# Intergenic accumulation of RNA polymerase II maintains the potential for swift transcriptional restart upon release from quiescence

Manuela Baquero Pérez, Gertjan Laenen, Isabelle Loïodice, Mickaël Garnier, Ugo Szachnowski, Antonin Morillon, Myriam Ruault, Angela Taddei

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## ► To cite this version:

Manuela Baquero Pérez, Gertjan Laenen, Isabelle Loïodice, Mickaël Garnier, Ugo Szachnowski, et al. Intergenic accumulation of RNA polymerase II maintains the potential for swift transcriptional restart upon release from quiescence. Genome Research, 2025, 35 (10), pp.2226-2239. 10.1101/gr.279874.124. hal-05369356

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Submitted on 17 Nov 2025

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Intergenic accumulation of RNAPII maintains the potential for swift

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transcriptional re-start upon release from quiescence

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Baquero Pérez, Manuela¹

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Laenen, Gertjan¹**

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Loïodice, Isabelle¹**

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Mickaël Garnier¹

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Szachnowski, Ugo²

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Morillon, Antonin²

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Ruault, Myriam¹

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Taddei, Angela¹

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Affiliations

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¹ Institut Curie, Université PSL, Sorbonne University, CNRS, UMR3664 Nuclear

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Dynamics, Paris, France

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² Institut Curie, Université PSL, Sorbonne University, CNRS, UMR3244 DIG-Cancer,

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Paris, France

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** Equally contributing authors

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**Running title:** Transcription dynamics in and out quiescence

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**Key words:** Quiescence, return to growth, RNA Pol II, nuclear organization, Single-

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Cell RNA-Seq

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**Data accession numbers:** E-MTAB-14337 (ChIP-seq); E-MTAB-14980 (scRNA-seq);

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E-MTAB-14983 (RNAseq)

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27 ABSTRACT (224/250 words)

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29 Quiescent cells (Q) are seemingly inactive, developmentally arrested cells, whose
30 universal characteristic is the ability to promptly re-enter the cell cycle upon sensing
31 of external cues. Q cells are responsive to the environment and flexible enough to
32 adapt to available resources. In budding yeast, quiescent nuclear features are
33 drastically distinct from those observed in nutrient replete conditions: the nuclear
34 volume is reduced, the telomeres relocate from the nuclear periphery to the center of
35 the nucleus into a hypercluster, chromatin is found in a compacted, hypo-acetylated
36 state, and transcription is globally shutdown. Yet, Q cells can re-start transcription
37 within minutes of re-feeding. Here, we follow the global decrease of transcription in
38 sorted, developing Q populations, and its reactivation upon release. We find that
39 transcription and telomere clustering dynamics in and out of quiescence are
40 independent events. We report a genome-wide re-distribution of the transcription
41 machinery as cells progress into quiescence. Although most genes are shut down,
42 3% of coding genes remain active. Furthermore, RNAPII accumulates at one third of
43 gene promoters. The corresponding genes are highly enriched among those
44 showing a high level of transcription and high frequency of expression in individual
45 cells, shortly after cells are refed, as monitored by single-cell RNASeq. Our results
46 point toward a role for quiescent-specific RNAPII distribution to ensure a rapid and
47 robust transcriptional response upon return to growth.

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49 INTRODUCTION

50 Eukaryotic genome organization is dynamic, varying across cell types and conditions,
51 both functionally and spatially. These adaptations can be subtle, like individual gene
52 repositioning upon activation (Abruzzi et al. 2006; Brickner and Walter 2004; Casolari
53 et al. 2004; Taddei et al. 2006), or drastic, such as whole-genome reorganizations
54 during development or metabolic transitions (Guidi et al. 2015; Bourbousse et al. 2020;
55 Feodorova et al. 2020). In this study, we explore the transitions in and out of
56 quiescence in budding yeast, aiming to understand how the genome adapts to
57 environmental changes.

58 S. cerevisiae undergoes a number of metabolic transitions, favoring glucose
59 fermentation in rich medium and activating alternative metabolic pathways and stress-
60 response genes as glucose depletes. When glucose runs out, cells enter the diauxic
61 shift, transitioning to ethanol metabolism, with a growth arrest and a twofold decrease
62 in transcription (DeRisi 1997; Gasch and Werner-Washburne 2002; McKnight et al.
63 2015; Miles et al. 2013; Young et al. 2017). Cells will then progressively exhaust their
64 medium, reaching stationary phase within 3 to 7 days (Gray et al. 2004; Miles et al.
65 2021; Opalek et al. 2023; Werner-Washburne et al. 1996). From the early post-diauxic
66 shift phase, two populations of cells develop, with highly dense quiescent (Q) cells that
67 can be separated from lowly dense cells non quiescent (NQ) along a density gradient
68 (Allen et al. 2006). Q cells exhibit remarkable stress resistance and longevity,
69 compared to the rest of the population that is heterogeneous in terms of viability (Allen
70 et al. 2006; Aragon et al. 2008; Davidson et al. 2011). Transcriptome analysis shows
71 a 2- to 30-fold reduction in transcription in quiescent cells compared to cycling cells
72 (McKnight et al. 2015; Young et al. 2017). Yet, upon nutrient restoration, a robust and
73 swift transcriptional re-ignition is observed within minutes.
The RSC remodeling complex was proposed to play a crucial role in this rapid re-ignition of transcription upon nutrient addition (Cucinotta et al. 2021). However, the extent to which the transcription machinery is associated with the quiescent genome is still debated, with early studies indicating poised RNAPII upstream of genes (Radonjic et al. 2005) and recent studies reporting either a different distribution, towards the 3' end of coding sequences (Young et al. 2017), or no association of RNAPII to intergenic regions (Cucinotta et al. 2021; Young et al. 2017).

Changes in transcriptional activity are accompanied by a global change in chromatin and nuclear organization in Q cells. The quiescent genome is compacted (Schäfer et al. 2008; Swygert et al. 2019, 2021), and globally de-acetylated at promoters targeted by Rpd3 (McKnight et al. 2015). Telomeres in quiescent cells hypercluster in a SIR3-dependent manner (Guidi et al. 2015). Although it has been suggested that condensin-dependent compaction plays a role in transcriptional shutdown (Swygert et al. 2019), the interplay between hyperclustering and transcriptional activity has not been explored.

Here we aim to shed light on the state of transcription upon quiescence entry and return to growth, in relation to spatial constraints and rearrangements in the nucleus.

## RESULTS

### Transcriptional shutdown during quiescence entry occurs independently of telomere hypercluster formation

We sought to dissect the process of transcriptional shutdown as cells enter quiescence and understand its functional relationship with telomere hypercluster formation. We mapped the genome-wide transcription machinery occupancy by quantitative chromatin-immunoprecipitation of the Rpb3 subunit fused to a FLAG tag (methods).
In parallel, using the same strain, we followed the subnuclear distribution of the telomere-bound protein Rap1 fused to GFP. Cells from post-diauxic shift cultures onwards were purified using a density-based gradient, as described in (Allen et al. 2006), to mainly retrieve the developing Q population. We followed entry into quiescence in two nutrient depletion conditions (Fig. 1A). (i) We grew cells in glucose-rich medium and examined samples at different stages as the population grows into stationary phase (SP): cells growing exponentially by fermenting glucose (Log), undergoing diauxic shift (DS), and purified highly dense (HD) cells from post-diauxic cultures at 1 (day 1-HD), 2 (day 2-HD), and 6 days-HD (Q) after inoculation. (ii) Alternatively, we induced quiescence by abrupt starvation of post-diauxic shift cells for 24 hours (Q-like).

As previously described (Guidi et al. 2015), clustering of telomeres changes as cells transition from exponential growth by fermenting glucose (Log), through slowed-down growth in the post-diauxic shift phase (PDS, day 1 HD). The proportion of nuclei with more than 3 clusters is reduced, and foci become brighter. Hyperclusters (Guidi et al. 2015), defined here as clusters that are 4 times brighter than the median of clusters in Log phase, increase in proportion from day 2 onwards (Fig 1B).

Total RNAPII levels show little variation upon growth into stationary phase and quiescence entry (Jona et al. 2000; Radonjic et al. 2005; Young et al. 2017). In contrast, genome-wide counts of spike-in calibrated RNAPII ChIP-seq show varying levels of the transcription machinery associated with the genome in these kinetics (Fig. 1B, bottom). First, RNAPII genome association decreases 2 and 1.4-fold respectively in diauxic-shift (DS) and early respiration (day 1 HD) samples relative to exponential phase (Log). All samples after 2 days of growth show more than a 3-fold decrease relative to Log. We previously showed that both Q (6 days of nutrient exhaustion) and
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Q-like (abrupt starvation) cells display telomere hyperclusters (Guidi et al. 2015). Here we show that these two stages are associated with very low Rpb3-FLAG occupancy genome-wide. Furthermore, while decrease of RNAPII occupancy and increase of telomere hypercluster follow a similar trend over the kinetics of quiescence entry, the former precedes the latter, as minimal RNAPII occupancy (6.2-fold decrease relative to Log) is observed after 2 days of growth and telomere hyperclustering keeps increasing up to 6 days.

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To test whether hypercluster formation is required for transcriptional shutdown, we examined RNAPII binding in a *sir3Δ* mutant, defective for hypercluster formation, but able to form highly dense, small cells, morphologically similar to WT (Guidi et al. 2015) and Fig. 1C). To avoid pseudo-diploidy effects owing from the derepression of cryptic mating type loci in *sir3Δ* strains (Galitski et al. 1999), we worked in an *hmlΔ* background. As expected, RNAPII binding is higher at subtelomeric loci in *sir3Δ* cycling cells (Log) (Supplemental Fig. S1A). Yet, Rpb3-FLAG ChIP-seq samples in *sir3Δ* background highly correlate with WT (Fig. 1C; Supplemental Fig. S1B) in Log and Q samples, including at 297 genes with significant Rpb3-FLAG binding in quiescent chromatin (Supplemental Fig. S1C), demonstrating that shutdown of transcription does not depend on telomere hyperclustering. Therefore, during quiescence entry, transcriptional shutdown precedes and occurs independently of telomere hyperclustering.

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**Dismantling of telomere hypercluster follows but is independent of transcriptional activation upon return to growth.**

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We next asked when telomere hyperclusters dismantle upon return to growth and whether it depends on transcriptional activation that occurs within 3-10 minutes after refeeding (Cucinotta et al. 2021; Martinez et al. 2004; Radonjic et al. 2005). We

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followed Rap1-GFP in released quiescent cells and found hyperclusters disassembling within 15-30 minutes, one hour before S-phase starts as monitored by bud emergence (Fig 2A). De-clustering is particularly apparent in the decrease in brightness of clusters, which reach intensities comparable to those found in cycling cells after 30 minutes of release in glucose (Fig 2B; Supplemental Fig S2A). Thus, hypercluster dismantling occurs swiftly but it only comes after re-ignition of transcription.

To test whether transcriptional activation could be the cause of telomere hypercluster dismantling, we followed telomere clusters after releasing the thermosensitive *rbp1-1* strain at restrictive temperature. RNA-seq confirmed a severe defect of transcriptional activation (Supplementary Fig. S2B) and a close to wild-type kinetic of declustering in these conditions. Similar results were observed in cells pre-treated with the RNAPII inhibitor phenanthroline (Supplemental Fig. S2C). In contrast, quiescent cells treated with thiolutin did not dismantle their telomere hyperclusters, confirming the complex effect of this drug (Eshleman et al. 2020; Qiu et al. 2024; Lauinger et al. 2017). We thus conclude that hypercluster dismantling upon quiescence exit is largely independent of RNAPII activity.

**Scant transcription of poorly characterized genes remains in the quiescent population**

We examined the genome-wide distribution of RNAPII through our spike-in calibrated Rpb3-FLAG mappings, which shows no bias in distribution along the chromosome arms, except for a two-fold increase of counts of Rpb3-FLAG at the rDNA locus observed both in WT and *sir3Δ* cells (Supplemental Fig. S3A,B) that is accompanied by a decrease in RNAPI occupancy (Supplemental Fig. S3C), consistent with a smaller nucleolus and reduced protein synthesis (Guidi et al. 2015; Fuge et al. 1994; Laporte
174 et al. 2013). These observations suggest a competitive binding of RNAPII and RNAPI
175 as was previously proposed (Cioci et al. 2003).

176 As cells progress into quiescence, Rpb3-FLAG signal is depleted over genes and
177 intergenes alike (Supplemental Fig. S3D). To address the extent of shutdown, we
178 examined RNAPII annotations. Since non-coding annotations do not particularly show
179 a quiescence-specific increase in Rpb3-FLAG binding (Supplemental Fig. S3E), we
180 focused our analyses on the transcription of protein-coding genes. Importantly, Rpb3-
181 FLAG presence over gene bodies correlates very well with previous mappings of
182 untagged Rpb3 (pearson and spearman R=0.8) and of the Ser2 phosphorylation of
183 the C-terminal domain (CTD) of Rpb1 (pearson R= 0.87, spearman R=0.85) in
184 quiescence (Swygert et al. 2019), associated with its elongating form (Fig. 3A). Based
185 on the presence or absence of Rpb3-FLAG and newly synthesized transcripts (4tU-
186 seq) (Cucinotta et al. 2021) (methods) we define 4 classes of genes.

187 Amongst the 297 genes that are bound by RNAPII in Q, more than half show newly
188 synthesized transcripts in Q cells (class I, 161 genes), while the rest, which are shorter
189 (Supplemental Fig. S3F), do not (class II, 136 genes). Of note sir3Δ strain shows
190 similar RNAPII binding profile at these genes (Supplemental Fig. S1C).

191 One-third of all protein coding genes show detectable transcripts but Rpb3-FLAG
192 counts below the threshold (class III, 1916), while about half do not meet the threshold
193 for Rpb3-FLAG or 4tU-seq counts (class IV, 3602). Class III genes show on average
194 less recently synthesized transcripts than class I and more RNAPII than class IV
195 (Supplemental Fig. S3F), thus showing consistency between 4tU and RNAPII
196 detection. The presence of 4tU signal at these genes with no detectable RNAPII in
197 their gene body could reflect the difference in sensitivity between ChIP and 4tU
198 detection.
Class I and II show comparable levels of Rpb3-FLAG binding but differ in 4tU-seq counts. Class II genes being particularly small (Supplemental Fig. S3F), size bias and/or reduced stability of transcripts might account for less efficient capture of 4tU-seq reads assigned to these genes. In addition, differences between 4tU incorporation and RNAPII binding could stem from small variations between growth conditions. While both class I and class II are enriched in uncharacterized ORFs, class II genes are also enriched in dubious ORFs (Supplemental Fig. S3G). Thus, class II genes show overall less evidence for bona fide transcription in quiescent cells than class I genes. Curiously, 55/136 class II genes do not show RNAPII binding in fermentation or respiration. In contrast, almost all class I genes were bound by RNAPII during growth: most during all growth phases (112), some (49) specifically after the diauxic shift (Supplemental Fig. S3H). Only few class I genes are annotated as verified ORFs (62 genes) and show an enrichment for catabolic processes and carboxylic acid transport (Supplemental Fig. S3G). Such little functional enrichment is consistent with class I genes being significantly enriched in uncharacterized ORFs (Supplemental Fig. S3G), in agreement with the scant characterization of quiescence. Only two verified ORFs, with little functional characterization, RTS2 and BSC4, are bound exclusively in Q cells (Supplemental Fig. S3H).

## Single cell RNA-seq confirms active transcription in Q cells.

Density gradients allow to enrich for the highly-dense population, also referred to as quiescent (Q)(Allen et al. 2006). To rule out that class I transcripts resulted from transcription in leftover low-density cells, or a subpopulation of Q cells, we examined the transcriptome of the population at the single-cell level, after adapting the 10× Genomics SC3' encapsulation protocol to quiescent cells ((Jariani et al. 2020; Vermeersch et al. 2022), methods). The correlation between pseudo-bulk scRNA-seq
and 4tU-seq counts (Pearson R=0.6, Spearman's R=0.64), indicates that single cell
RNA seq in quiescent cells detects mainly recently synthesized transcripts. This is in
good agreement with the reported difficulty to extract stored mRNAs in quiescent cells
(Aragon et al. 2006). UMAP dimensionality reduction of single-cell RNA-seq data
revealed that our gradient-sorted population at 6 days of medium exhaustion show a
bilobate structure (Fig. 3B). Nevertheless, class I transcripts are well-represented
throughout both subpopulations (Fig. 3B), independently of read counts
(Supplemental Fig. S3I). Indeed, these 161 genes make up one-third (35.6 %) of the
transcriptome of the average cell (Supplemental Fig. S3J). Our single-cell analysis
thus reveals a widespread transcription of 3% of protein-coding genes for the Q
population.

Of note, seven class I genes are detected in more than 80% of the cells and collectively
make up 18.8% of the average transcriptome, amongst which is GRE1, expressed in
over 99% of the 6d HD (Q) population (Fig 3C). Some of these are previously identified
stress-response genes (SPG1, and two orthologs, GRE1 and SIP18 (Dang and
Hincha 2011; Martinez et al. 2004; Miralles and Serrano 1995)) and four with partial
(TMA10, DDR2 (Fleischer et al. 2006; Kobayashi et al. 1996)), or no characterization
(YKL065W-A, YER084W). Individual deletion of these 7 genes did not affect growth
into quiescence, return to the cell cycle, telomere hypercluster formation and
dismantling, or expression of the Hxt1 hexose transporter upon return to growth
(Supplemental Fig. S3K). We hypothesize that these genes exert redundant functions
or are important to resist specific stress conditions. These genes either have less
RNAPII binding in abruptly starved cells, or they show a 5'-biased profile, as seen for
GRE1 (Fig. 3C). Thus, despite showing a genome-wide decrease in transcription, and
similar nuclear morphology, the remaining transcription in quiescence can distinguish
different methods of induction, as previously reported (Klosinska et al. 2011). Taken together, these results provide strong evidence for active transcription that is homogeneously retained in the highly dense population at 6 days for a set of 161 genes, with only a handful being widely and relatively strongly transcribed.

**RNAPII re-distributes to a subset of promoter-bearing intergenes when cells enter quiescence**

We noticed that entry into quiescence is accompanied by a redistribution of RNAPII away from gene bodies (Fig. 3A, C, gray boxes). When first induced at respiration or fermentation, class I genes show a canonical profile, with a TSS-proximal accumulation followed by increasing RNAPII enrichment towards the 3' end of coding sequences (Bataille et al. 2012; Mayer et al. 2010; Tietjen et al. 2010). But, as the culture approaches SP (day 2-HD) and increasingly as cells reach either quiescent state (Q or Q-like), Rpb3-FLAG is partially retained within the gene body while accumulating outside the ORF specifically in Q cells (Fig. 3A, C, gray boxes and below).

To ask whether RNAPII was enriched in Q in all intergenes alike, we sorted them based on the orientation of their surrounding annotations (convergent, divergent, tandem) and examined their enrichment in RNAPII (Fig. 4A). As previously reported (Bataille et al. 2012; Mayer et al. 2010; Ocampo et al. 2019; Tietjen et al. 2010), during exponential growth (Log), RNAPII is highly enriched at convergent intergenes, and near the transcription start sites in tandem and divergent intergenes.

In sharp contrast, in Q samples, RNAPII is mostly enriched within the boundaries of those intergenes containing a promoter: tandem and divergent, the latter one being particularly striking, as these show virtually no enrichment in growing cells. Peak
273 calling analysis, using the IP on untagged samples (FLAG on Rpb3 WT) as controls
274 (methods), corroborates the significance of the observed enrichment (Supplemental
275 Fig. S4A). Input and untagged IP (anti-FLAG on Rpb3 WT) chromatin coverage tracks
276 show no enrichment or depletion at these locations, ruling out a potential artefact
277 (Supplemental Fig. S4B). This was further confirmed by ChIP q-PCR (see below, Fig.
278 4B). Thus, RNAPII is depleted from convergent intergenes, while accumulating at
279 1773 promoter-bearing intergenes, upstream of 2478 genes in quiescent chromatin
280 (Supplemental Fig. S4A, methods).

281 Promoter-bound RNAPII bears signs of canonical transcription

282 All epitopes of the RNAPII complex that we targeted using ChIP-qPCR follow the same
283 trend of enrichment at promoters (Fig. 4B; Supplemental Fig. S4C), thereby indicating
284 presence of the whole catalytic RNAPII. This was not the case for the FLAG-tagged
285 Rpa135 subunit of RNAPI. As our ChIP-seq was performed against FLAG-tagged
286 Rpb3 this allows to rule out a potential artefact owing from the *hyper-chippability* of the
287 FLAG epitope at these loci (Teytelman et al. 2013). Furthermore, we detect the Rpb1
288 CTD phosphorylations (Ser2, 3E8; Ser5, 3E10), associated with elongation and
289 initiation (Komarnitsky et al. 2000) in cycling cells, suggesting that RNAPII may be
290 active at these sites specifically in quiescence. Indeed, Ser5P is enriched at the
291 *DCP2/MLS1* intergenic region where no Ser5P enrichment could be detected during
292 log phase, though Rpb3 was clearly detected over the *DCP2* gene body. Ser5P was
293 also found enriched in the promoter of the *ADY2* class I gene (Supplemental Fig. S4C)
294 with levels three to six times higher than in the *DCP2/MLS1* intergene, possibly linked
295 to the stronger activity of *ADY2* over *DCP2* or *MLS1*.

296 We next compared Rpb3-FLAG ChIP signal at the summits of peaks within promoter-
297 containing intergenic regions (1442 upstream of 1940 genes, methods) with those of
298 other factors previously mapped in quiescent chromatin (Cucinotta et al. 2021;
299 Swygert et al. 2019; Guidi et al. 2015). As expected, the silencing factor Sir3 does not
300 co-localize with Rpb3-FLAG (Fig. 4C), while ChIP-seq signal obtained with a native
301 Rpb3 antibody does (Swygert et al., 2019). In contrast, Sth1 (RSC) showed low
302 enrichment at these sites, while Rpb1-Ser2P, Brn1, Msn2, and especially TFIIB (Sua7)
303 showed higher correlation with Rpb3-FLAG peaks. Notably, TFIIB binding suggests
304 the presence of the pre-initiation complex (PIC) and closely follows RNAPII enrichment
305 in quiescent chromatin. This pattern differs in cycling (Jeronimo and Robert 2014) (Fig.
306 4C, gray box) and refed quiescent cells (Cucinotta 2021) (Supplemental Fig. S4C),
307 where TFIIB localizes near to but not at Rpb3-FLAG peaks.

308 The detection of Rpb1-Ser5P and TFIIB subunit Sua7 at intergenic Rpb3 peaks is
309 indicative of active transcription. However, recently synthesized transcripts (4tU-seq,
310 (Cucinotta et al. 2021)) are rarely found directly under the strongest summits (42/513).
311 The absence of 4tU signal could stem from the lack of sensitivity to detect short or
312 unstable transcripts. Yet, amongst all 1940 promoters showing Rpb3-FLAG peak
313 summits in quiescence, half (945) are found upstream of genes with detectable levels
314 of newly synthesized transcripts (class I, p-value 2.23×10⁻³³, and class III genes, p-
315 value 1.23×10⁻⁴³, Fig 4B, 4D).

316 In summary, RNAPII is found associated with PIC components and bearing marks
317 indicative of activity at non-canonical sites, in the quiescent genome.

318 **RNAPII accumulates at upstream activating sequences (UAS) during**
319 **quiescence entry**

320 Summits of strong peaks fall more often than expected within promoters bearing a
321 fuzzy and or fragile nucleosome positioning (hypergeometric test, p-value= 2.1×10⁻³⁶,
322 (Tirosh and Barkai 2008; Kubik et al. 2015)). Amongst the different classes of TF
bound promoters defined by Rossi et al, 2021, strong peaks are enriched at those endowed with SAGA, Mediator or TUP cofactors (STM) (hypergeometric test, p-value=3.7×10⁻¹⁹⁰), and at Ribosomal Protein (RP) genes (hypergeometric test, p-value=6.2×10⁻²⁰). This was not the case at TFO promoters (TF organization lacking STM cofactors, and bound by insulators, p-value=0.19). RNAPII is thus enriched at inducible promoters in quiescent cells.

The binding of RNAPII skews towards the 3' end of all three classes (RP, STM and TFO) after cells undergo Diauxic Shift, reminiscent of the pattern reported for stationary phase cells by the Osley lab (Young et al. 2017). This is followed by a virtual depletion over the gene body that is accompanied by a progressive accumulation at the promoter of STM and RP genes but not at TFO genes from late post-diauxie to quiescence (Fig. 5A).

The strongest RNAPII enrichment lies at 250 bp from TSS in quiescence, where upstream activating sequences (UAS) are often found (Fig. 5B, Supplemental Fig. S5 A). Indeed, about 25% (125/513) of summits of strong peaks falling in promoters coincide with annotated UAS. Similarly, 111 binding motifs, encompassing a variety of functions (methods), are found to be enriched in the vicinity (100 bp) of the strongest RNAPII summits.

Thus, RNAPII quiescent enrichment does not seem to result from a previously known program driven by a small set of regulators. Those motifs with a fold-enrichment over control greater than 3 are shown in Fig. 5C. Some sites are well-positioned near but not immediately under the RNAPII summit in quiescence (e.g., Rap1, Pdr3, Fig. S5C), and correspond to known regulators of genes highly expressed during growth (Rap1) or to factors involved in chromatin modifications associated with the shutdown of transcription upon quiescence entry (e.g. Snt2, recruiting factor for Rpd3, (Baker et al.
348 2013; McKnight et al. 2015)) or the remodeling of chromatin/maintenance of the NFR
349 (e.g. Rsc3, Rsc30, (Badis et al. 2008)).

350 Some of the TFBS we found are puzzling, like those recognized by proteins of
351 uncharacterized function (YPR196W, YKL222C, YER184C, YNR063W and
352 YER130C), but also that of Gal4, as cells were presumably not exposed to galactose.

353 In total, 220 genes with a significantly enriched RNAPII peak show virtually no sign of
354 transcription during quiescence and quiescence entry. These include GAL genes
355 (Supplemental Fig. S5A).

356 We next used *k*-means clustering to study the dynamics of RNAPII around all summits
357 in promoter-bearing intergenes during quiescence entry (methods). The first group
358 comprises 437 summits in Q-cells (Fig. 5D; supplemental Fig. S5B) where polymerase
359 is enriched throughout the kinetics, starting from exponentially growing cells. The
360 second group (601 summits) show weaker levels of RNAPII at the summit in log and
361 early respiration phase (day 1). Note that one such Rpb3-FLAG summit is found at the
362 intergene upstream of ADY2, together with the initiation Ser5-P modification
363 (Supplemental Fig. S4C). The third group (404 summits) is depleted of signal in all
364 growing phases (Log through day 1), and only starts increasing after day 2, as
365 illustrated with GAL7 (Fig. 5D; Supplemental Fig. S5C). Binding motifs highly enriched
366 under summits of the third group of peaks include binding factors known to be active
367 upon glucose de-repression (e.g. Adr1, Mig1,2,3 (Carlson 1999; Schüller 2003)), but
368 also include a variety of factors involved in various responses, including, again, Gal4
369 (Fig. 5C; Supplemental Fig. S5C). Abruptly starved cells either show a lower
370 enrichment of RNAPII at these regions (e.g. at DDR2 promoter, Fig. 5C, Supplemental
371 Fig. S5B), or none (e.g. at GAL7 promoter, Fig. 5D; Supplemental Fig. S5A-B).
372 In summary, in contrast to genic binding of RNAPII in Q, that was already present as
373 cells were growing, a third of the intergenic enrichment found in starved cells is Q-
374 specific, increasing only after cells have been in culture for more than two days.

375 **Upstream RNAPII predicts strength of transcriptional re-start upon release**

376 We next asked whether promoter-bound RNAPII in quiescent chromatin would relate
377 to the swift re-start of transcription observed upon return to growth. We performed k-
378 means clustering to categorize 5778 genes based on the dynamics of newly
379 synthesized transcripts abundance 10 minutes after nutrient restoration (Cucinotta et
380 al. 2021) (Fig. 6A), thus identifying four groups: the first one comprises class I and
381 class III genes with transcripts in Q that show a decrease in relative labels of 4tU
382 incorporation upon release (Ø, 101 genes Fig. 6A; Supplemental Fig. S6A).

383 The other three groups (RI, 287 genes; RII, 2030 genes; RIII 3218 genes) show
384 decreasing levels of release probability (Supplemental Fig. S6B). The genes that more
385 robustly re-start (categories RI and RII) show the highest enrichment for genes with
386 an upstream RNAPII summit, amongst which are found most ribosomal protein (RP)
387 genes (Fig. 6A). Conversely, genes with a stronger upstream enrichment (with a
388 RNAPII peak overlapping with their promoter) show higher 4tU counts upon release
389 compared to those with a milder one or no detectable enrichment (Supplemental Fig.
390 S6C).

391 Although 738 genes with upstream RNAPII summit show a milder upregulation 10
392 minutes after release with glucose, it is stronger than those without an RNAPII summit
393 (cluster RIII, Fig. 6A; Supplemental Fig. S6D). These are mostly enriched in functions
394 related to the metabolism of galactose and various alcohols, to which cells are not
395 exposed in these feeding conditions (Supplemental Fig. S6E). In short, while not
396 predicting the transcriptional program upon release, promoter-bound RNAPII predicts
397 the strength of potential re-ignition of downstream genes.

398 To assess the heterogeneity of transcriptional activation across the cells and across
399 release conditions, we performed single cell transcriptomes 30 min after diluting cells
400 either in rich glucose (2%) or lactate (0.5 %) and ethanol (2%) medium. For both
401 conditions, we found that genes with an RNAPII summit in their promoter in Q cells
402 are highly enriched amongst the 10% most frequently expressed genes (Fig. 6B;
403 Supplemental Fig. S6F) corresponding to genes detected in 32-98% of cells in glucose
404 and 22-97% of cells in lactate-ethanol (Supplemental Fig. S6G). This suggests that
405 promoter-bound RNAPII marks genes that can be pervasively transcribed in the
406 population when quiescent cells are refed.

407 Pervasively transcribed genes with an RNAPII peak in their promoter highly overlap
408 (Fig 6B) between the two conditions, suggesting that, regardless of the available
409 sugar, cells engage in a common program in the early stages of quiescence release.

410 These common genes are enriched in protein synthesis functions (Supplemental Fig
411 S6H), including Ribi and RP genes. However, 24% of the promoter-bound RNAPII
412 showing pervasive transcription upon release in lactate ethanol are not pervasively
413 expressed in glucose. These are enriched for functions related to respiration
414 (Supplemental Fig. S6H) and include the gluconeogenesis gene PYC1. Therefore,
415 RNAPII peaks in promoter regions marks genes that can be mobilized differently to
416 adapt to distinct environments.

417

418
419 DISCUSSION

420 We investigated the relationship between telomere clustering and transcriptional
421 dynamics during quiescence entry and return to growth. We show that transcriptional
422 shut-down is partial and precedes telomere hyperclustering during quiescence entry.

423 Similarly, transcriptional upregulation occurs before the dismantling of telomere
424 hyperclustering upon return to growth. Yet we show that these two dynamics are
425 independent. This is reminiscent of rod cell nuclei of nocturnal mammals, that show a
426 similar internalization of heterochromatin that is not accompanied by notable effects
427 on genome expression (Solovei et al. 2009).

428 We also report a distinct, transcriptionally idle quiescent chromatin landscape, holding
429 at promoters the potential for prompt restart in the form of RNAPII accumulation.

430 Quiescent chromatin is not transcriptionally inert

431 We report a two-fold increase in RNAPII binding at the rDNA, together with a three-
432 fold decrease in the rest of the genome. Our calibrated ChIP-seq study, integrated with
433 metabolically labeled transcriptomic data (Cucinotta et al. 2021), provides strong
434 evidence for active transcription of 3% of protein-coding genes (class I), thus showing
435 that quiescent cells are not transcriptionally inert. The sensitivity of 4tU-labeled
436 transcript detection and RNAPII ChIP might cause an underestimation, and the actual
437 figure could be up to 35,7% if considering only 4tU-signal.

438 RNAPII re-distributes to promoter-bearing intergenes over several days

439 Although transcriptional shut down is observed after 2 days of culture, RNAPII binding
440 continues to evolve at least until day 6, with different gene body association of RNAPII
441 and its progressive accumulation at UAS of RP genes and inducible STM genes.
442 Consistently, we observe a stronger intergenic RNAPII accumulation in quiescent cells
resulting from medium exhaustion than in quiescent cells resulting from post-diauxic shift starvation.

Previous assessments of polymerase binding to quiescent chromatin were conflicting, with recent studies reporting a lack of enrichment at intergenes. Unlike Radonjic et al., who detected intergenic enrichment in unsorted stationary phase populations, Young et al. found a bias towards the 3' of CDS. This distribution correlates best with our early post-diauxic shift samples, suggesting a difference in the kinetics of quiescence entry, likely due to differences in growth conditions such as aeration and medium pH (Greenlaw et al. 2024; Opalek et al. 2023). Data from the Tsukiyama laboratory, however, agree with our findings (Swygert et al. 2019 and Supplemental Fig. S4C).

Some of the reported discrepancies (Cucinotta et al. 2021) could be attributed to differential efficiency in immunoprecipitation, as tagged epitopes can lead to better enrichments, as previously reported (Hu et al. 2015; Jeronimo and Robert 2014).

Finally, we observed the enrichment of the condensin subunit Brn1 (Swygert et al. 2019) at RNAPII Q peaks, in good agreement with published data showing the accumulation of condensin at RNAPII bound sites in other conditions (D'Ambrosio et al. 2008).

## Bookmarking genes in quiescent yeast

Evidence for RNAPII pausing in budding yeast is limited, leading to the general belief that it does not occur. Although the Negative Elongation Factor (NELF) — absent in budding yeast — is linked to stable transcriptional pausing, it is not essential as shown in *S. pombe* (Booth et al. 2018; Core and Adelman 2019). To our knowledge, few reports of promoter-proximal seemingly inactive RNAPII, with the potential to reactivate, exist in budding yeast. RNAPII remains at the *INO1* promoter for hours post-repression, enhancing reactivation upon reinduction and indicating epigenetic
468 memory (DG Brickner 2007, Brickner & Walter 2004, Light et al., 2010). Such
469 bookmarking may explain RNAPII presence upstream of genes transcribed during
470 quiescence entry.

471 However, 220/1910 genes with an upstream RNAPII peak are not transcribed during
472 quiescence entry, including GAL genes. This is reminiscent of RNAPII found together
473 with PIC components, at promoters made accessible by the absence of Hda1 and
474 Rpd3 deacetylases, in a state favorable for activation under Snf1 control (Tachibana
475 et al. 2007, 2005).

476 Such an activation-prone state is consistent with our observation of RNAPII mostly
477 upstream of genes seemingly ready to be highly transcribed within minutes of
478 refeeding in at least two different conditions.

479 Using ATP analog-sensitive mutants (kin28-as) (Bataille et al. 2012; Jeronimo and
480 Robert 2014; Tietjen et al. 2010) or anchor-away depletion (Wong et al. 2014) to inhibit
481 Kin28 results in RNAPII accumulation at core promoters. RNAPII accumulation in
482 quiescence, however, tends to occur 250 bp upstream of the TSS, further upstream
483 than that typically observed in cycling cells (Bataille et al. 2012; Jeronimo and Robert
484 2014; Kubik et al. 2019; Mayer et al. 2010; Pelechano et al. 2014; Steinmetz et al.
485 2006), and rather coinciding with reported locations for Mediator at UAS (Andrau et al.
486 2006; Ansari et al. 2009; Fan et al. 2006; Jeronimo and Robert 2014). Possibly related
487 to our finding, Mediator subunits also accumulate at UAS of RP genes upon heat-
488 shock (Sarkar et al. 2022).

489 Although our crosslinked chromatin mapping cannot exclude some enzyme proximity
490 rather than DNA binding, recent studies using improved detection methods for
491 engaged polymerase and small transcripts have mapped RNAPII at similar distances
492 from the canonical PIC assembly (Xi et al. 2024), suggesting RNAPII recruitment may
occur at UAS in cycling cells. Consistently, RNAPII intergenic signal can be detected at two thirds of the quiescent peaks in growing cells, although not forming well-defined peaks. Furthermore, we detect some Ser5-P CTD phosphorylation at some of these intergenic peaks in Q cells, suggesting that at least some RNAPII might be active at these loci.

These observations are in agreement with *in vitro* studies showing transient RNA pol II interaction at activator-bound UAS before loading into the core promoter (Baek et al. 2021). Although RNAPII binding is not detected at UAS by ChIP in exponentially growing cells it is clearly detected by ChEC-Seq2 at active STM genes (VanBelzen et al. 2024). The weak general levels of transcription in quiescent cells may uncover binding sites otherwise difficult to detect by ChIP in growing cells.

RSC was proposed to enable newly recruited RNAPII to progress into gene bodies upon release (Cucinotta et al. 2021). Furthermore, re-entry into cell cycle is delayed (Cucinotta et al. 2021) in the absence of the general transcription elongation factor TFIIS (Dst1) (Kettenberger et al. 2003; Kireeva and Kashlev 2009; Wery et al. 2004).

Our results indicate that these factors favor transcription from RNAPII accumulated upstream of genes during quiescence.

We hypothesize that the odd RNAPII ChIP profile at these genes stems from altered dynamics of the transcription cycle, wherein RNAPII would bind UAS, only rarely engaging in transcription along the gene body. This idea is well supported with the recently published kinetic model of RNAPII transcription (VanBelzen et al. 2024).

Such idle, minimal, activity would occur stochastically in the population, contributing to maintain a responsive quiescent chromatin, that can be mobilized differently to adapt to distinct environments.
518 METHODS

519 Strains and growth conditions

520 All S. cerevisiae strains used in this study, haploid (mating type a) and isogenic to the
521 W303 background, are listed in Supplemental Table S1. Gene targeting for gene
deletion and gene tagging was performed as described in (Longtine et al. 1998). Cells
522 were inoculated in YPD (yeast-peptone-dextrose 2%) and grown overnight before
523 diluting to 0.1 OD600/mL to start the kinetics of entry into quiescence. Cells were grown
524 at 30°C (except rpb1-1 strains that were grown at 25°C), shaking at 250 rpm, at 1/10
525 volume in glass flasks with aeration caps. Diauxic shift samples were harvested two
526 hours after glucose was no longer detectable in the medium, when transcriptional
527 changes are detectable (Brauer et al. 2008). To induce quiescence by nutrient
528 exhaustion (Q 6d-HD), cells were grown to reach stationary phase (50-60 OD600/mL)
529 at 30 °C, then gradient-enriched in HD cells (see below). To induce quiescence by
530 abrupt starvation (Q-like), cells were grown in YPD as described above for 24h, then
531 gradient-sorted (see below), and the recovered HD fraction was resuspended in sterile
532 H2O, then incubated at 30 °C, shaking, for another 24 hours. The S. pombe strain
533 used as spike-in for ChIP-seq was grown in YES medium (yeast extract
534 supplemented) to mid-log phase, in parallel to mid-log phase S. cerevisiae samples
535 (0.4-0.7 OD600/mL). For release from quiescence experiments, HD cells were enriched
536 from a 6 days stationary phase culture, washed in H2O and incubated overnight at
537 30°C, under shaking. Then, they were re-suspended at 1 OD/mL in fresh YPD at 1:10
538 volume:air ratio for the indicated times. For single cell experiments, cells were
539 released in either YPD (2% glucose) or YPLE (0.5%Lactate, 2% Ethanol). To recover
540 mainly the highly dense population as cells progress into stationary phase in the
541 nutrient exhaustion kinetics, all samples after diauxic shift (24h, 48h, Q-like, Q6d) were
542
gradient sorted, based on the principle described in (Allen et al. 2006). A detailed
procedure is described in Supplemental Materials and Methods

545 Transcription inhibition experiments

546 Transcription inhibition experiments were carried out on stationary phase culture (7 d)
547 or HD-enriched (quiescent) culture, as indicated in the corresponding figure. *rpb1-1*
548 cells were grown to stationary phase at 25°C. Cells were pretreated for 2 hours with
549 the inhibitor (thiolutin SIGMA, final concentration at 10µg/mL, or phenanthroline
550 100µg/ml final, stock solutions in DMSO) or preincubated for 1 hour at 37°C for the
551 *rpb1-1* strain. For each condition, 6×10⁷ cells (1 OD/mL) were released in fresh YPD
552 only, or in the presence of the corresponding inhibitor or temperature. Then, cultures
553 were sampled after 30, 60 and 120 minutes of incubation, shaking, at 25°C, 30°C or
554 37°C as indicated in the figure legend. For wash-out, all samples, including controls,
555 were washed 3 times in PBS1× and re-suspended in fresh YPD only, then imaged
556 after 30, 90, 120 and 240 minutes.

557 Imaging and Rap1-GFP foci quantification

558 Samples were harvested at the indicated timepoints, rinsed in H₂O and placed on a
559 4% agarose patch before imaging. Image acquisition was performed as described in
560 Guidi et al. 2015, with modifications described in Supplemental Materials and
561 Methods. Image analysis relied on our custom-made software
562 (https://github.com/mgPICT/NuFoQ), which was adapted from Guidi et al. 2015 to
563

enable three-dimensional (3D) segmentation and quantification of nuclear foci. Further
details can be found in the Supplemental Materials and Methods section.

564

565

Sample harvesting for chromatin immunoprecipitation

566

For every timepoint in the kinetics of quiescence entry, 100 OD₆₀₀ of cells were
harvested for chromatin immunoprecipitation. An aliquot from the same sample was
washed in H₂O and placed on a 4% agarose patch before imaging. For each of the
two biological replicates, WT Rpb3 (yAT1684) and FLAG-tagged (yAT3239, yAT3555)
strains were grown with the same batch of YPD and processed together for chromatin
immunoprecipitation and imaging. Chromatin immunoprecipitation experiments,
described in detail in Supplemental Materials and Methods, were performed as
described in (Guidi et al. 2015; Ruault et al. 2021). qPCR primers are listed in
Supplemental Table S2.

567

568

569

570

571

572

573

574

575

ChIP-seq data processing

576

Rpb3-FLAG ChIP-seq libraries were sequenced (single-end 50 (HiSeq-Rapid Run)) at the Institut Curie NGS platform. WT samples (yAT1684 and yAT3239) were sequenced together. WT IP samples for Log and Q (6dHD) were re-sequenced together with sir3Δ samples in a different run. Thus, they were treated separately for analysis. For mapping, reads were competitively aligned to a hybrid S. pombe (ASM294v2.28) and S. cerevisiae genome (SGD Project 2015), with Bowtie 2 (bowtie2-align-s -p 8, (Langmead and Salzberg 2012)). To visualize sub-telomeric genes, filtering was performed to allow up to three mismatches (grep -E "(^@|XM:i:[0-3])").

577

578

579

580

581

582

583

584

To calibrate the ChIP signal, an occupancy ratio (OR) was calculated based on the
number of uniquely mapped reads to either genome (experimental: S.cerevisiae,
spike-in: S. pombe), present in IP and INPUT samples of the Rpb3-FLAG tagged

585

586
587 (yAT3239) and Rpb3 untagged (yAT1684) strains, as described in (Hu et al. 2015).

588 OR was used as a scaling factor to generate coverage tracks from IP samples (shown

589 in all genome browser snapshots, and average profile plots, except when compared

590 to other mappings, in Fig. 4D and Supplemental Fig. S4C, see below), using

591 deepTools (3.5.0) (Ramírez et al. 2016) BamCoverage with RPKM normalization. The

592 coverage for all IP and INPUT samples is depicted in Supplemental Fig. S4B.

593 **scRNA-seq experiments**

594 Single-cell encapsulation and libraries preparation were performed using the

595 SingleCell3'-10× technology. For cell harvesting and counting, we adapted the 10×

596 demonstrated protocol for methanol fixation (available upon request from 10×

597 Genomics, and described in more detail in Supplemental Materials and Methods). For

598 cell encapsulation, steps 1.2d-e of the 10× Genomics single cell protocol version v3.1

599 were modified as follows: a gel beads and zymolyase suspension was prepared. A

600 100× stock of zymolyase (100 mg/mL, MP Biomedicals, 8320932) solution was added

601 to the gel beads suspension to reach 1.7× final concentration before loading on the

602 chip. Note that previous studies in growing cells (Vermeersch et al. 2022; Jariani et al.

603 2020) have used 1× concentration, but that, for quiescent cells, this was not sufficient.

604 Then, 50 µL of gel beads and zymolyase suspension were dispensed as indicated into

605 row labeled 2 of the chip, before proceeding with library preparation according to

606 protocol. Samples were sequenced in a NovaSeq (PE28-10-10-90).

607 **RNA-seq experiments**

608 25 OD600nm equivalent of cells were harvested, pelleted, flash-frozen and stored at

609 -70° before processing with Ribopure Yeast RNA (invitrogen AM1926). Directional

610 mRNA libraries (poly(A) enrichment) were processed and paired-end sequenced by

611 Novogene Europe.
612 **Data processing**

613 ChIP-seq, scRNA-seq and RNA-seq data processing details are described in
614 Supplemental Materials and Methods. Promoter-bearing intergenes (Fig 4)
615 overlapped by RNAPII peaks are listed in Supplemental Table S3. Rpb3-FLAG peaks'
616 summits falling in promoter-bearing intergenes (Fig 4) are listed in Supplemental Table
617 S4. TFBS enriched in the vicinity (100 bp) of the strongest RNAPII summits (Fig 5) are
618 listed in Supplemental Table S5. R packages used for analyses are listed in
619 Supplemental Table S6.

620

621 DATA ACCESS

622 The Rpb3-FLAG ChIP-seq, scRNA-seq and RNA-seq data generated in this study
623 have been submitted to EMBL-EBI ArrayExpress

624 https://www.ebi.ac.uk/biostudies/arrayexpress under accession numbers E-MTAB-
625 14337, E-MTAB-14980, E-MTAB-14983.

626

627 COMPETING INTEREST STATEMENT

628 The authors declare that they have no known competing interests.

629 ACKNOWLEDGEMENTS

630 The authors thank the members of the Taddei laboratory and of UMR3664 for helpful
631 discussions. The A.T. team was financially supported by funding from the Labex DEEP
632 (ANR-11-LABEX-0044 DEEP and ANR-10IDEX-0001-02 PSL), from the Agence
633 Nationale de la Recherche DNA-Life (ANR-15-CE12-0007) and DeSynLE (ANR-22-
634 CE12-0013-01). M.B.P. was supported in part by a fellowship from Institut Curie (IC3i
635 2019) and by the Labex DEEP (ANR-11-LABEX-0044 DEEP).
636 Most bioinformatic analyses were performed on the Core Cluster of the Institut
637 Français de Bioinformatique (IFB) (ANR-11-INBS-0013), using an estimated 4762.68
638 hours per CPU. The authors also thank the PICT-IBISA@Pasteur Imaging Facility of
639 the Institut Curie, a member of the France Bioimaging National Infrastructure (ANR-
640 10-INBS-04).

641 AUTHOR CONTRIBUTIONS

642 I.L., M.B.P. and M.R. generated strains. M.B.P and M.R performed ChIP-seq
643 experiments. M.B.P, M.R., and I.L. carried out imaging and ChIP-qPCR experiments.
644 M.G. generated the software to quantify microscopy images. M.B.P. and U.S.
645 processed ChIP-seq data. M.B.P and G.L performed scRNA-seq experiments. G.L.
646 carried out scRNA-seq analyses. G.L. performed RNA-seq experiments. M.B.P.
647 carried out all other bioinformatic analyses. A.T., M.R., A.M., G.L., and M.B.P
648 contributed to the design of the experiments. A.T. and A.M. obtained funding. A.T., and
649 M.B.P. contributed to the interpretation of the data, the drafting of the figures, and the
650 writing/revision of the manuscript.
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895
896 FIGURE LEGENDS

897 **Figure 1. Transcriptional shutdown during quiescence entry occurs**
898 **independently of telomere hypercluster formation**

899 A. Timeline of events occurring during quiescence entry. Samples in the nutrient
900 exhaustion kinetics are shown as a function of time after inoculation in rich liquid
901 medium (YPD). All cells following diauxic shift (DS) are sorted through a density
902 gradient (Allen et al., 2006) to recover the highly dense (HD) fraction. For abrupt
903 starvation, yielding Q-like cells, gradient-enriched cells grown for +24h to post-diauxic
904 shift (day1-HD cells) are re-suspended in water for 24h hours.

905 B. Proportion of nuclei with hyperclusters during quiescence entry compared to spike-
906 in calibrated RNAPII ChIP-seq (Rpb3-FLAG) counts genome-wide. (top)
907 Representative images of Rap1-GFP sampled during quiescence entry. Bar = 1µm.
908 (middle) Rap1-GFP cluster intensities relative to total nuclear signal classified
909 according to the number of clusters found per nucleus. Hyperclusters, shown in blue,
910 are defined as clusters with a relative intensity over four-fold higher than the average
911 cluster in Log phase. Pie charts represent the proportion of nuclei with a hypercluster.
912 (bottom) Rpb3-FLAG spike-in calibrated IP counts averaged over 1kb bins genome-
913 wide.

914 C. Comparison of telomere hyperclustering and Pol II (Rpb3-FLAG) binding in
915 quiescence (6dHD, Q). (Left) Representative images of Rap1-GFP in WT and *sir3Δ*
916 samples in quiescent cells. Small cells, with a Feret's diameter < 4.5 µm are marked
917 with an asterisk (*) in transmitted light images. Bar = 1µm. (Right) Rpb3-FLAG counts
918 over genes normalized by gene size in WT vs *sir3Δ* samples.

919 **Figure 2. The dismantling of telomere hyperclusters during return to growth**
920 **follows but is independent of transcriptional activation.**

921 A. Telomere hypercluster dismantling upon quiescence exit. Representative images
922 of Rap1-GFP sampled during quiescence release. Brightness for all Rap1-GFP
923 images is set at 1600 maximum.
924 B. Telomere hypercluster dismantling in rich medium (YPD) after shifting cells grown
925 at 25°C to restrictive temperature (37°C, methods) to inactivate RNAPII (rpb1-1) or not
926 (WT) for RNAPII transcription: representative Rap1-GFP images after 15 or 30 min of
927 refeeding of stationary phase cells. Brightness for all Rap1-GFP images is set at 1600
928 maximum.

929 **Figure 3. Transcriptional output is scant, but homogeneous, in the quiescent**
930 **population**

931 A. Representative genome browser views of genes classified according to binarized
932 Rpb3-FLAG binding and recently synthesized transcripts detection within the coding
933 region. Thresholds are set at 50 average counts for Rpb3-FLAG and 1 average count
934 for 4tU-seq (see Supplemental Fig. 3 C, D, F). *This study. **Rpb1 CTD-Ser2P and
935 recently synthesized transcripts mappings are from the Tsukiyama laboratory (Swygert
936 et al., 2019, Cucinotta et al., 2021).

937 B. UMAP representation of cells in the quiescent population assessed by scRNA-seq.
938 Colour scale represents the percentage of the transcriptome per cell for the sum of
939 class I genes.

940 C. (left) List of top 7 class I genes, representing 18.8% of the transcriptome of the
941 average cell. (right) Genome browser view of GRE1, detected in >99% of 6d-HD (Q)
942 cells, during early respiration (Day 1 HD) and in two types of quiescence induction
943 (abrupt starvation from post-diauxic shift, Q-like; nutrient exhaustion after 6 days, Q).
944 *This study. **Rpb1 CTD-Ser2P and recently synthesized transcripts mappings are
945 from the Tsukiyama laboratory (Swygert et al., 2019, Cucinotta et al., 2021).
946 Intergenic accumulation of Rpb3-FLAG upstream of genes in Q samples (6d-HD) is
947 highlighted in gray to allow comparison across conditions.

948 **Figure 4. RNAPII re-distributes to a subset of promoter-bearing intergenes when**
949 **cells enter quiescence**

950 A. Rpb3-FLAG spike-in calibrated signal piled-up at the center of intergenes classified
951 based on the orientation of surrounding coding annotations. Rows are sorted based
952 on intergene size.

953 B. Mapping of RNAPII (Rpb1 C-terminal domain, Rpb1 CTD; Rpb1 CTD Serine 2
954 phosphorylation, Rpb1 CTD-Ser2P; Rpb1 CTD Serine 5 phosphorylation, Rpb1 CTD
955 Ser5P) and RNAPI (Rpa135-FLAG) epitopes in and around the RNAPII (Rpb3-FLAG)
956 quiescent peak at the divergent intergene between *DCP2* and *MLS1* (class III genes).
957 (Top) Genome browser view of spike-in calibrated Rpb3-FLAG (this study), Rpb1 CTD
958 Serine 2 phosphorylation (Rpb1 CTD-Ser2P, Swygert et al., 2019) and recently
959 synthesized transcripts (4tU-seq, both strands merged, Cucinotta et al., 2021).
960 (Bottom) ChIP-qPCR IP/INPUT signal normalized to an intergenic region in Chr1
961 (methods). Antibodies used are written in parentheses.

962 C. Average profile plot of ChIP-seq or ChIP-array mappings (z-scores) of various
963 nuclear proteins in exponential growth (Log) and quiescent (Q 6d-HD) cells: RNAPII
964 (Rpb3-FLAG, this study), TFIIB (Sua7 in Log, Jeronimo et al., 2014; Sua7 in quiescent
965 (6d-HD) cells, Cucinotta et al., 2021) and Sir3 (Guidi et al., 2015). Signals are centered
966 at the summits of RNAPII (Rpb3-FLAG) strongest peaks in quiescence (MACS2 signal
967 Value > 2.4), falling in promoter-bearing intergenes, or at random locations in
968 promoter-bearing intergenes without RNAPII peak overlap. (Right) Coverage tracks of
969 samples shown in left panel at *DCP2/MLS1* intergene.

970 D. Pie charts depicting the proportion of genes bearing an RNAPII summit in the
971 vicinity of their promoter, stratified by transcription status in quiescence, as described
972 in Fig. 3A. (p-values for hypergeometric test: I**, 2.23×10⁻⁰³; III***, 1.23×10⁻⁴³)

973 **Figure 5. Quiescence-specific intergenic RNAPII accumulates at non-canonical**
974 **binding sites in promoters**

975 A. RNAPII distribution over coding sequences at 137 RP (Ribosomal Protein), 938
976 STM (SAGA, Mediator or TUP-bound) and 1783 TFO (TF organization lacking STM
977 cofactors but bound by insulators) genes (Rossi et al. 2021). Rpb3-FLAG signal (z-
978 score) is re-scaled over TSS-TES.

979 B. Distribution of summits falling in promoter-bearing intergenes relative to
980 transcription start sites (TSS). TSS positions are corrected using CRAC-seq from
981 cycling cells (Candelli et al. 2018). Locations of mapped upstream activated
982 sequences (Schofield and Hahn 2023) are shown as dark red lines.

983 C. Transcription factor binding sites (TFBS from JASPAR 2022 fungi database
984 (Castro-Mondragon et al. 2022)) found in the vicinity (100 bp) of strongest RNAPII
985 intergenic summits, with a fold enrichment over control > 3 (SEA, MEME suite (Bailey
986 and Grant 2021)).
987 D. Clustering of Rpb3-FLAG at Q summits +/- 1kb (Top) Average profile plot of RNAPII
988 ChIP-seq (Rpb3-FLAG, this study). Signals are centered at the summits of RNAPII
989 (Rpb3-FLAG) in quiescence falling in promoter-bearing intergenes. Summits are
990 clustered (k-means) into three groups: 437 summits with signal present throughout the
991 kinetics of Q entry, 601 summits with signal locally depleted during growth phase and
992 404 summits appearing de novo in the later stages of quiescence entry. (bottom)
993 Snapshots of Rpb3-FLAG mapping (*this study) in fermentation (Log), respiration
994 (post-diauxie, day1 HD; day 2 HD) or quiescence induced by abrupt starvation (Q-like)
995 or nutrient exhaustion (Q, 6d HD). Typical examples are shown below each group.
996 Dotted lines indicate the position of the peak summit for each intergene and grey boxes
997 indicate the region over which the signal was clustered.

998 **Figure 6. Upstream RNAPII predicts strength of transcriptional re-start upon**
999 release

1000 A. k-means clustering of recently synthesized transcripts data (**Cucinotta et al., 2021)
1001 over TSS-TES. Shown are the number of genes per cluster. Pie charts show the
1002 proportion of genes per cluster bearing an upstream RNAPII summit (black). Outer
1003 ring shows the proportion of ribosomal protein genes (blue). *** gene clusters
1004 significantly enriched in RNAPII at promoter (hypergeometric test, R-I, 1.14×10⁻⁴; R-II,
1005 2.32×10⁻²⁷; R-III, 3.75×10⁻³⁹; R-IV, ns)

1006 B. Heatmap of RNAPII enrichment at promoter per decile of expression in single cell
1007 RNA transcriptomes performed 30 min after release in media containing different
1008 carbon sources. RNAPII enrichment at promoter was estimated by a hypergeometric
1009 test on the proportion of genes with a Rpb3-FLAG peak summit falling in the upstream
1010 intergene (p-values shown in S6F). Deciles of expression are based on the number of
1011 cells expressing the gene per sample (cells released on media containing either
1012 glucose (2%) or lactate and ethanol (0.5 %, 2%) as carbon source). Pie charts
1013 represent the proportion of genes with (black) or without (grey) a summit in their
1014 promoter amongst the 10% most frequently expressed genes in either condition. Outer
1015 ring shows the proportion of ribosomal protein genes (blue), as in panel A. *** indicate
1016 significant enrichment in RNAPII summits at promoter.
<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Figure 5</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Figure 6</figcaption></figure>

<figure><img src="image_11.png" /></figure>
Intergenic accumulation of RNAPII maintains the potential for swift transcriptional re-start upon release from quiescence

Baquero Pérez, Manuela ¹, Laenen, Gertjan ¹ **, Loïodice, Isabelle ¹ **, Mickaël Garnier ¹, Szachnowski, Ugo ², Morillon, Antonin ², Ruault, Myriam ¹, Taddei, Angela ¹

Affiliations

¹ Institut Curie, Université PSL, Sorbonne University, CNRS, UMR3664 Nuclear Dynamics, Paris, France

² Institut Curie, Université PSL, Sorbonne University, CNRS, UMR3244 DIG-Cancer, Paris, France

Supplemental Material

<table><tr><td>Supplemental Figure S1</td><td>2</td></tr><tr><td>Supplemental Figure S2</td><td>3</td></tr><tr><td>Supplemental Figure S3</td><td>5</td></tr><tr><td>Supplemental Figure S4</td><td>13</td></tr><tr><td>Supplemental Figure S5</td><td>16</td></tr><tr><td>Supplemental Figure S6</td><td>18</td></tr><tr><td>Supplemental Materials and Methods</td><td>21</td></tr><tr><td>HD gradient enrichment</td><td>21</td></tr><tr><td>Imaging and Rap1-GFP foci quantification</td><td>21</td></tr><tr><td>Sample harvesting for chromatin immunoprecipitation</td><td>22</td></tr><tr><td>ChIP-seq</td><td>23</td></tr><tr><td>ChIP-qPCR experiments</td><td>24</td></tr><tr><td>ChIP-seq data processing</td><td>25</td></tr><tr><td>scRNA-seq experiments</td><td>30</td></tr><tr><td>scRNA-seq data processing</td><td>30</td></tr><tr><td>RNA-seq data processing</td><td>32</td></tr></table>