016">http://dx.doi.org/10.7554/eLife.03635.016</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635fs006"/></fig></fig-group></p></sec><sec id="s2-7"><title><italic>HMS2</italic> transcription represses the <italic>SUT650</italic> antisense transcript and <italic>BAT2</italic></title><p>To address whether <italic>HMS2</italic> transcription influences <italic>SUT650</italic> antisense transcription, we replaced the <italic>HMS2</italic> promoter with the inducible <italic>GAL1</italic> promoter (p<italic>GAL1</italic>) (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). When cultured in glucose, the <italic>GAL1</italic> promoter is repressed but becomes induced about 2 h after transfer to galactose (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). <italic>SUT650</italic> is normally induced during the GLU-GAL shift (see <xref ref-type="fig" rid="fig1">Figure 1</xref>). However in the p<italic>GAL1:HMS2</italic> strain, an antisense transcript (B<sup>G</sup>) is evident in GLU and remains until the sense transcript is induced by GAL (<xref ref-type="fig" rid="fig5">Figure 5C,D</xref>). This suggests that sense transcription represses the antisense transcript. We used the inducible p<italic>GAL1:HMS2</italic> system in conjunction with the Anchor Away technique (<xref ref-type="bibr" rid="bib13">Haruki et al., 2008</xref>) to deplete the nucleus of the Gal4p activator during growth in GAL. Removal of Gal4p by treatment with rapamycin (Rap) for 1 h, after activation of p<italic>GAL1:HMS2</italic> for 3 h with GAL, abolishes the <italic>HMS2</italic> transcript (A<sup>G</sup>) and leads to restoration of the antisense transcript (B<sup>G</sup>) (<xref ref-type="fig" rid="fig5">Figure 5E,F</xref>). This confirms that the reduction in the antisense transcript is a consequence of sense transcription, rather than the change in condition (GLU to GAL). To determine whether antisense transcription had any effect on p<italic>GAL1:HMS2</italic> sense transcript induction rates, the antisense--less <italic>HMS2:URA3</italic> construct was placed under the control of the <italic>GAL1</italic> promoter (<xref ref-type="fig" rid="fig5">Figure 5A,B</xref>). The sense transcript profile in poly(A)<sup>&#x2b;</sup>-enriched RNA was examined using a probe for the <italic>HMS2:BAT2</italic> intergenic region (probe IG), a region transcribed in both constructs. No changes in induction rate were apparent, with the sense transcript only detectable after 2 h in both strains, signifying that antisense transcription does not impair sense activation kinetics (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). Rather, this finding is compatible with our observation that without antisense, more cells would express the sense transcript (see <xref ref-type="fig" rid="fig3">Figure 3F,G</xref>). Activation of the p<italic>GAL1:HMS2</italic> sense transcript results in up to 3.5-fold repression of <italic>BAT2</italic> (<xref ref-type="fig" rid="fig5">Figure 5F</xref>), consistent with p<italic>GAL1:HMS2</italic> sense polyadenylation at 426 nt, just upstream of the <italic>BAT2</italic> ATG, interfering with <italic>BAT2</italic> transcription initiation in galactose (see <xref ref-type="fig" rid="fig2s1">Figure 2&#x2014;figure supplement 1</xref>). We conclude that formation of the <italic>HMS2</italic> sense transcription unit represses the formation of the <italic>SUT650</italic> and <italic>BAT2</italic> transcription units, regardless of conditions.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.03635.017</object-id><label>Figure 5.</label><caption><title><italic>HMS2</italic> sense transcription represses the <italic>HMS2</italic> antisense transcript and <italic>BAT2.</italic></title><p>(<bold>A</bold>) Schematic showing transcripts when expression of <italic>HMS2</italic> is regulated by the <italic>GAL1</italic> promoter (p<italic>GAL1</italic>) in glucose (antisense-dominant state) or galactose (sense-dominant state) or when the <italic>HMS2</italic> coding region is substituted with the <italic>URA3</italic> coding region. (<bold>B</bold>, <bold>C</bold>, <bold>E</bold>, <bold>G</bold>&#x2013;<bold>I</bold>) Northern blots showing total RNA (<bold>C</bold>, <bold>E</bold>, <bold>G</bold>, <bold>I</bold>) or poly(A)<sup>&#x2b;</sup> RNA (<bold>B</bold>, <bold>H</bold>) from cells expressing p<italic>GAL1:HMS2</italic> cultured in glucose (GLU) or induced for the times indicated in galactose (GAL) in the genetic backgrounds indicated. (<bold>D</bold>, <bold>F</bold>) Quantitation of the experiments exemplified in (<bold>C</bold>) and (<bold>E</bold>) respectively, n &#x3d; 2, error are SEM, <xref ref-type="supplementary-material" rid="SD5-data">Figure 5&#x2014;source data 1A,B,C</xref>. (<bold>E</bold>, <bold>F</bold>) Anchor Away of Gal4 is achieved after incubation with rapamycin in DMSO for 1 h, added 3 h after induction with galactose (240 Rap). The control culture (240 DMSO) was treated with DMSO. See also <xref ref-type="fig" rid="fig5s1">Figure 5&#x2014;figure supplement 1</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.017">http://dx.doi.org/10.7554/eLife.03635.017</ext-link></p><p><supplementary-material id="SD5-data"><object-id pub-id-type="doi">10.7554/eLife.03635.018</object-id><label>Figure 5&#x2014;source data 1.</label><caption><p>(<bold>A</bold>) Source data for <xref ref-type="fig" rid="fig5">Figure 5D</xref>, (<bold>B</bold>) Source data for <xref ref-type="fig" rid="fig5">Figure 5F</xref>, (<bold>C</bold>) Source data for <xref ref-type="fig" rid="fig5">Figure 5F</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.018">http://dx.doi.org/10.7554/eLife.03635.018</ext-link></p></caption><media xlink:href="elife03635s005.xlsx" mimetype="application" mime-subtype="xlsx"/></supplementary-material></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03635.019</object-id><label>Figure 5&#x2014;figure supplement 1.</label><caption><title>Mapping the 3&#x2032; ends of the p<italic>GAL.HMS2</italic> antisense transcript isoforms.</title><p>(<bold>A</bold>) Schematic of the <italic>pGAL1.HMS2</italic> transcription unit. (<bold>B</bold>) 3&#x2032;RACE performed on the galactose-inducible <italic>HMS2</italic> strain in glucose displays evidence of two poly(A) tails, one consistent with the short, B<sup>G2</sup>, AS isoform (poly(A) tail<sup>2</sup>) terminating at a region downstream of the UAS<sub><italic>GAL</italic></sub> (underlined in orange) &#x2248;195 nt upstream of the <italic>GAL1</italic> ATG and the long isoform, B<sup>G1</sup>, terminating at a region upstream of the UAS<sub><italic>GAL</italic></sub> (poly(A) tail<sup>1</sup>), &#x2248;500 nt upstream of the <italic>GAL1</italic> ATG. (<bold>C</bold>) Evidence for an intermediate species, B<sup>G3</sup>, not resolvable using Northern analysis, was detected after 3&#x2032; RACE was performed on cells grown in galactose for 3 hr. Potential end sites are marked with an asterisk.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.019">http://dx.doi.org/10.7554/eLife.03635.019</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635fs007"/></fig></fig-group></p></sec><sec id="s2-8"><title>Antisense transcript isoforms are condition-specific and inherent to <italic>HMS2</italic></title><p>There are two species of antisense transcript during induction of p<italic>GAL1:HMS2</italic>; a long species (B<sup>G1</sup>), dominant until 30 min after the shift to GAL, and a shorter species (B<sup>G2</sup>), also detectable after 30 min of growth in GAL and which becomes the dominant antisense transcript form after 1 h (<xref ref-type="fig" rid="fig5">Figure 5C</xref>). 3&#x2032;end RACE revealed alternative polyadenylation sites as one cause for the observed size difference, with the long form terminating upstream of the Gal4p binding sites at approx. 500 nt upstream of the ATG in the <italic>GAL1</italic> promoter and the shorter form terminating midway through the <italic>GAL1</italic> promoter, 195 nt upstream of the <italic>GAL1</italic> ATG, and downstream of the Gal4p binding sites (<xref ref-type="fig" rid="fig5s1">Figure 5&#x2014;figure supplement 1</xref>). Upon glucose repression of induced p<italic>GAL1:HMS2</italic>, levels of the long form of antisense transcript were recovered (<xref ref-type="fig" rid="fig5">Figure 5G</xref>). Antisense transcript shortening after transfer to GAL is an inherent property of the natural <italic>SUT650</italic> antisense transcript (<xref ref-type="fig" rid="fig5">Figure 5H</xref>). To ask if this was a function of transcription factor binding to the promoter, we used the p<italic>GAL1:HMS2</italic> construct and showed that shortening of the antisense transcript is independent of Gal4 binding at p<italic>GAL1</italic> and remains for 3 h in GAL in the absence of sense transcription (<xref ref-type="fig" rid="fig5">Figure 5I</xref>). This rules out a potential roadblock by Gal4 as an explanation for the use of different antisense polyadenylation/termination sites in p<italic>GAL1</italic>. We conclude that <italic>SUT650</italic> antisense transcription is normally repressed by <italic>HMS2</italic> transcription but remains stably expressed in GLU or GAL, varying in the use of polyadenylation sites, when sense transcription is inhibited.</p></sec><sec id="s2-9"><title>Transcription factors contribute to sense/antisense switching at <italic>HMS2:SUT650</italic></title><p>To see if loss of transcription factors associated with the native <italic>HMS2</italic> gene, Cbf1 (<xref ref-type="bibr" rid="bib24">MacIsaac et al., 2006</xref>), and Fkh1 (<xref ref-type="bibr" rid="bib55">Venters et al., 2011</xref>), influence the balance of sense and antisense transcription at <italic>HMS2</italic>, we used <italic>cbf1</italic>&#x394;, <italic>fkh1</italic>&#x394;, and <italic>fkh2</italic>&#x394; strains, regulators of nutrient availability, oxidative growth, and the cell cycle (<xref ref-type="bibr" rid="bib29">Mitchell and Magasanik, 1984</xref>; <xref ref-type="bibr" rid="bib28">Mellor et al., 1990</xref>; <xref ref-type="bibr" rid="bib17">Kent et al., 1994</xref>; <xref ref-type="bibr" rid="bib60">Zhu et al., 2000</xref>; <xref ref-type="bibr" rid="bib31">Morillon et al., 2003</xref>; <xref ref-type="bibr" rid="bib48">Tsankov et al., 2011</xref>). While under standard growth conditions in GLU these mutant strains showed normal expression of <italic>HMS2</italic> and <italic>SUT650</italic>, growth in nutrient-limited GLU-based medium depleted for tryptophan resulted in loss of the <italic>HMS2</italic> transcript and accumulation of the short isoform of the <italic>SUT650</italic> antisense transcript (B<sub>2</sub>) in the <italic>cbf1&#x394;</italic> and <italic>fkh1&#x394;</italic> strains (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). In contrast, the <italic>fkh2</italic>&#x394; strain showed reduced levels of <italic>SUT650</italic> and increased <italic>HMS2</italic> under these growth conditions. This supports transcription factors controlling the balance of sense, and thus antisense transcription, at this locus during the cell cycle (Fkh factors) (<xref ref-type="bibr" rid="bib11">Granovskaia et al., 2010</xref>) and during oxidative growth in metabolic cycles (Cbf1) (<xref ref-type="bibr" rid="bib48">Tsankov et al., 2011</xref>). We ablated a number of non-essential transcription factors, including Gln3, with putative binding sites at the 3&#x2032; region of <italic>HMS2</italic> (<xref ref-type="bibr" rid="bib24">MacIsaac et al., 2006</xref>) but observed no significant change in levels of <italic>SUT650</italic>, <italic>HMS2</italic>, or <italic>BAT2</italic> transcripts with the exception of a small increase in the <italic>HMS2:BAT2</italic> read-through transcript (C) in a <italic>gln3&#x394;</italic> strain in minimal medium (<xref ref-type="fig" rid="fig6s1">Figure 6&#x2014;figure supplement 1</xref>). We propose that transcription factors signal to the <italic>HMS2</italic> promoter (Input) to mediate state-changing (antisense-dominant to sense-dominant) in response to environmental conditions (<xref ref-type="fig" rid="fig6">Figure 6B</xref>). As we could find no direct evidence for regulation of <italic>SUT650</italic> either by TFs (<xref ref-type="fig" rid="fig6">Figure 6</xref> and <xref ref-type="fig" rid="fig6s1">Figure 6&#x2014;figure supplement 1</xref>) or by the GLU to GAL shift (see <xref ref-type="fig" rid="fig5">Figure 5</xref>) but only by transcriptional interference resulting from activation of <italic>HMS2</italic> transcription, we examined OX and RC genes genome-wide to look for common themes in their regulation. We asked what factors are significantly enriched (p &#x3c; 0.01) at promoters of RC and OX genes, using a large data set of 202 factors (<xref ref-type="bibr" rid="bib55">Venters et al., 2011</xref>). We observed marked differences (<xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1K</xref>). Of particular interest are the predominance of chromatin modulators and components at RC gene promoters, suggesting that RC promoters are particularly sensitive to chromatin-mediated repression (<xref ref-type="bibr" rid="bib46">Tirosh and Barkai, 2008</xref>). These features, coupled with the distinct behaviour of OX and RC genes in GLU and GAL, particularly OX.RC genes in tandem (<xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1F</xref>), lead us to propose chromatin-mediated repression of RC genes by transcriptional interference. In addition, antisense transcription into OX promoters, for example by <italic>SUT650</italic> at <italic>HMS2</italic>, may also limit OX gene transcription.<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.03635.020</object-id><label>Figure 6.</label><caption><title>General and specific transcription factors control state switching at <italic>HMS2</italic>.</title><p>(<bold>A</bold>, <bold>C</bold>, <bold>D</bold>) Northern blots showing sense or antisense-dominant state at <italic>HMS2</italic> in strains lacking general or specific transcription factors. (<bold>A</bold>) Strains were cultured in YPD depleted for tryptophan. (<bold>B</bold>) Model for state-switching between a sense-dominant state and an antisense-dominant state by transcription factors (Input) at the divergent promoters between <italic>RPS4A</italic> and <italic>HMS2</italic> (blue double arrow) and <italic>HMS2</italic> and <italic>BAT2</italic> (orange double arrow). During growth in glucose (high input), cells cycle between the sense-dominant state and the antisense-dominant state with the majority of cells existing in the sense-dominant state. During growth in galactose (low input), cells cycle between the sense-dominant state and the antisense-dominant state with the majority of cells existing in the antisense-dominant state. (<bold>C</bold>) Anchor Away of TFIIB (Sua7) is achieved after incubation with rapamycin (Rap) (&#x2b;) in DMSO for 1 h or DMSO alone (&#x2212;). (<bold>D</bold>) Biological replicates for the WT strain BY4741 and the isogenic strain expressing the <italic>sua7-1</italic> allele are shown.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.020">http://dx.doi.org/10.7554/eLife.03635.020</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03635.021</object-id><label>Figure 6&#x2014;figure supplement 1.</label><caption><title>The effect of deletion or ablation of transcription factors with putative binding sites at the <italic>HMS2:BAT2</italic> intergenic region on <italic>HMS2:BAT2</italic> transcripts.</title><p>(<bold>A</bold>) Schematic of transcripts. (<bold>B</bold>, <bold>C</bold>) Northern blot analysis in the strains indicated. (<bold>B</bold>) In the top panel strains were grown in glucose (&#x2212;) or for 1 hr in galactose (GAL, &#x2b;). In the bottom panel, strains were cultured in YDP (Y) or CSM (-C). (<bold>C</bold>) Anchor Away (AA) analysis of Gln3 and Sua7 (repeated experiment) after 5 min or 60 min treatment with Rapamycin (Rap) in the conditions shown and probed for <italic>HMS2</italic>, <italic>SUT650</italic>, and <italic>BAT2</italic> transcripts.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.021">http://dx.doi.org/10.7554/eLife.03635.021</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635fs008"/></fig></fig-group></p></sec><sec id="s2-10"><title>TFIIB (Sua7) is required to limit the sense-dominant state at <italic>HMS2</italic></title><p>We assessed a role for a number of essential transcription factors using the Anchor Away technique (<xref ref-type="bibr" rid="bib13">Haruki et al., 2008</xref>) but only upon nuclear depletion of TFIIB (Sua7) did we observe an increase in the <italic>HMS2:BAT2</italic> read-through transcript (C) (<xref ref-type="fig" rid="fig6">Figure 6C</xref>). However, this occurred together with reduced levels of <italic>HMS2</italic> (A), <italic>BAT2</italic> (D), and <italic>SUT650</italic> (B) transcripts, as expected upon depletion of an essential transcription factor. To explore this further, we used a well-characterised allele of <italic>SUA7</italic> (<italic>sua7-1</italic>) (E62K) (<xref ref-type="bibr" rid="bib38">Pinto et al., 1994</xref>), associated specifically with the 3&#x2032; region of genes, polyadenylation/transcription termination and higher order structures (gene loops) in chromatin (<xref ref-type="bibr" rid="bib27">Medler et al., 2011</xref>). Remarkably, we observed an increase in <italic>HMS2</italic> transcript (A) and sense read-through transcripts (C), coupled with a decrease in the <italic>SUT650</italic> antisense transcript (B) (<xref ref-type="fig" rid="fig6">Figure 6D</xref>). Thus, TFIIB may have a specific role in determining the antisense transcription unit by establishing directionality of transcription over the region (<xref ref-type="bibr" rid="bib44">Tan-Wong et al., 2012</xref>). For instance, by determining an antisense transcription unit, TFIIB aids in sense transcription termination, preventing &#x2018;read-through&#x2019; over the intergenic region between <italic>HMS2</italic> and <italic>BAT2</italic>. The capacity to read through transcription regulatory regions such as promoters and terminators is an essential component of a model for gene regulation by transcriptional interference such as that developed for the <italic>HMS2:BAT2</italic> locus. To test this experimentally, we inserted a new transcription unit into <italic>HMS2</italic> to disrupt the <italic>HMS2</italic> and <italic>SUT650</italic> transcription units and asked what mutations would restore transcription between the beginning and end of these TUs.</p></sec><sec id="s2-11"><title>Engineering <italic>HMS2</italic> reveals the balance between sense and antisense states and demonstrates the plasticity of transcription units</title><p>The <italic>HMS2</italic> locus was engineered by insertion of the p<italic>TEF.KanMX.TEF</italic>t expression cassette (<xref ref-type="bibr" rid="bib23">Longtine et al., 1998</xref>) at position &#x2b;650 bp (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). In this construct (<italic>HMS2:TEF:Kan</italic>), there are two promoters in tandem (<italic>HMS2</italic> and <italic>TEF</italic>) and two terminators in tandem (<italic>TEF</italic> and <italic>HMS2</italic>). This is sufficient to disrupt the <italic>HMS2</italic> and <italic>SUT650</italic> transcription units. The <italic>SUT650</italic> antisense promoter is functional and produces a truncated antisense transcript (B<sup>K</sup>) that ends in the vicinity of <italic>TEF</italic>t (<xref ref-type="fig" rid="fig7">Figure 7A,B</xref>). Thus <italic>TEF</italic>t functions bi-directionally to terminate <italic>SUT650</italic> (B<sup>K</sup>), the sense transcript from p<italic>TEF</italic>, expressing the kanamycin resistance (<italic>KanMX</italic>) transcript (K), and the sense transcript (A<sup>K</sup>) initiated at the <italic>HMS2</italic> promoter and terminating at <italic>TEF</italic>t (<xref ref-type="fig" rid="fig7">Figure 7A,B</xref>). p<italic>TEF</italic> is a well-characterised (<xref ref-type="bibr" rid="bib43">Steiner and Philippsen, 1994</xref>) strong bi-directional promoter and produces an antisense transcript (E) extending into p<italic>HMS2</italic>. To confirm this, we inserted <italic>ADH1</italic>t directly upstream of p<italic>TEF</italic> (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). This resulted in increased levels of a truncated sense transcript (A<sup>A</sup>) and considerably reduced levels of antisense transcript (E) (<xref ref-type="fig" rid="fig7">Figure 7B</xref>).<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.03635.022</object-id><label>Figure 7.</label><caption><title>The plasticity of transcription units.</title><p>(<bold>A</bold>, <bold>C</bold>) Schematic of constructs showing transcripts and position of probes. Transcripts resulting from the p<italic>TEF:kanMX:TEFt</italic> insertion, the same construct with the <italic>ADH1</italic> terminator (pink box), or the <italic>TEF:kanMX:TEF</italic> insertion with a deleted <italic>TEF</italic> promoter (p) are identified with a superscript K, A, or p<italic>TEFdel</italic>, respectively (see <xref ref-type="fig" rid="fig7s1">Figure 7&#x2014;figure supplement 1</xref> for more details). (<bold>B</bold>, <bold>D</bold>, <bold>E</bold>) Northern blots in the strains indicated showing the conditional nature of the <italic>TEF</italic> terminator (t) when the p<italic>TEF</italic> is compromised. (<bold>F</bold>) Modelling transcription in an interleaved genome demonstrating an increase in complexity of the transcriptional landscape as the strength of terminators and promoters decreases. Constructs representing each state are indicated; grey and white boxes are downstream TUs. Solid directional boxes are promoters; squares are terminators. Deeper colour indicates increased strength. Arrows on top of diagrams represent the transcripts initiating or terminating at p<italic>HMS2</italic> and <italic>HMS2t</italic> (black sense; red antisense). Curved lines under diagrams show the extent of each individual transcription unit. Transcription units are envisaged as dynamic with any one region only involved in one TU at any time in an individual cell (see <xref ref-type="fig" rid="fig7">Figure 7G</xref>). Weakening of p<italic>TEF</italic> in the <italic>HMS2:TEF:Kan</italic> construct results in a level of transcription complexity (number of different TUs) similar to the native <italic>HMS2</italic> locus. (<bold>G</bold>) Schematic showing the dynamic alternative transcription units at the <italic>HMS2:SUT650:BAT2</italic> locus. Directional boxes are promoters, squares are terminators coloured coded according to the nature of the TU to which they belong (blue, <italic>HMS2</italic>; black, <italic>BAT2</italic>; Orange, <italic>SUT650</italic>; Grey, <italic>RPS4A</italic> and <italic>YJR149W</italic>). <italic>HMS2</italic> forms alternative TUs with its own terminator (sense-state 1) or with the <italic>BAT2</italic> terminator (sense-state 2&#x2014;a di-cistronic transcription unit) excluding the <italic>SUT650</italic>, <italic>BAT2,</italic> and <italic>YJR149W</italic> promoters from a region where transcription can occur (green shading). <italic>RPS4A</italic>, an OX gene expressed divergently from <italic>HMS2</italic> is active. The sense state predominates in the OX phase of the YMC and during growth on GLU. Any promoter or terminator excluded from the transcription machinery is neutral to the transcription process, explaining how both promoters and terminators are subject to extensive read-through transcription. The sense-state toggles to the alternative antisense-state, predominant in GAL and during the RC phase of the YMC. Here, the formation of the <italic>SUT650</italic> transcription unit excludes the <italic>HMS2</italic> and <italic>RPS4A</italic> promoters from the transcription machinery. This relieves interference of <italic>BAT2</italic> and <italic>YJR149W</italic> by <italic>HMS2</italic> sense transcription. One transcription unit is envisaged to exist in one cell at any one time. The antisense state is the default, requiring transcription factor-dependent activation at <italic>HMS2</italic> to switch to the sense state.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.022">http://dx.doi.org/10.7554/eLife.03635.022</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03635.023</object-id><label>Figure 7&#x2014;figure supplement 1.</label><caption><title>Creating and characterizing an interleaved locus around <italic>HMS2</italic>.</title><p>(<bold>A</bold>) Schematic (i) and estimated sizes (ii) of transcripts in the <italic>HMS2.kan, HMS2 TEF&#x0394;1.kan,</italic> and <italic>HMS2.ADH</italic> disruption strains. (<bold>B</bold>) Dissection of the 5&#x2032; region of the <italic>TEF</italic> promoter. The <italic>TEF</italic> promoter can be divided into 5 arbitrary regions based on the presence of putative transcription factor binding sites. Schematic of <italic>TEF</italic> promoter dissection depicting the remaining promoter sequence after deletions (del or &#x0394;) were introduced. 5&#x2032;<sub><italic>P</italic></sub><italic>TEF</italic>del-2 excluded regions C, which contained no sequence of particular interest, and regions A and B, which contained the putative Reb1 binding sites, REB1A and REB1B. In 5&#x2032;<sub><italic>P</italic></sub><italic>TEF</italic>del-3, regions A and B were excluded, effectively deleting all of the putative Reb1 binding sites but leaving most of the <italic>TEF</italic> promoter intact. A 217 bp deletion was introduced at the 5&#x2032; region of the <italic>TEF</italic> promoter to create the 5&#x2032; <sub><italic>P</italic></sub><italic>TEF</italic>del-<italic>1</italic>. The correct deletions were confirmed by DNA sequencing. (<bold>C</bold>) Northern analysis of 5&#x2032;<sub><italic>P</italic></sub><italic>TEF</italic> mutant strains. The deletions in the 5&#x2032;<italic>TEF</italic> are reflected by the shift in size of the <sub><italic>P</italic></sub><italic>HMS2-kan-TEFt</italic> sense transcript (transcript <bold>A</bold>). (<bold>B</bold>) In the antisense direction, only the 5&#x2032;<sub><italic>P</italic></sub><italic>TEF</italic>del-1 deletion results in the reduction of the AS transcript (transcript <bold>E</bold>) and the generation long antisense transcripts (B<sup>pTEFdel</sup>). In contrast, the 5&#x2032;<sub><italic>P</italic></sub><italic>TEF</italic> del-2 and 5&#x2032;<sub><italic>P</italic></sub><italic>TEF</italic> del-3 mutations resulted in an increase in AS transcript E levels. (<bold>D</bold>) Using the K1 sense probe, the increase in transcript E was reflected by an increase in detectable levels of <italic>Kan</italic><sup>r</sup> transcript (transcript K). Transcripts of interest are also depicted in the miniature transcript maps below the blots.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.023">http://dx.doi.org/10.7554/eLife.03635.023</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03635fs009"/></fig></fig-group></p><p>We reasoned that by introducing mutations into the <italic>TEF</italic> promoter, we would be able to ask how disabling p<italic>TEF</italic> affects the <italic>HMS2</italic> and <italic>SUT650</italic> transcription units. Using the <italic>HMS2:TEF:Kan</italic> construct as a starting point, mutations were introduced into the <italic>TEF</italic> promoter, including deletions of putative transcription factor binding sites (<xref ref-type="fig" rid="fig7">Figure 7C</xref> and <xref ref-type="fig" rid="fig7s1">Figure 7&#x2014;figure supplement 1</xref>). A long deletion (<italic>HMS2.TEFdel1:Kan</italic>), removing all the <italic>TEF</italic> promoter sequences upstream of the TATA box, results in a significant reduction in expression of the <italic>KanMX</italic> cassette itself (transcript K) and in levels of the antisense transcript (E) (<xref ref-type="fig" rid="fig7">Figure 7D,E</xref> and <xref ref-type="fig" rid="fig7s1">Figure 7&#x2014;figure supplement 1</xref>). Remarkably, in this strain, we observed a new antisense transcript from the 3&#x2032; end of <italic>HMS2</italic> that reads through <italic>TEF</italic>t (B<sup>pTEFdel</sup>) (<xref ref-type="fig" rid="fig7">Figure 7C&#x2013;E</xref>). We confirmed that the B<sup>pTEFdel</sup> transcript spans the 3&#x2032; region of <italic>HMS2</italic> using the H3AS probe (<xref ref-type="fig" rid="fig7">Figure 7D</xref>). Thus, the disabled weak p<italic>TEFdel1</italic> resulted in reduced functionality at <italic>TEF</italic>t allowing B<sup>pTEFdel</sup>, initiating at the <italic>SUT650</italic> antisense promoter, to read through <italic>TEF</italic>t, the <italic>KanMX</italic> cassette and into the <italic>HMS2</italic> promoter (<xref ref-type="fig" rid="fig7">Figure 7C&#x2013;E</xref>). This experiment illustrates (i) the conditional nature of the bi-directional <italic>TEF</italic> terminator and (ii) the remarkable plasticity of interleaved transcription units. We suggest that the p<italic>TEF.KanMX.TEF</italic>t expression cassette is strongly expressed (compare levels of transcripts A<sup>K</sup> and K in <xref ref-type="fig" rid="fig7">Figure 7E</xref>), effectively preventing the formation of the <italic>HMS2:SUT650</italic> transcription units. When the <italic>TEF</italic> promoter is disabled, reducing expression of the p<italic>TEF.KanMX.TEF</italic>t expression cassette, the <italic>SUT650</italic> antisense transcription unit can form, resulting in the appearance of the long antisense transcript. Alternatively, reduced <italic>KanMX</italic> sense transcription decreases the ability of a terminator to stop antisense transcription from the <italic>SUT650</italic> antisense promoter. This illustrates the complex relationships that control levels of expression in the interleaved yeast genome (<xref ref-type="fig" rid="fig7">Figure 7F</xref>).</p></sec></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p>We propose a two-state model for transcription at <italic>HMS2</italic> and <italic>BAT2</italic> that combines the effects of temporal separation, overlapping interfering transcription able to bypass typical termination and promoter signals, and insulation from interference by the formation of an antisense transcription unit (<xref ref-type="fig" rid="fig7">Figure 7G</xref>). There are two states: a sense-dominant state (blue transcripts) and an antisense-dominant state (orange transcripts) with respect to <italic>HMS2</italic> (see <xref ref-type="fig" rid="fig6 fig7">Figures 6B and 7G</xref>). Transcription factors (Input) at the <italic>HMS2</italic> promoter regulate sense transcription and loss of input results in the antisense-dominant state. <italic>SUT650</italic> transcription insulates <italic>BAT2</italic> from interference by <italic>HMS2</italic>, reinforcing the antisense dominant state-switch. We propose that transcription in the <italic>HMS2</italic> sense orientation transmits the state-defining regulatory signals to neighbouring genes through mechanisms of <italic>cis</italic>-acting transcriptional interference, thus repressing <italic>BAT2, YJR149W,</italic> and <italic>SUT650</italic>. By these means, reciprocal regulation in cycles or on environmental change could be achieved.</p><p>More generally, how far a transcription event extends into the regulatory sequences of its SAP, or the regulatory sequences of an upstream or downstream gene may determine whether that region is competent to make a state-switch or not and respond to environmental or metabolic cues. Our genome-wide analysis supports many more examples of the type of organisation we observe at <italic>HMS2:BAT2</italic>. Moreover, the <italic>HMS2:BAT2</italic> cluster is conserved in a range of yeast strains expected of a functional linkage. There are many loci at which di-cistronic read-through transcripts are evident (<xref ref-type="bibr" rid="bib35">Pelechano et al., 2013</xref>), raising the possibility that transcriptional interference leading to reciprocal expression of genes in tandem arrays is a much more widespread phenomenon in yeast.</p><p>A di-cistronic transcript means that RNA polymerase has to read through the region determining polyadenylation and termination (the terminator). Moreover, antisense transcription arising from divergent promoters may have to bypass the terminator of the upstream gene. This work indicates the capacity of a terminator to function is conditional on the strength of the proximal promoter. We suggest that this reflects the role for promoters in defining the beginnings and ends of TUs (<xref ref-type="bibr" rid="bib9">El Kaderi et al., 2009</xref>; <xref ref-type="bibr" rid="bib34">O'Sullivan et al., 2004</xref>); stronger promoters preferentially selecting the proximal terminator.</p><p>The role of Sua7 in preventing read-through transcription is intriguing given its association with the 3&#x2032; region of genes, polyadenylation/transcription termination, and higher order structures in chromatin (<xref ref-type="bibr" rid="bib27">Medler et al., 2011</xref>). This raises the possibility of an antisense transcription unit-specific function for TFIIB at the 3&#x2032; ends of genes, characterised here using the <italic>sua7-1</italic> allele. A simple scenario in which 3&#x2032;end TFIIB directs transcription by promoting both antisense transcription and physically terminating sense transcription is compatible with the interference/insulation model proposed here. It is also plausible that alternative higher order structures linking the sense or antisense promoters to one of a possible number of terminators could also explain dynamic state-switching between sense and antisense or multi-cistronic transcripts (<xref ref-type="bibr" rid="bib33">Murray et al., 2012</xref>) (<xref ref-type="fig" rid="fig7">Figure 7G</xref>). Only one structure would exist in each cell at any one time leading to temporal separation of the different TUs. By this model only the juxtaposed regions would function in transcription initiation and termination, allowing other promoters or terminators between these regions to be ignored (<xref ref-type="fig" rid="fig7">Figure 7F,G</xref>). In either case, our data support the idea that the functions of these linked regulatory elements are coupled through the act of transcription. Thus, the interleaved architecture of the yeast genome not only lends itself to high degrees of transcriptional overlap, but more importantly, to an equally high degree of transcription unit interdependency by co-opting transcription itself as a regulatory mechanism.</p><p>While this study has been predominantly conducted using a single locus approach in yeast, the implications of this work could be far reaching. For instance, establishing wider, biologically relevant transcriptional landscapes through sense/antisense state-switching could be pertinent to aspects of chronobiology such as circadian rhythms (<xref ref-type="bibr" rid="bib19">Kramer et al., 2003</xref>; <xref ref-type="bibr" rid="bib10">Feng and Lazar, 2012</xref>), the mitotic/meiotic cell cycles (<xref ref-type="bibr" rid="bib32">Morris and Vogt, 2010</xref>), and other developmental processes in higher eukaryotes. This work places the sense/antisense relationships in a broader biological context and sheds light on how transcription might work as the nexus in more extensive networks that are organized in both spatial and temporal dimensions.</p></sec><sec sec-type="materials|methods" id="s4"><title>Materials and methods</title><p>All experiments were performed at least in duplicate to ensure that the trends observed were reproducible. Briefly, unless otherwise stated, cells were grown to exponential phase in rich medium (YP) supplemented with 2% glucose (YPD) or 2% galactose (YPGal). The strains of <italic>S. cerevisiae</italic> used for this study are shown in <xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1L</xref>. The Anchor Away technique exploits the high affinity interaction between the FK506 Binding Protein (<italic>FKBP12</italic>) and <italic>FKBP12</italic> Rapamycin binding (FRB) domain of human mTOR protein in order to deplete target proteins from the nucleus in a time-dependent manner (<xref ref-type="bibr" rid="bib13">Haruki et al., 2008</xref>). Control and treated cells were harvested after 1 h of DMSO or rapamycin exposure. Total RNA extracts were acquired by hot phenol extraction and enriched for polyadenylated transcripts using Oligotex&#x2010;dT as directed by the manufacturer (QIAGEN NV.). The concentration of RNA extract was measured and strandardised to 1 &#xb5;g/&#x3bc;l using a nanodrop spectrophotometer. The protocol for rapid amplification of cDNA 3&#x2032; ends (3&#x2032; RACE) used in this study was modified from the methods provided by the 3&#x2032; RACE System for Rapid Amplification of cDNA Ends kit (Invitrogen Carlsbad, CA). Primers used are shown in <xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1M</xref>. Northern blotting was done as described in <xref ref-type="bibr" rid="bib33">Murray et al. (2012)</xref>. In vitro transcription with T7 RNA polymerase and radiolabelling was used to create strand-specific probes for Northern blotting. Primers are shown in <xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1N</xref>. Hybridization of RNA to strand-specific, high resolution tiling arrays was performed as described in <xref ref-type="bibr" rid="bib36">Perocchi et al. (2007)</xref>. Data analysis was performed by members of the Lars Steinmetz group at the European Molecular Biology Labs in Heidelberg, Germany (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://steinmetzlab.embl.de/cgi-bin/viewMellorLabArray.pl?showSamples=502_Glu1Vs503_Gal1%26type=heatmap%26gene=hms2">http://steinmetzlab.embl.de/cgi-bin/viewMellorLabArray.pl?showSamples&#x3d;502_Glu1Vs503_Gal1&#x26;type&#x3d;heatmap&#x26;gene&#x3d;hms2</ext-link>). Native elongating transcript sequencing, (NET-seq) was done and analysed as described in <xref ref-type="bibr" rid="bib7">Churchman and Weissman (2011)</xref>. To assess the genome-wide significance of our observations, all genes in <xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1A</xref> were characterised according to their orientation with respect to their upstream (5P divergent, 5P tandem) or their downstream gene (3P, convergent), their own, and their neighbouring genes&#x2032; gene type (ORF, CUT, SUT, other) and YMC status (OX, oxidative; RB, reductive building; RC, reductive charging; NA/NC, non-cycling) and the number of genes in each category and median expression level in glucose and galactose determined. Genes that were located more than 1,000 bp from their neighbouring genes were excluded from the analysis. Computer simulations were performed by shuffling the strand location, gene type, YMC status, and expression levels of individual genes on every chromosome while keeping distances between genes and the total number of genes on each strand and of each type constant (<xref ref-type="supplementary-material" rid="SD7-data">Source code 1</xref>&#x2013;MATLAB codes). Expression levels were simulated by randomly selecting recorded expression levels. After each simulation run, the number of genes and the median expression levels were calculated for each category. Simulations were run 10,000 times. Overlapping genes were defined as being positioned on the opposite strand and having 3&#x2032; ends covering the same region of the genome. Enrichment analysis for factors was performed using the published data set by <xref ref-type="bibr" rid="bib55">Venters et al. (2011)</xref>. The dual-labelled single molecule RNA Fluorescence <italic>In Situ</italic> hybridization (FISH) protocol was adapted from <xref ref-type="bibr" rid="bib59">Zenklusen and Singer (2010)</xref>. Fluorescently labelled DNA probes to detect sense or antisense transcripts are shown in <xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1O</xref>. Image acquisition was performed with a DeltaVision CORE: Wide-field fluorescence deconvolution imaging microscope. Using a 100x objective lens, 31 &#x2018;z-stacks&#x2019; were collected. For each stack, an exposure time of 0.5 s for DAPI and 1 s for Cy3 (TRITC) and Cy5 filters was applied. Images were captured using a CoolSNAP HQ camera (Photometrics, Tucson, AZ). To facilitate image analysis, 3D data sets were compressed into 2D images by using a maximum projection, using <italic>hms2&#x394;</italic> cells to determine threshold signal for expression. Images displayed as 2D compressions of z-stacks 12&#x2013;22 (11 stacks), which included most of the nucleus and cytoplasm. Error bars represent standard error of the mean. Chromatin immunoprecipitation (ChIP) was performed as described by <xref ref-type="bibr" rid="bib31 bib30">Morillon et al. (2003, 2005)</xref>. Primers used for RT qPCR are displayed in <xref ref-type="supplementary-material" rid="SD6-data">Supplementary file 1P</xref>.</p></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>The authors would like to thank Ilan Davis, Micron Oxford, and Dan Larsen for help with RNA FISH, Athar Ansari, and Mike Hampsey for strains containing the <italic>sua7-1</italic> allele and helpful advice, Stirling Churchman for help and advice with the NET-seq technique, Anitha Nair for excellent laboratory support, Benjamin Schuster-B&#xf6;ckler for advice regarding the computer simulations, and the following for funding; Keble College and the Clarendon Fund (to T.N.) and a Wellcome Trust Strategic Award (091911) supporting advanced microscopy at Micron Oxford (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://micronoxford.com/">http://micronoxford.com</ext-link>)</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>JM: Adviser to Oxford Biodynamics Ltd and Sibelius Ltd and sits on the board of Chronos Therapeutics. OBD provided funding for this work but like all the funders, had no say in the design or outcome of the research and do not benefit in any way from this research.</p></fn><fn fn-type="conflict" id="conf2"><p>The other authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>TN, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>FSH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>RW, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>HF, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con5"><p>ASB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con6"><p>ZX, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con7"><p>DB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con8"><p>SCM, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con9"><p>SH, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con10"><p>JMH, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con11"><p>LO'C, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con12"><p>GS, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con13"><p>LMS, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con14"><p>JM, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD6-data"><object-id pub-id-type="doi">10.7554/eLife.03635.024</object-id><label>Supplementary file 1.</label><caption><p>(<bold>A</bold>) Genome-wide NET-seq (NET) and poly(A)<sup>&#x2b;</sup> RNA hybridised to microarray (mi). (<bold>B</bold>) Data for Pie Charts in <xref ref-type="fig" rid="fig1">Figure 1</xref>. (<bold>C</bold>) YMC genes (Coloured in columns A, C, and E) that overlap with genes whose transcription changes &#x3e;threefold on the GLU to GAL shift (Column G&#x2014;complete list colour coded). (<bold>D</bold>) Gene Ontology (GO) associated with genes that change &#x3e;threefold on the GLU to GAL shift. (<bold>E</bold>) Annotated CUTs and SUTs that change &#x3e;threefold on GLU to GAL shift (from Supplementary file 1A). (<bold>F</bold>) Extracted data from genome-wide simulation of gene type, orientation, and regulation. (<bold>G</bold>). Gene Groups from genome-wide annotations of OX.RC, RC.OX, and non-cycling (NC) pairs in tandem&#x2014;used to provide information for Supplementary file 1J and to derive the distinct environments surrounding pairs of cycling or non-cycling genes. (<bold>H</bold>) Selected genes from Supplementary file 1A and analysis of their environment. (<bold>I</bold>) Genes that change &#x3e;threefold on the GLU/GAL shift that also have an annotated antisense CUT or SUT that also changes &#x3e;threefold on the GLU/GAL shift. (<bold>J</bold>) Selected gene clusters resembling <italic>HMS2:BAT2</italic>. (<bold>K</bold>) Transcription-related factors enriched at the promoters of RC or OX genes. (<bold>L</bold>) Genotype of yeast strains used in this study. (<bold>M</bold>) Primers used for 3&#x2032;RACE. (<bold>N</bold>) Primers used to generate strand-specific probes for Northern blot analysis. The T7 promoter sequence is shown in parentheses. (<bold>O</bold>) RNA FISH probes. (<bold>P</bold>) Primers used for real-time PCR.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.024">http://dx.doi.org/10.7554/eLife.03635.024</ext-link></p></caption><media xlink:href="elife03635s006.xlsx" mimetype="application" mime-subtype="xlsx"/></supplementary-material><supplementary-material id="SD7-data"><object-id pub-id-type="doi">10.7554/eLife.03635.025</object-id><label>Source Code 1.</label><caption><p>Source Codes in MATLAB 'simulation_gene_orientation_and_expression'.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03635.025">http://dx.doi.org/10.7554/eLife.03635.025</ext-link></p></caption><media xlink:href="elife03635s007.m" mimetype="application" mime-subtype="m"/></supplementary-material></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Batada</surname><given-names>NN</given-names></name><name><surname>Urrutia</surname><given-names>AO</given-names></name><name><surname>Hurst</surname><given-names>LD</given-names></name></person-group><year>2007</year><article-title>Chromatin remodelling is a major source of coexpression of linked genes in yeast</article-title><source>Trends in Genetics</source><volume>23</volume><fpage>480</fpage><lpage>484</lpage><pub-id pub-id-type="doi">10.1016/j.tig.2007.08.003</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Berretta</surname><given-names>J</given-names></name><name><surname>Pinskaya</surname><given-names>M</given-names></name><name><surname>Morillon</surname><given-names>A</given-names></name></person-group><year>2008</year><article-title>A cryptic unstable transcript mediates transcriptional trans-silencing of the Ty1 retrotransposon in <italic>S. cerevisiae</italic></article-title><source>Genes &#x26; 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letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Espinosa</surname><given-names>Joaquin M</given-names></name><role>Reviewing editor</role><aff><institution>Howard Hughes Medical Institute, University of Colorado</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled &#x201c;Transcription mediated insulation and interference direct gene cluster expression switches during biological rhythms&#x201d; for consideration at <italic>eLife</italic>. Your article has been favourably evaluated by James Manley (senior editor), a Reviewing editor, and 3 reviewers.</p><p>After a thorough discussion, the consensus among the reviewers and the editors was that this is an excellent paper reporting some remarkable findings, which would be worthy of publication in <italic>eLife</italic> after addressing the concerns listed below.</p><p>The metabolic cycle has emerged as a fundamental biological phenomenon that is critical for proper cell function. It has been appreciated that oxidative conditions are in conflict with reducing reactions, and vice versa. Consequently, cells have evolved mechanisms to temporarily separate these conflicting conditions, by oscillating both the oxygen consumption as well as the expression of &#x223c;3000 genes. The remarkable precision of the synchrony in maintaining oscillating levels of mRNAs must involve intracellular events and communication, the basis of which remains obscure. This paper provides some solutions to this enigma by demonstrating how transcription of tandemly arrayed genes can regulate a bi-modal switch so that activation of one gene would repress the other and vice versa. This paper will serve the scientific community in various aspects. First, it will drive the attention of the readers to the complexity of the YMC, an issue that is still underrepresented in the literature. Second, it illustrates the critical roles played by transcription terminators in both terminating transcription and initiating transcription in the anti-sense direction. Third, transcription of anti-sense (AS) as a key modulator of gene expression, in general, and its involvement in the bi-modal switch, in particular. Fourth, and most importantly, the paper demonstrates, in many ways, the intricate relationship between transcription of sense, anti-sense and neighbouring genes in a manner that supports the bimodal switch.</p><p>The major concerns raised by the reviewers are:</p><p>1) Generality of findings. The impact of this work would certainly be higher upon defining the prevalence of the key regulatory mechanisms described. The paper focuses on two tandemly arrayed genes (HMS2/BAT2) whose expression switches during the YMC. They provide a detailed mechanism of the switching whereby transcription read-through into the downstream promoter downregulates transcription of the downstream gene and how AS transcription is involved. Their conclusion that the sense and AS transcription are in conflict, and that cells tend to separate the &#x201c;sense state&#x201d; from the &#x201c;AS state&#x201d; temporarily is by itself remarkable. However, how general is this mechanism? What fraction of YMC-regulated genes is adjacent or anti-sense? More than expected by chance? And then what fraction of adjacent or anti-sense transcripts is co-regulated in the YMC? Moreover, if the mechanism is more prevalent than just HMS2/BAT2, do the anti-sense transcripts always fall into opposite YMC categories, i.e., oxidative vs reductive? Is this locus even representative of other glu-gal clusters? If 10 loci are chosen at random (based on having one strong glu-gal-regulated transcript), do the neighbouring genes show potent regulation like the one well-explored region? Reviewers agreed that a more global assessment of the prevalence of the mechanism described would raise the impact significantly. However, it was noted that the bioinformatics involved for a global analysis would not be trivial. Thus, reviewers request that the authors manually mine the NET-seq data regarding transcript isoforms (like the data shown in <xref ref-type="fig" rid="fig2s1">Figure 2&#x2013;figure supplement 1D</xref>) and examine whether transcription of one gene invades the promoter of its neighbouring gene, and whether the AS transcription is anti-correlated. The control should be the same number of tandemly positioned gene pairs that are chosen at random. If the authors have the bioinformatic means to take a whole genome view, all the better.</p><p>2) Weak links to YMC and circadian rhythms. Although the reviewers appreciated the novelty and elegance of the molecular mechanisms discovered, there was agreement that in its current form the manuscript does not make a convincing argument about general relevance to YMC or circadian rhythms and it was agreed that the writing, including the title, should be tempered to focus more on the mechanistic aspects and less on the YMC and circadian biology. It is proposed that the title be changed to &#x201c;Transcription mediated insulation and interference direct gene cluster expression switches&#x201d;.</p><p>3) The choice of sua7-1 is not well communicated. The reader of the &#x201c;Results&#x201d; section is puzzled why TFIIB was chosen to begin, and why this specific allele was used, in particular. Only in the Discussion the reader learns that sua7-1 is defective in loop formation.</p><p>4) <xref ref-type="fig" rid="fig3">Figure 3B</xref> shows that the insertion of ADH1t abolished the signal detected by probe H2. From the point of view of the AS transcription, this probe is located downstream of ADH1t. The authors concluded that ADH1t blocks SUT650 AS transcription. However, it is possible that ADHt acts in both orientation and simply terminated transcription and therefore H2 could not detect any transcript. If the authors know that ADH1t does not act as terminator in the reverse orientation, they should indicate it. Moreover, even if the ADH1t is not acting as terminator of the AS, it can introduce a destabilization element that leads to its rapid degradation. In order to support this important conclusion, the authors could use a decay mutant strain (e.g., rrp6) and probe the membrane to detect the region between the ADHt and SUT650 promoter.</p><p>5) The replacement of HMS2 ORF with URA3 requires some clarifications. Why didn&#x27;t the authors simply remove the region around the TSS, leaving all other sequences unchanged? Why did they prefer to introduce the entire URA3? There are two issues that need to be addressed. (1) The possibility that URA3 sequences, in both orientations, introduce stability biases onto the RNAs. (2) The possibility that the URA3 sequence contains a terminator in the AS direction, explaining why the AS RNA was not detected by H1 probe, located downstream of this hypothetical terminator. These issues can be discussed or addressed experimentally.</p><p>6) In <xref ref-type="fig" rid="fig6s1">Figure 6&#x2013;figure supplement 1</xref>, the authors used TFs with putative binding sites at intergenic region between HMS2 and BAT2 (no references were included). The membranes were probed only with the H1 probe. This case might be an opportunity to examine if any of these factors play a role in the transcription of BAT2, by probing the membrane with an appropriate probe. URA3 ORF can be replaced back to HMS2 ORF by homologous recombination and selection on 5-FOA, thus resulting in a precise deletion of just the region around the TSS. Maybe one of these TFs is required for BAT2 transcriptional repression by C1 transcription (e.g., transcription through the binding site might displace the TF).</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03635.027</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) Generality of findings. The impact of this work would certainly be higher upon defining the prevalence of the key regulatory mechanisms described</italic>.</p><p>We agree with the reviewers about this point and had, in fact, already embarked on a computational genome-wide analysis. Part of this very large dataset has been analysed for this work and is presented in the revised version of the paper.</p><p><italic>The paper focuses on two tandemly arrayed genes (HMS2/BAT2) whose expression switches during the YMC. They provide a detailed mechanism of the switching whereby transcription read-through into the downstream promoter downregulates transcription of the downstream gene and how AS transcription is involved. Their conclusion that the sense and AS transcription are in conflict, and that cells tend to separate the &#x201c;sense state&#x201d; from the "AS state" temporarily is by itself remarkable. However, how general is this mechanism</italic>?</p><p>From the point of switching sense and antisense states there is now one experiment in a recent paper from the Zenklusen lab supporting the idea of separation of the sense and antisense states in individual cells, in this case at <italic>PHO84</italic> (Castelnuovo M, Rahman S, Guffanti E, Infantino V, Stutz F and Zenklusen D: Bimodal expression of PHO84 is modulated by early termination of antisense transcription. Nat Struct Mol Biol 20: 851-8). We also have as yet unpublished evidence that at <italic>GAL10</italic>, the sense and antisense transcripts are present in different cells during early induction. Given the FISH evidence for YMC regulated genes from the Singer and Botstein labs, we think that switching is likely to be common.</p><p><italic>What fraction of YMC-regulated genes is adjacent or anti-sense? More than expected by chance? And then what fraction of adjacent or anti-sense transcripts is co-regulated in the YMC?</italic></p><p>We have data in the revised manuscript addressing these issues (Tables S5 to S9). The antisense question is hard to address adequately due to the relatively poor annotation of the antisense transcriptome &#x2013; being limited to CUTs and SUTs. There are 1772 annotated CUTs and SUTs but much more transcription antisense to genes is evident from the NET-seq analysis. A separate analysis suggests that 75% of all yeast genes are subject to some antisense transcription in the vicinity of the sense promoter that influences the chromatin organization around that promoter. Nevertheless, our analysis for this work suggests that YMC genes are much more likely to have antisense transcription, although the number of genes is relatively small because of the high thresholds (&#x3e;3-fold change in GLU/GAL both the YMC gene and the SUT or CUT). 71 of the 206 genes (34.4%) with regulated sense and antisense have SUTs/CUTs whose transcription also changes 3-fold in the GLU/GAL shift. 26% of all SUTs and CUTs show a &#x3e;3-fold change on the GLU/GAL shift. Thus regulated SUTs and CUTs are enriched opposite to ORFs that also show a &#x3e; 3-fold shift.</p><p><italic>Moreover, if the mechanism is more prevalent than just HMS2/BAT2, do the anti-sense transcripts always fall into opposite YMC categories, i.e., oxidative vs reductive</italic>?</p><p>Our selected gene study shows that for the 10 OX.RC tandem genes (like <italic>HMS2.BAT2</italic>), 9 (90%) of the OX genes have an antisense (annotated or not) that shows reciprocal transcription to the OX gene.</p><p><italic>Is this locus even representative of other glu-gal clusters</italic>?</p><p>Yes, see Table S9 and <xref ref-type="fig" rid="fig1s1 fig1s2 fig1s3 fig1s4">Figure 1&#x2013;figure supplements 1&#x2013;4</xref>.</p><p><italic>If 10 loci are chosen at random (based on having one strong glu-gal-regulated transcript), do the neighbouring genes show potent regulation like the one well-explored region</italic>?</p><p>Yes, see Table S9 and <xref ref-type="fig" rid="fig1s1 fig1s2 fig1s3 fig1s4">Figure 1&#x2013;figure supplements 1&#x2013;4</xref>.</p><p><italic>Reviewers agreed that a more global assessment of the prevalence of the mechanism described would raise the impact significantly</italic>.</p><p>I hope that we have persuaded the reviewers that this is likely to be a more general phenomenon.</p><p><italic>However, it was noted that the bioinformatics involved for a global analysis would not be trivial. Thus, reviewers request that the authors manually mine the NET-seq data regarding transcript isoforms (like the data shown in</italic> <xref ref-type="fig" rid="fig2s1"><italic>Figure 2&#x2013;figure supplement 1D</italic></xref><italic>) and examine whether transcription of one gene invades the promoter of its neighbouring gene, and whether the AS transcription is anti-correlated. The control should be the same number of tandemly positioned gene pairs that are chosen at random. If the authors have the bioinformatic means to take a whole genome view, all the better</italic>.</p><p>We have used both approaches to provide the data in order to conclude this is a wider phenomenon.</p><p><italic>2) Weak links to YMC and circadian rhythms. Although the reviewers appreciated the novelty and elegance of the molecular mechanisms discovered, there was agreement that in its current form the manuscript does not make a convincing argument about general relevance to YMC or circadian rhythms and it was agreed that the writing, including the title, should be tempered to focus more on the mechanistic aspects and less on the YMC and circadian biology. It is proposed that the title be changed to &#x201c;Transcription mediated insulation and interference direct gene cluster expression switches&#x201d;</italic>.</p><p>We are happy to do this and have removed this from the title and tempered our references to these mechanisms in the text.</p><p><italic>3) The choice of sua7-1 is not well communicated. The reader of the &#x201c;Results&#x201d;" section is puzzled why TFIIB was chosen to begin, and why this specific allele was used, in particular. Only in the Discussion the reader learns that sua7-1 is defective in loop formation</italic>.</p><p>We agree with this point and have included more details in the Results section.</p><p><italic>4)</italic> <xref ref-type="fig" rid="fig3"><italic>Figure 3B</italic></xref> <italic>shows that the insertion of ADH1t abolished the signal detected by probe H2</italic>.</p><p>This is correct for the H2AS probe.</p><p><italic>From the point of view of the AS transcription, this probe is located downstream of ADH1t</italic>.</p><p>This is correct.</p><p><italic>The authors concluded that ADH1t blocks SUT650 AS transcription. However, it is possible that ADHt acts in both orientation and simply terminated transcription and therefore H2 could not detect any transcript</italic>.</p><p>This is correct.</p><p><italic>If the authors know that ADH1t does not act as terminator in the reverse orientation, they should indicate it</italic>.</p><p>We do know that the ADH1t acts as a terminator in both directions. It does so in its native context (terminating the YOL086AW TU) and when used out of context in <italic>GAL10</italic> (see <xref ref-type="bibr" rid="bib33">Murray et al. 2012</xref> NAR).</p><p><italic>Moreover, even if the ADH1t is not acting as terminator of the AS, it can introduce a destabilization element that leads to its rapid degradation. In order to support this important conclusion, the authors could use a decay mutant strain (e.g., rrp6) and probe the membrane to detect the region between the ADHt and SUT650 promoter</italic>.</p><p>We have included two additional pieces of data in <xref ref-type="fig" rid="fig3">Figure 3</xref> to address this. First we have data showing the effect of the decay mutant strain (<italic>rrp6</italic>) using probe H2AS. There are no unstable transcripts detectable in the <italic>rrp6</italic> strain.</p><p>Second we use probe H3AS to show that SUT650 is prematurely terminated. Thus both <italic>HMS2</italic> sense transcription and <italic>SUT650</italic> antisense transcription are prematurely terminated in this strain.</p><p><italic>5) The replacement of HMS2 ORF with URA3 requires some clarifications. Why didn&#x27;t the authors simply remove the region around the TSS, leaving all other sequences unchanged? Why did they prefer to introduce the entire URA3</italic>?</p><p>We chose to completely replace the <italic>HMS2</italic> ORF with the <italic>URA3</italic> ORF so as to avoid problems with changing codons, deleting bases, or altering transcript stability while leaving the <italic>HMS2</italic> flanking sequences unaltered.</p><p><italic>There are two issues that need to be addressed. (1) The possibility that URA3 sequences, in both orientations, introduce stability biases onto the RNAs. (2) The possibility that the URA3 sequence contains a terminator in the AS direction, explaining why the AS RNA was not detected by H1 probe, located downstream of this hypothetical terminator. These issues can be discussed or addressed experimentally</italic>.</p><p>We cannot address this using experimental data as BY4741 is <italic>ura3&#x0394;</italic> and thus the <italic>URA3</italic> gene is not in our datasets to test for the presence of an early terminator. Given that the promoter of a gene is reported to determine sense RNA stability (Bregman A, Avraham-Kelbert M, Barkai O, Duek L, Guterman A and Choder M: Promoter elements regulate cytoplasmic mRNA decay. Cell 147: 1473-83; Trcek T, Larson DR, Moldon A, Query CC and Singer RH: Single-molecule mRNA decay measurements reveal promoter- regulated mRNA stability in yeast. Cell 147: 1484-97), we felt the <italic>URA3</italic> ORF replacement was an appropriate strategy. We have discussed these potential problems in the text and how we have interpreted our data given these provisos.</p><p><italic>6) In</italic> <xref ref-type="fig" rid="fig6s1"><italic>Figure 6&#x2013;figure supplement 1</italic></xref><italic>, the authors used TFs with putative binding sites at intergenic region between HMS2 and BAT2 (no references were included). The membranes were probed only with the H1 probe. This case might be an opportunity to examine if any of these factors play a role in the transcription of BAT2, by probing the membrane with an appropriate probe. URA3 ORF can be replaced back to HMS2 ORF by homologous recombination and selection on 5-FOA, thus resulting in a precise deletion of just the region around the TSS. Maybe one of these TFs is required for BAT2 transcriptional repression by C1 transcription (e.g., transcription through the binding site might displace the TF)</italic>.</p><p>We apologise for not including references to the putative TF binding sites &#x2013; this is now done. Putative factor binding sites, and even ChIP data with tagged factors, does not provide evidence that a particular factor is functional at a particular site, for a variety of reasons (Lenstra TL, Benschop JJ, Kim T, Schulze JM, Brabers NA, Margaritis T, van de Pasch LA, van Heesch SA, Brok MO, Groot Koerkamp MJ, Ko CW, van Leenen D, Sameith K, van Hooff SR, Lijnzaad P, Kemmeren P, Hentrich T, Kobor MS, Buratowski S and Holstege FC: The specificity and topology of chromatin interaction pathways in yeast. Mol Cell 42: 536-49, 2011). Indeed, many of the experiments linking transcription factors to changes in particular transcriptional responses at genes are likely to be artefacts resulting simply from altered growth rate as a result of the genetic intervention (O&#x27;Duibhir E, Lijnzaad P, Benschop JJ, Lenstra TL, Leenen D van, Groot Koerkamp MJA, Margaritis T, Brok MO, Kemmeren P and FCP H: Cell cycle population effects in perturbation studies. Mol Syst Biol. 10: 2014; Slavov N, Airoldi EM, van Oudenaarden A and Botstein D: A conserved cell growth cycle can account for the environmental stress responses of divergent eukaryotes. Mol Biol Cell 23: 1986-97, 2013), (and the different times spent in the OX and RC phases of the YMC). We think this criticism applies to all the experiments we have done with TF KO strains in <xref ref-type="fig" rid="fig6">Figure 6</xref> and <xref ref-type="fig" rid="fig6s1">Figure 6&#x2013;figure supplement 1</xref> and have been careful not to over-interpret the data to infer that these TFs actually bind to the DNA. Rather, the data in <xref ref-type="fig" rid="fig6">Figure 6A</xref> showing the switching of the balance of <italic>HMS2</italic> sense and <italic>SUT650</italic> antisense suggest that ablation of some factors coupled to mild nutrient limitation leads to cells where transcription in the <italic>HMS2:SUT650</italic> region is predominantly in sense or antisense orientations. This provides evidence for reciprocal switching between <italic>HMS2</italic> sense and <italic>SUT650</italic> antisense and suggests a signalling input regulates this only at the <italic>HMS2</italic> promoter. We have a number of experiments in the paper to support this, particularly those in <xref ref-type="fig" rid="fig5">Figure 5</xref>. In summary these experiments show that expression of the antisense state (<italic>SUT650</italic> and <italic>BAT2</italic>) is constitutive in the absence of sense transcription, regardless of growth conditions (GLU or GAL). Normally <italic>SUT650</italic> transcription increases on the GLU to GAL shift but this reflects the reduction in <italic>HMS2</italic> sense transcription in GAL. Without sense, SUT650 levels remain high in GLU. Similar arguments apply to <italic>BAT2</italic>. Elimination of sense transcript in the p<italic>GAL1:HMS2</italic> anchor away experiment in <xref ref-type="fig" rid="fig5">Figure 5</xref>, by addition of Rapamycin to deplete nuclear Gal4 from cells in GAL, restores <italic>BAT2</italic> transcript levels. Thus the primary <italic>BAT2</italic> regulation is transcriptional interference by the sense transcription in GAL (in this experiment only as the inserted <italic>GAL1</italic> promoter is GAL-regulated).</p><p>Our view is that the (semi-) quiescent RC phase in the YMC is likely to be the default and RC genes would be expressed unless a specific growth signal is received. <italic>HMS2</italic> switches on and interferes with the transcription of <italic>SUT650</italic> and <italic>BAT2</italic> as long as the growth signal remains. If there is no growth signal, transcription of <italic>BAT2</italic> and <italic>SUT650</italic> is constitutive, as if cells are in the quiescent state that occurs naturally when nutrients are limited. In fact all the changes that occur when yeast naturally enter stationary phase due to nutrient depletion also occur in RC cells (Shi L, Sutter BM, Ye X and Tu BP: Trehalose is a key determinant of the quiescent metabolic state that fuels cell cycle progression upon return to growth. Mol Biol Cell 21: 1982-90). This does not rule out TFs in the regulation of RC genes, but it does suggest they are not condition-specific. This is supported by our two new genome-wide analyses now included in the manuscript (Table S10). We have examined the nature of the transcription factors&#x2019; association with the promoters of RC or OX genes (in GLU) from the Venters and Pugh work (2011) (now included in reference list). They looked at the association of 202 factors at yeast promoters. We used their dataset to ask whether OX and RC genes are enriched for particular factors, as both will be expressed in GLU-grown cells. We show significantly different (p&#x3c;0.01) ChIP-Chip binding patterns for factors at the two groups of genes. The RC genes are enriched for cohesion, the CCR4/NOT complex, elongator, chromatin and chromatin remodelling factors and four TFs, (Fhl1, Ino4, Ume6 and Skn7). By contrast, OX genes are associated with GTFs, TEFs, TFIID, and the TFs Ifh1 and Rap1. Although &#x201c;stress&#x201d; related transcription factors may play a role at the RC genes, these data suggest chromatin-mediated regulation as a central feature for RC genes. Our data support TI-mediated chromatin changes at the <italic>BAT2</italic> promoter.</p><p>So to the question of whether the putative TF binding sites between <italic>HMS2</italic> and <italic>BAT2</italic> regulate <italic>BAT2</italic> expression or more simply, contribute to limiting <italic>HMS2</italic> read-through (<xref ref-type="fig" rid="fig6s1">Figure 6&#x2013;figure supplement 1</xref>). As it stands, our data, albeit quite crude, provides no evidence for a role for these factors in activation of either <italic>HMS2</italic> or <italic>SUT650</italic>. We rule out TFs as roadblocks (at least Gal4 for <italic>SUT650</italic> transcribed into the <italic>GAL</italic> promoter) but do see a role for Gln3 (only in minimal medium) in preventing transcript C (<italic>HMS2:BAT2</italic> read through) and a reduction in <italic>SUT650</italic> levels (supporting increased TI at the <italic>HMS2:BAT2</italic> intergenic region). Given the literature supports a role for TFs and promoters in determining transcript stability, another explanation for the effect of loss of Gln3 is that transcript C is stabilised, but there is no change in transcription <italic>per se</italic> and thus no change in the transcript ratios. How RC genes are regulated is clearly complicated and beyond the scope of what we can do for a 2 month revision. We think the suggestion of the referee is excellent but have a number of reservations about this type of approach, especially as some of the putative TF binding sites are within the <italic>HMS2</italic> ORF (similar arguments to the <italic>URA3</italic> question above). Instead we have provided an Anchor-Away experiment to deplete nuclear Gln3 in Galactose (conditions expected to increase <italic>BAT2</italic> and <italic>SUT650</italic> expression). In the same experiment nuclear depletion of Sua7 results in loss of both <italic>BAT2</italic> and <italic>SUT650</italic> transcripts. We find no evidence that Gln3 is required for <italic>BAT2</italic> or <italic>SUT650</italic> expression in these conditions and the Sua7 control suggests this is not simple a result of the <italic>BAT2</italic> transcript being particularly stable in GAL. These data are now included in <xref ref-type="fig" rid="fig6s1">Figure 6&#x2013;figure supplement 1</xref>.</p></body></sub-article></article>