1 Transcription mediated insulation and interference direct gene 2 cluster expression switches 3 Tania Nguyen1, Harry Fischl1#, Françoise S. Howe1#, Ronja Woloszczuk1#, Ana Serra 4 Barros1*, Zhenyu Xu2*, David Brown1, Struan C. Murray1, Simon Haenni1, James M. 5 Halstead1, Leigh O’Connor1, Gergana Shipkovenska1, Lars M. Steinmetz2 and Jane Mellor1§ 6 1Department of Biochemistry, South Parks Road, Oxford, OX1 3QU, UK 7 2European Molecular Biology Laboratory (EMBL), Genome Biology Unit, 69117 Heidelberg, 8 Germany 9 10 . 11 # , * Equal contribution 12 § Corresponding author: [email protected] 13 14 Competing interests statement: JM is an advisor to Oxford Biodynamics Ltd and Sibelius Ltd and sits 15 on the board of Chronos Therapeutics Ltd. OBD supported this work but have no commercial 16 interest in the study design, data collection and interpretation, or the decision to submit the work 17 for publication. 1 18 Abstract 19 In yeast, many tandemly arranged genes show peak expression in different phases of the 20 metabolic cycle (YMC) or in different carbon sources, indicative of regulation by a bi- 21 modal switch, but it is not clear how these switches are controlled. Using native 22 elongating transcript analysis (NET-seq), we show that transcription itself is a component 23 of bi-modal switches, facilitating reciprocal expression in gene clusters. HMS2, encoding a 24 growth-regulated transcription factor, switches between sense- or antisense-dominant 25 states that also coordinate up- and down-regulation of transcription at neighbouring 26 genes. Engineering HMS2 reveals alternative mono-, di- or tri-cistronic and antisense 27 transcription units (TUs), using different promoter and terminator combinations, that 28 underlie state-switching. Promoters or terminators are excluded from functional TUs by 29 read-through transcriptional interference, while antisense TUs insulate downstream genes 30 from interference. We propose that the balance of transcriptional insulation and 31 interference at gene clusters facilitates gene expression switches during intracellular and 32 extracellular environmental change. 33 34 Impact Statement 35 The formation of mutually exclusive coding and non-coding transcription units contributes 36 to transcriptional interference and insulation at gene clusters and manages state- 37 switching in response to environmental change. 38 39 Introduction 40 Genome-wide mapping of RNA transcripts in the budding yeast Saccharomyces cerevisiae 41 has revealed an extensive array of coding and non-coding transcripts, giving rise to a 2 42 genome that is heavily interleaved. Individual genes can possess multiple, overlapping 43 transcripts, in the sense and the antisense orientations with respect to the pre-mRNA, as 44 well as poly-cistronic transcripts, which span neighbouring genes (Berretta et al, 2008; 45 Kapranov et al, 2007; Pelechano et al, 2013). Genome-wide mapping of nascent 46 transcription, using techniques such as NET-seq, shows that transcription commonly 47 extends into and over the intergenic regions of both convergent and tandemly arranged 48 genes (Churchman & Weissman, 2011). For tandemly arranged genes, transcription into the 49 promoter of the downstream gene would be expected to interfere with its expression by a 50 variety of mechanisms, including modifying the local chromatin environment and 51 interference by removal of transcription factors (Hainer et al, 2011; Martens et al, 2004; 52 Martianov et al, 2007). Furthermore, extensive transcription antisense to (Murray et al, 53 2012; Venters & Pugh, 2009), and into the promoter of (Perocchi et al, 2007; Xu et al, 2009), 54 the canonical coding transcript is also implicated in modulating gene expression by similar 55 mechanisms (Camblong et al, 2007; Castelnuovo et al, 2013; Hongay et al, 2006; Houseley et 56 al, 2008; Pinskaya et al, 2009; Uhler et al, 2007; van Werven et al, 2012; Xu et al, 2011). An 57 interleaved genome with overlapping transcription units requires that polyadenylation and 58 transcription termination signals in the sense and antisense orientations are by-passed but 59 it is not clear how this is achieved. In addition, questions are commonly raised about 60 whether transcription of these overlapping transcription units is contemporaneous. 61 It is now clear that much transcription is organised into biologically-relevant 62 temporal windows within phenomena such as the metabolic cycle (Tu & McKnight, 2006). 63 Indeed, periodic or cycling expression of genes can be detected using fluorescent reporters 64 or dual-labelled RNA FISH in cultures of asynchronous cells (Laxman et al, 2010; Silverman et 65 al, 2010), or in the absence of cell division (Slavov et al, 2011). This periodic expression is a 3 66 result of synchronization of respiratory and glycolytic activities into robust oscillations in 67 oxygen consumption, characterized by phase-specific transcript signatures involving over 68 3,000 genes, known as the Yeast Metabolic Cycle (YMC) (Cai & Tu, 2012; Klevecz et al, 2004; 69 Slavov et al, 2011; Soranzo et al, 2009; Tu et al, 2005; Tu et al, 2007). In the long-period 70 YMC, a single cell alternates between periods of high (oxidative (OX) phase) or low oxygen 71 consumption (reductive building (RB) and charging (RC) phases), the residence time in each 72 phase being nutrient-dependent. For exponentially growing cells in batch culture, the 73 majority of cells in the population will be in the OX phase of the YMC (Slavov et al, 2011). 74 Transcript levels for genes that cycle in the YMC will change as the cell moves through these 75 phases; at any time, some cells in an asynchronous population will contain a transcript and 76 some will not. These shifts through transcriptional states are robust but not invariable, as 77 YMC-regulated genes switch on and off in response to cues prompted by both regulated and 78 erratic changes in the intracellular and extracellular environment. 79 In addition to the partitioned gene expression patterns during the YMC, genomic 80 spatial arrangements also contribute to regulatory relationships between genes, albeit on a 81 smaller scale (Batada et al, 2007; Cohen et al, 2000; Lee & Sonnhammer, 2003). While genes 82 displaying functional relatedness, such as the GAL genes, are regulated similarly by 83 possessing the same cis-acting sequences (i.e. the UASGAL), co-expression of clustered genes 84 in S. cerevisiae is largely independent of similarly controlled transcription, gene orientation, 85 and/or shared regulatory sequences. However, members of adjacent gene pairs or clusters 86 are more likely to belong to the same functional pathway than expected by chance (Batada 87 et al, 2007; Cohen et al, 2000; Lee & Sonnhammer, 2003). The mechanism by which these 88 clustered genes are regulated remains largely elusive. 4 89 Here we show that overlapping transcription, in both the sense and antisense 90 orientations, constitutes an additional layer of regulation at clustered genes by managing 91 state-switching in response to environmental change. We use a simple carbon source shift 92 coupled with NET-seq to define genes whose transcription increases or decreases >3-fold, 93 after transfer from glucose- (GLU) to galactose- (GAL) containing media and show a 94 remarkable enrichment for genes whose transcripts cycle during the YMC. The majority of 95 these genes have no functional associations with transcription factors that might mediate 96 repression or induction in GLU or GAL, but are organised in clusters and subject to 97 overlapping sense and antisense transcription. We exemplify this mode of gene regulation, 98 state-switching by transcriptional interference and insulation, at the HMS2:BAT2 tandem 99 gene cluster. By engineering promoters and terminators at HMS2:BAT2, we demonstrate 100 the formation of alternative transcription units underlies state-switching. We suggest that 101 overlapping transcription and the formation of alternative transcription units, associated 102 with temporally segregated gene expression, will be a general feature of gene regulation. 103 104 Results 105 Genome-wide response to a change in carbon source reveals genes whose transcripts 106 cycle in the YMC 107 The question we address in this work is whether transcriptional interference can explain the 108 switching on and off of genes and thus altered gene expression in response to 109 environmental change. We used a change in carbon source by shifting exponentially 110 growing cells from glucose- (GLU) to galactose–containing media (GAL) for 3 hours and 111 analysed the native transcripts associated with elongating RNA polymerase II (NET-seq) 112 (Churchman & Weissman, 2011) to identify genes whose transcription is altered during this 5 113 environmental change (Supplementary File 1A). 10.45% (551) of ORF-Ts (open reading 114 frame – transcripts) showed a >3-fold increase and 9.99% (527) showed a >3-fold decrease 115 in transcription in GAL relative to GLU (Supplementary File 1B). By comparing the NET-seq 116 output with a microarray of Poly(A)+ RNA, we show that for the majority (≈88%) of genes, 117 the change in transcript levels on the GLU to GAL shift reflects altered transcription rather 118 than altered transcript stability (Supplementary File 1A). Gene ontology (GO) analysis 119 revealed highly significant associations to growth, quiescence and transcripts that cycle in 120 the YMC, particularly during the oxidative (OX) and the reductive charging (RC) phases of the 121 YMC (Fig. 1A; Supplementary File 1C,D). The genes whose transcription decreases in GAL are 122 enriched (467; 88.6% p<1x10-5) for OX phase genes that are mainly involved in ribosome 123 biosynthesis and growth (Fig. 1B). By contrast, for genes whose transcription increases in 124 GAL, there is significant enrichment (392; 71.1%, p<1x10-5) for genes whose expression 125 peaks in the reductive charging phase (RC) of the YMC (Fig. 1B). These genes are associated 126 with stress resistance, metabolism and quiescence. 33.1% (891) of the 2,691 annotated OX- 127 and RC-regulated genes also show >3-fold change on the GLU to GAL shift, supporting a 128 shared regulatory mechanism between the YMC and the GLU to GAL shift (Fig.