bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Gala et al, Pol II pausing in G0
RNA polymerase II pausing regulates a quiescence-dependent transcriptional program, priming cells for cell cycle reentry
Hardik P. Gala1,2, Debarya Saha1, Nisha Venugopal1,2, Ajoy Aloysius 1,2,3 and Jyotsna Dhawan1, 2 1Centre for Cellular and Molecular Biology, Hyderabad, 500 007 India 2 Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065 India 3National Center for Biological Sciences, Bangalore, 560065 India
Running title: Promoter-proximal RNA Pol II pausing in G0
Keywords: Quiescence, myoblast, Adult stem cells, G0, RNA Pol II, ChIP-seq, promoter proximal RNA polymerase pausing, myotube, cell cycle re-entry, reversible arrest Address correspondence to: Jyotsna Dhawan Center for Cellular and Molecular Biology Uppal Road, Hyderabad - 500 007 India. Tel. (40)-2719-2754 Fax. (40)-2716-0591 Email [email protected]
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Gala et al, Pol II pausing in G0
Abstract Adult stem cells persist in mammalian tissues by entering a state of reversible arrest or quiescence associated with low transcription. Using cultured myoblasts and primary muscle stem
cells, we show that RNA synthesis is strongly repressed in G0, returning within minutes of activation. We investigate the underlying mechanism and reveal a role for promoter-proximal RNAPol II pausing: by mapping global Pol II occupancy using ChIP-seq, in conjunction with
RNA-seq to identify repressed transcriptional networks unique to G0. Strikingly, Pol II pausing
is enhanced in G0 on genes encoding regulators of RNA biogenesis (Ncl, Rps24, Ctdp1), and release of pausing is critical for cell cycle re-entry. Finally, we uncover a novel, unexpected
repressive role of the super-elongation complex component Aff4 in G0-specific stalling. We
propose a model wherein Pol II pausing restrains transcription to maintain G0, preconfigures
gene networks required for the G0-G1 transition, and sets the timing of their transcriptional activation.
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Gala et al, Pol II pausing in G0
Introduction In mammalian tissues, adult stem cells exist in a state of reversible arrest or quiescence.
This “out of cycle” or G0 phase is characterized by the absence of DNA synthesis, highly condensed chromatin, and reduced metabolic, transcriptional and translational activity. By contrast to their terminally differentiated counterparts, quiescent cells retain the ability to re- enter cell cycle. Indeed, it is this property of reversible arrest that is crucial for stem cell functions such as self-renewal and regeneration. Mounting evidence indicates that the transition
into G0 includes not only the induction of a specific quiescence program (Coller et al., 2006; Fukada et al., 2007; Liu et al., 2013; Subramaniam et al., 2013), but also suppression of alternate arrest programs such as differentiation, senescence and death (García-Prat et al., 2016; Sousa- Victor et al., 2014). Recent studies highlight the role of metabolic adaptations, epigenetic regulation and signaling mechanisms that enable cells to establish and/or maintain quiescence (reviewed in (Cheung and Rando, 2013)). Collectively, these insights have promoted the view that rather than a passive decline due to the absence of nutrients, mitogens or signaling cues, the
quiescent state is actively maintained. Parallels with G0 in yeasts (Martinez et al., 2004; Sajiki et al., 2009; Yanagida, 2009) have shown that the quiescence program is evolutionarily ancient (reviewed in (Dhawan and Laxman, 2015)). Among the adult stem cell populations that are amenable to comprehensive analysis, the skeletal muscle stem cell or satellite cell (Mauro, 1961) holds a special position as it is readily isolated, easily imaged and can be cultured along with its myofiber niche (Bischoff, 1990; Beauchamp et al., 2000; Siegel et al., 2009). However, there is a growing appreciation that perturbing the stem cell niche leads to activation and loss of hallmarks of quiescence, and that
freshly isolated stem cells while not yet replicating, have triggered the G0-G1 transition (Kami et al., 1995; Fukada et al., 2007; Pallafacchina et al., 2010; Zhang and Anderson, 2014; Fu et al., 2015). Thus, culture models employing C2C12 myoblasts that recapitulate the reversibly arrested stem cell state (Milasincic et al., 1996; Sachidanandan et al., 2002; Yoshida et al., 2013) are
useful adjuncts for genome-wide analysis of G0 as distinct from early G1 (Sebastian et al., 2009; Subramaniam et al., 2013; Cheedipudi et al., 2015) that is inaccessible in vivo. Importantly,
genes isolated on the basis of induction in G0 myoblasts in culture mark muscle satellite cells in vivo (Sachidanandan et al., 2002; Doles and Olwin, 2015), strengthening their utility as a model of quiescent muscle stem cells.
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Gala et al, Pol II pausing in G0
Several lines of evidence suggest that quiescent cells are held in readiness for cell cycle re-entry by mechanisms that keep the genome poised for action. For example there is an increase in epigenetic modifications (H4K20me3) that promote the formation of facultative heterochromatin and chromatin condensation, and trigger transcriptional repression (Evertts et al., 2013; Boonsanay et al., 2016), while other histone modifications help to maintain the quiescence program (Srivastava et al., 2010; Juan et al., 2011; Mousavi et al., 2012; Woodhouse
et al., 2013; Liu et al., 2013). Chromatin regulators induced specifically in G0 hold genes encoding key cell cycle regulators in a poised state (Cheedipudi et al., 2015) by preventing
silencing. Importantly, epigenetic regulation in G0 ensures that the repression of key lineage determinants such as MyoD is reversible, helping to sustain lineage memory (Sebastian et al., 2009). There is little information on mechanisms that regulate the expression of quiescence- specific genes in the context of global transcriptional repression. The regulation of RNA polymerase II (Pol II) is an integral part of the transcription cycle, with cell type and cell state- specific control (reviewed in (Adelman and Lis, 2012; Puri et al., 2015)). Pol II in quiescent adult stem cells exhibits reduced activation (Freter et al., 2010), Mediator (MED1) is associated with maintenance of quiescence (Nakajima et al., 2013) and transcription factor complex II (TFII) proteins have been implicated in differentiation (Deato and Tjian, 2007; Malecova et al., 2016). However, while components of the Pol II complex contribute to cell state changes, Pol II regulation in reversible quiescence is poorly explored. In recent years, the view that global regulation of Pol II occurs largely at the level of transcription initiation has undergone a major revision. Elongation control at the level of promoter-proximal Pol II pausing has been implicated in developmental, stress-induced genes, neuronal immediate early genes (IEG) and mitogen-induced activation of groups of genes (Adelman and Lis, 2012; Saha et al., 2011; Levine, 2011; Gaertner et al., 2012). Further, Pol II pausing allows genes to be poised for future expression (Kouzine et al., 2013), and appears to mark regulatory nodes prior to a change in developmental state, allowing coordinated changes in gene networks. The temporal dynamics of Pol II pausing during development have been investigated (Kouzine et al., 2013), but the role of Pol II pausing in regulating cell cycle state is not well understood. We hypothesized that withdrawal of cells into alternate arrested states might preconfigure different subsets of genes by Pol II pausing, and that these genes may play critical
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Gala et al, Pol II pausing in G0
roles in the subsequent cell fates. Here, we investigated the role of promoter proximal RNA Pol II pausing in regulating the
quiescent state and maintaining G0 cells in a primed condition that might enable cell fate transitions. Using ChIP-seq and RNA-seq, we elucidated Pol II occupancy and the extent of transcriptional repression during different types of cell cycle arrest (reversible vs. irreversible),
thereby identifying poised transcriptional networks characteristic of G0. We used knockdown
analysis of G0-specific stalled genes identified by our analysis, as well as known regulators of Pol II activity, to delineate the role of pausing in quiescence-dependent functions. Our results suggest a model wherein promoter-proximal Pol II pausing regulates a specific class of repressed
genes in G0 and creates a state that is primed for cell cycle re-activation. We propose that Pol II stalling may preconfigure gene networks whose repression aids entry into and maintenance of
G0, and confers appropriate timing to their transcriptional activation during the G0-G1 transition
and subsequent G1-S progression.
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Gala et al, Pol II pausing in G0
Results Altered RNA metabolism in quiescent cells: reduced RNA content and low RNA synthesis Global transcription mediated by all three RNA polymerases is dynamic across the cell cycle (Yonaha et al., 1995; White et al., 1995; Russell and Zomerdijk, 2005), and dampened during cell cycle exit (Hannan et al., 2000; Scott et al., 2001; Russell and Zomerdijk, 2005). To determine if this suppression is common to cells entering alternate states of arrest, we measured total cellular RNA content of C2C12 skeletal myoblasts in different states: proliferating
undifferentiated myoblasts (MB), reversibly arrested undifferentiated myoblasts (quiescent/G0) and permanently arrested differentiated myotubes (MT). First, using quantitative in situ imaging of cells stained with SYTO RNA-select we found that total RNA content per cell was lower in
G0 than in MB (Figure 1a,b). Interestingly, despite exiting the cell cycle, MT showed no significant reduction in total RNA content, providing evidence that global transcriptional activity distinguishes quiescence from differentiation. A time course of quiescence reversal (30’-18 hr
reactivation out of G0) showed that a rapid, robust increase in RNA content occurred within 30’ of cell cycle reentry (Figure 1b), and was sustained as cells enter S phase. Flow cytometry of cells stained simultaneously for DNA and RNA confirmed that cells with 2N DNA content in suspension-arrested cultures showed less than half the RNA content per cell than 2N cells in proliferating cultures, validating the results of quantitative imaging and distinguishing between
G0 and G1 (Figure 1c). Flow cytometric analysis of DNA synthesis during reentry is shown for comparison (Figure 1d): cells begin to enter S phase between 12-16 hr after replating. Taken together, the results show that a rapid rise in cellular RNA content occurs minutes after exit from
quiescence (marking the G0-G1 transition) and greatly precedes the G1-S transition.
To determine whether reduced steady state levels of RNA in G0 result from reduced transcription, we measured active RNA synthesis using pulse-labeling with 5-ethynyl uridine (EU) (Jao and Salic, 2008) (Figure 1e,f). EU incorporation into newly synthesized RNA was
strongly suppressed in G0 compared to MB and sharply increased within 30’ of activation (Figure 1f) correlating with the rise in RNA content (Figure 1b), and indicating a rapid restoration of the transcriptional machinery concomitant with cell cycle reentry. To evaluate the transcriptional output of muscle stem cells in situ, we isolated single myofibers with associated satellite cells and determined their EU incorporation. We found that reversibly arrested satellite cells (SC) ex vivo already show robust RNA synthesis within 30’ of isolation, while
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Gala et al, Pol II pausing in G0
differentiated myofiber nuclei (MN) in the same sample showed low incorporation of EU (Figure 1g, Figure 1-figure supplement 1a). This observation is consistent with evidence that SC on freshly isolated fibers have already exited from quiescence during myofiber isolation (Kami et al., 1995; Fukada et al., 2007; Pallafacchina et al., 2010; Zhang and Anderson, 2014; Fu et al., 2015; Zammit et al., 2006). Taken together, the comparison of steady state RNA levels and active RNA synthesis per
cell confirm that in G0, low global RNA levels result directly from depressed transcriptional activity, which is rapidly reversed in a transcriptional burst within minutes of cell cycle reactivation. Further, since total RNA levels are maintained in irreversibly arrested myotubes, strong repression of RNA biogenesis is not a general function of cell cycle cessation, but rather, a feature of a specific quiescence program.
Revisiting the quiescent transcriptome using RNA-seq Identifying genes regulated in quiescence using cell number based normalization methods Previous estimates of altered gene expression in quiescence using differential display (Sachidanandan et al., 2002) in myoblasts, or comparative microarray analysis in a variety of cell types (Venezia et al., 2004; Coller et al., 2006) including myoblasts (Subramaniam et al., 2013) and satellite cells (Fukada et al., 2007; Pallafacchina et al., 2010; Liu et al., 2013) have identified quiescence-induced genes. However, all these studies were based on normalization to equivalent amounts of total RNA which can lead to misinterpretation of global gene expression data (Lovén et al., 2012), particularly when there is sharply varying cellular RNA content across compared samples (as documented above). To quantitatively re-investigate the transcriptome of quiescent myoblasts in contrast to proliferative or differentiated cells, we used RNA-seq analysis, where gene expression was measured by normalization to equal cell number and not equal RNA (enabled by ERCC spike-in analysis as described in Materials and Methods). Post cell number normalization, the similarities between replicates and dissimilarities between samples were computed using Euclidean Distance (Figure 1-figure supplement 1b), confirming that no bias was generated by data processing. To assess the extent of effects of normalization methods (equal RNA vs. equal cell number) on biological interpretation, we compared gene lists identified as differentially
expressed between MB and G0 derived from the same RNA-seq dataset but using the two
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Gala et al, Pol II pausing in G0
different normalization methods. The Venn diagram (Figure 1h) shows that equal RNA normalization over-represented up-regulated genes compared to the equal cell number normalization (1583 vs. 497) and greatly under-represented down-regulated gene numbers (1564 vs. 9449) (Figure 1 - source data 1). Thus, quiescence is characterized by repression of a much larger number of transcripts than previously appreciated.
Repressed RNA biogenesis pathways distinguish quiescent from differentiated cells To identify distinguishing features between two mitotically inactive states (reversibly
arrested G0 cells and irreversibly arrested MT), we analyzed gene ontology (GO) terms of differentially regulated genes derived from equal cell number normalization, using gProfiler tools. Consistent with our earlier microarray study (Subramaniam et al., 2013), compared to MB,
genes identified as up-regulated in G0 are distinct from those in MT (Figure 1-figure supplement 3). GO terms representing response to external stimuli, stress and extracellular matrix
organization are over-represented in G0, whereas skeletal muscle contraction and ion transport are highlighted in MT. These contrasting terms reflect the suppression of myogenic
differentiation in G0 myoblasts. Expectedly, the down-regulated genes identified from both mitotically arrested states were enriched for cell cycle processes, DNA replication and cell proliferation (Figure 1-figure supplement 4). Commonly down-regulated gene families that were
repressed in both G0 and MT included replication-dependent histone transcripts bearing a 3’ - stem-loop and lacking a polyA tail (Figure 1i). Slbp which binds the 3’ stem-loop in polyA histone mRNAs regulating their stability and translation during S phase (Whitfield et al., 2000), was also repressed in both arrested states (Figure 1i).
We focused on the ontologies that were uniquely down-regulated in G0 but not in MT, which intriguingly, involved RNA biogenesis itself (tRNA and rRNAs), mitochondria & electron transport chain, proteasome and carbon metabolism. Thus, reversible arrest is uniquely associated with suppression of RNA metabolism, protein turnover and bioenergetics (Figure 1- figure supplement 4). Further, GO terms involved in stem cell proliferation, epithelial differentiation, neurogenesis and cellular growth, were exclusively down-regulated in MT, consistent with the repression of lineage-inappropriate genes in terminally differentiated myotubes (Ali et al., 2008), and retention of progenitor features and plasticity in quiescent myoblasts. Indeed, as has been previously reported, while the satellite cell specification factor 8 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Gala et al, Pol II pausing in G0
Pax7 was induced in G0 and repressed in MT, the lineage determinant MyoD was suppressed in
G0 and induced in MT.
Further, genes identified here in our cultured quiescence model as differentially expressed (quiescent vs. cycling) were corroborated by comparison to genes reported as differentially expressed in freshly isolated quiescent satellite cells (QSCs) vs. activated satellite cells (ASCs) (Liu et al., 2013) (Figure 1-figure supplement 5-a, b). More than 98% of genes
expressed in ASCs at 60 hr were expressed in proliferating MB and not in G0, consistent with the derivation of C2C12 cells from adult SCs and confirming the global similarity in expression profiles between this cultured cell line and SCs. By contrast with near identity of the ASC- specific gene set, only 17% of the “QSC-specific” gene set of freshly isolated SC were also
identified as up-regulated in G0 in culture. This observation suggests either that G0 in vivo employs markedly different programs than in vitro, or that the reported “QSC-specific” gene set
defines cells that are no longer in G0. Taken together with the strong RNA synthesis seen in freshly isolated SC (Figure 1g), this comparative expression data strongly supports the view that
disturbance of the SC niche during fiber isolation activates a departure from G0. We conclude that the reported “QSC-specific” gene set likely reflects an early-activated cell state, which while still clearly pre-replicative, does not represent undisturbed quiescence.
The RNA Pol II transcriptional machinery is specifically altered in G0 Since we observed strong repression of total RNA levels, reduced RNA synthesis and
repression of a large class of Pol II-transcribed genes in G0, we hypothesized that Pol II expression itself may also be repressed, to control transcription globally. Indeed, RPB1 the
largest subunit of RNA Pol II (N20 pAb) showed decreased levels in both G0 and MT (Figure 2a,
b, Figure 2-figure supplement 1). Quantitative imaging also revealed that while both G0 and MT
showed decreased nuclear levels of Pol II (Figure 2b,c), G0 cells exhibited more cytoplasmically localized Pol II. Phosphorylation of the CTD of RNA Pol II marks distinct stages in the transcriptional activation cycle, where Ser5-p and Ser2-p reflect initiation and elongation stages
respectively (Zhang et al., 2012). G0 cells displayed the lowest level of active Pol II marks compared to MB and MT (Figure 2b, c), indicating that overall Pol II processivity in the 3 states
would be G0 < MT < MB. Taken together, these observations show that despite reduced Pol II
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Gala et al, Pol II pausing in G0
levels in both mitotically inactive states, MT exhibit a nuclear localized enzyme which is
transcriptionally active, whereas G0 cells show predominantly cytoplasmically localized Pol II, and is transcriptionally less active, consistent with reduced EU incorporation.
Identification of stage-specific Pol II recruitment in myogenic cells To identify stage-specific programs of gene activation we used Pol II enrichment profiling. We first validated the N20 antibody using targeted ChIP analysis of the Rgs2 locus
(called as G0-induced by RNA-seq (Figure 1 - source data 1) and by microarray analysis (Subramaniam et al, 2013)), interrogating enrichment at 4 locations across the gene using qPCR (Figure 2d). Specific enrichment of Pol II across the Rgs2 gene was detected in all 3 cellular states, with peak enrichment at the TSS.
To estimate the global recruitment of Pol II on the promoters of all genes, we used ChIP- seq. Libraries were prepared as detailed in Materials and Methods from duplicate samples of
MB, G0 and MT (Figure 2-figure supplement 2, 3). Only non-overlapping genes of length >2500 bases were used for this analysis (15,170 annotated mouse genes from mm9 build). A snapshot of the Rgs2 locus corroborates the finding from q-PCR based experiments confirming enrichment at the TSS (Figure 2e). Promoter-proximal engagement of Pol II on the Rgs2 gene
was strongest in G0 correlating with its high expression. Pol II enrichment at the TSS in all 3 states (Figure 2-figure supplement 4) is consistent with the “toll booth” model (Adelman and Lis, 2012) where initiated Pol II may pause, and represents a checkpoint for transcription, presaging stage-specific regulation of elongation.
To compare global cell state specific variations in Pol II recruitment ,we first categorized non-overlapping genes in decile groups based on enrichment score, separately for each of the three conditions, and further plotted the normalized enrichment of RNA Pol II around the TSS (Figure 2f). In all cases, enrichment peaked within 30-100 bases downstream of the TSS, consistent with initiated but paused polymerase complexes. Strikingly, the recruitment of Pol II
on promoters in G0 cells for the >90th percentile Pol II-enriched genes was significantly higher than that of either MB or MT, which were comparable. By contrast, profiles for the 70-90 percentile sub-groups behaved similarly for all 3 cellular states. Thus, a small group of promoters
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Gala et al, Pol II pausing in G0
in G0 are occupied with much higher abundance of Pol II than other genes, and may represent a
G0-specific class of genes, with elevated ongoing Pol II recruitment. Since promoter occupancy is observed not only on genes that are highly expressed (“active”), but also on genes that are not expressed and exhibit paused polymerases (“poised”), we used the extent of promoter clearance to distinguish these classes.
Calculation of polymerase stalling index (SI): more genes are stalled in G0 and MT than MB Promoter clearance can be quantified using the distribution of Pol II abundance across an entire locus. The ratio of Pol II enrichment at the promoter region to that across the gene body region is defined as the Pausing Index or Stalling Index (SI), where higher SI reflects a lower promoter clearance of Pol II and indicates a point of regulation (Zeitlinger et al., 2007; Min et al., 2011; Danko et al., 2013). All genes with annotated gene length >2500 bases were used for further analysis. Genes overlapping with neighboring genes were removed from the analysis. Pol II density is calculated by base pair enrichment in ChIP-seq data, at promoters (-500 to +300 relative to TSS) and gene body (+300 to +2500). To confirm that the SI is a function of specific enrichment of Pol II, we calculated stalling indices similarly for control input samples: each IP sample exhibited significantly more enrichment/stalling than that of its respective input (Figure 2g, Figure 2-figure supplement 5). Interestingly, >50% of analyzed genes display a SI >1 in any state, confirming that clearance of engaged Pol II from the promoter is a significant common regulatory mechanism. Comparative
GO analysis for cell state-specific stalled genes (SI>2.5 in MB, MT and G0) shows that metabolic and stress response genes are stalled in all three states (Figure 2-figure supplement 6).
Strikingly, the proportion of genes that are stalled in the two non-dividing states (G0 and MT) is higher than that in proliferating cells, suggesting that Pol II stalling represents a point of regulation associated with mitotic inactivity. Thus, halted cell division (either due to quiescence or differentiation) is associated with higher rates of polymerase pausing.
Short repressed replication-dependent histone genes exhibit Pol II occupancy in G0 but not MT The histone genes transcribed by Pol II were excluded from stalling index calculation, as they do not meet the length criteria set for the analysis. This important gene family comprises
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Gala et al, Pol II pausing in G0
~80 genes that map to chr13 and chr3 in clusters, with a few genes singly located on other chromosomes. To evaluate the relative transcriptional engagement of histone loci, the read density of Pol II across the entire gene region was used. Notably, strong enrichment of Pol II was
observed in both MB and G0 but not in MT (Figure 2h, Figure 2-figure supplement 7), indicating that the histone loci are transcriptionally silent in terminal differentiation, but not in reversible arrest. Thus, RNA Pol II occupancy on histone gene loci is indicative of transcriptional activity
but the low mRNA levels in G0 (Figure 1i) likely reflect rapid post-transcriptional turnover, and the sustained Pol II engagement across the entire histone gene family represents a primed gene network preconfigured for the return to proliferation.
G0-stalled genes are specifically repressed in quiescence Since promoter proximal stalling influences the transcriptional output from a given locus, the repertoire of genes regulated by this mechanism in a given state would yield insights into global Pol II-mediated regulation of that state. Our hypothesis was that control of quiescence might include uniquely stalled genes. Therefore, we analyzed the group of 727 genes that show
strong stalling (SI > 2.5) in G0 (hereafter referred to as ‘G0-stalled genes’ (Figure 2 - source data
1)). The cumulative distribution of SI for G0-stalled genes was compared across the three states
(SI>2.5 in G0 is shown in Figure 3a). Genes that were strongly stalled in G0 (high SI) display much lower SI in MB and MT states, indicating that they experience enhanced stalling specifically in quiescence (Figure 3a, Figure 3-figure supplement 1).
Expression levels enumerated by RNA-seq for G0-stalled genes showed that ~95% of
these genes were specifically repressed in G0 (Figure 3b, Figure 3-figure supplement 2). We
conclude that most of the 727 genes show very low transcriptional activity in G0 but continue to display high promoter proximal Pol II occupancy, indicative of a poised or primed state. Most of these genes are also repressed in MT but do not exhibit stalled Pol II, indicating that other mechanisms are responsible for their reduced expression in differentiated cells. Polymerase pausing has previously been associated with upstream divergent transcription (Flynn et al., 2011). Therefore, we evaluated the level of antisense RNA associated with promoters of genes
categorized as G0-stalled, G0 up-regulated (compared to MB) or a randomly selected group containing an equal number of genes. Interestingly, upstream antisense transcription was
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Gala et al, Pol II pausing in G0
observed only for G0-stalled genes (Figure 3c). Gene ontology-based clustering using DAVID
revealed distinct terms encompassing G0-stalled genes (Figure 3d). These include Pol II- transcribed genes involved in ribosomal machinery, mRNA biogenesis & RNA processing, as well as mitochondria-related genes. We conclude that RNA-pol II pausing is associated with high rates of antisense transcription, and distinguishes specific gene groups in G0.
Genes that display stalled polymerases in G0 are poised for reactivation during G1 Considering that quiescent cells display reduced levels of total RNA, the enrichment of GO terms relating to ribosomal machinery and RNA processing genes (Fig.1) led us to hypothesize that the genes regulated by RNA Pol II pausing might be key nodes for global control of RNA metabolism. To validate this promoter proximal stalling, we chose genes involved in RNA regulation which includes genes involved in rRNA biogenesis (nucleolin -Ncl) and ribosomal protein S24 -Rps24), Pol II regulation (Pol II recycling enzyme CTD phosphatase -Ctdp1), and post-transcriptional regulators (stem loop binding protein –Slbp, that controls histone mRNA stability, and Zinc Finger CCCH-Type Containing 12A -Zc3h12A), an RNase that modulates miRNA levels). Enhanced stalling is confirmed as depicted on genome browser snapshots of Pol II occupancy on these genes (Figure 3d, Figure 3-figure supplement 3,4,5,6).
Further, all these G0-stalled genes are marked by histone modifications typical of active (H3K4me3) and not repressive chromatin (H3K27me3) (Figure 3-figure supplement 7). Since these genes display active histone marks but are not actively expressed in G0
(Figure 3-figure supplement 7), we hypothesized that RNA Pol II stalling in G0 may poise genes for subsequent activation. Comparable RNA-seq data was not available for reactivation, so we used q-RTPCR to evaluate induction of specific genes during cell cycle reentry. Within the first
30’ of reactivation, we observed a sharp increase in normalized mRNA levels (referenced to G0)
of the immediate early gene Fos, a hallmark of the G0-G1 transition (Kami et al., 1995). Id2
(stalled but active in G0) was further induced during re-entry. By contrast, the G0-stalled active
gene Rgs2 showed a rapid decrease in expression during re-entry. Interestingly, only the G0- stalled, repressed genes (Ncl, Rps24, Ctdp1, Zc3h12A, Slbp) showed enhanced expression (Figure 3e) whereas genes repressed but not stalled (MyoD, L7, and YPLE5) failed to show rapid activation upon cell cycle re-entry (Figure 3-figure supplement 8). Further, the increased level of transcripts associated with exit from quiescence reflects in increased protein levels (Ncl
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Gala et al, Pol II pausing in G0
and Rps24; Figure 3-figure supplement 9 and 10) in both C2C12 model and primary satellite
cells. These data confirm that G0-stalled, repressed genes are functionally activated during the
G0-G1 transition. Together, these patterns of expression suggest that while these G0-stalled genes are differentially regulated at later stages of the cell cycle, their common early reactivation is consistent with the hypothesis that reversal of stalling contributes to their coordinate restoration to transcriptional competence.
Transcriptional induction during G0-G1 is associated with reduced stalling
Since expression of most G0-stalled genes was low in quiescence and induced during cell cycle reentry, one might expect a corresponding decrease in the proportion of promoter-proximal stalled Pol II, or an increase in the proportion of elongating Pol II (gene body). We investigated
potential changes of Pol II occupancy from G0 to early G1 (30’-2 hr, the window of induced expression), by computing SI for each candidate gene, using targeted ChIP-qPCR. Consistent
with their identification by ChIP-seq as G0-stalled, the SI calculated by this targeted analysis was
also highest in G0, and decreased within 2 hr of exit from quiescence, indicative of increased
promoter clearance as cells enter G1 (Figure 3f). The IEG Fos, that exhibits strong stalling in G0, registered a rapid drop in SI in the first 30’ after activation, at a time when its mRNA expression
is high (Figure 3e), consistent with a rapid increase in promoter clearance. All the selected G0-
stalled repressed genes showed similar trends. Notably, the Rgs2 and Id2 genes (G0 stalled but active) showed the opposite trend: the SI increased as cells exited quiescence, suggesting altered regulation at the level of pausing for repressed vs. active genes. Collectively, this analysis
confirms that activation of G0 stalled genes is accompanied by reduced stalling during the early
G1 transcriptional burst.
Alterations in RNA Pol II phosphorylation during the G0-G1 transition To analyze whether changes in global transcriptional activity of Pol II across cell states are associated with global alteration in its phosphorylation status, we used western blot analysis for total Pol II and its actively transcribing phosphorylated forms (Ser5-p and Ser2-p; Figure 3g), and calculated the ratio of phospho-Pol II to total Pol II in each state (Figure 3g). At R30’ we observed a sharp increase in the proportion of Ser5-p- and Ser2-p-modified polymerase,
indicating a rapid rise in transcriptional competence compared to G0 & correlating well with the
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Gala et al, Pol II pausing in G0
rapid rise of total RNA and active synthesis shown in Figure 1b and Figure 1f, which stabilizes in 2 hr.
Taken together, we conclude that these repressed G0-stalled genes undergo release of Pol II stalling upon exit from quiescence that correlates with their rapid induction during this transition. Further, the data so far suggest that the release of Pol II stalling is associated with exit
from quiescence, and this mode of regulation facilitates cell cycle re-entry by controlling G0- stalled genes encoding regulators of the protein synthesis machinery, mRNA biogenesis and transcriptional control.
RNA Pol II stalling mediates repression that controls the quiescent state
At the level of gene expression, the functional consequences of Pol II stalling in G0 are repression of transcription (resulting in depressed cellular activity), while stalled loci are poised
for activation in response to appropriate signals. Our observations thus far identified several G0- specific stalled genes with the appropriate functions to participate in the biology of quiescence and activation, but whether Pol II stalling is directly responsible for establishment or
maintenance of the quiescent state has not been demonstrated. Since Pol II pausing on G0-stalled genes results in lower mRNA production from the stalled transcription unit, we directly
decreased the expression level of individual G0-stalled genes in proliferating MB and analyzed the functional consequences for entry into quiescence. We used siRNA-mediated knockdown of
the selected G0-stalled genes (Ctdp1, Ncl, Rps24, Slbp, Zc3h12A) in cycling MB (Figure 4- figure supplement 1) and evaluated effects on RNA synthesis, DNA synthesis and the cell cycle. Knockdown cells were first examined for alterations in total RNA content and active RNA synthesis using SYTO-RNASelect staining and EU incorporation respectively (Figure 4a, Figure 4-figure supplement 2). Total RNA content was decreased in Ncl KD and increased for Zc3h12A KD compared to control siRNA treated cells, but not appreciably altered in Ctdp1, Rps24 or Slbp KD (Figure 4a, Figure 4-figure supplement 2). RNA synthesis was decreased by knockdown of four genes consistent with their roles in rRNA biogenesis (Ncl & Rps24), regulation of mRNA synthesis (Ctdp1) and miRNA-mediated mRNA turnover (Zc3h12A). Interestingly, nuclear area was decreased in Ncl & Ctdp1 siRNA conditions, consistent with the
increased chromatin compaction seen in G0 (Evertts et al., 2013) (Figure 1a, e). These observations demonstrate that in proliferating cells, repression of key genes that normally
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Gala et al, Pol II pausing in G0
experience a G0-specific stalling, is sufficient to trigger a quiescence-like state as assessed by reduction in RNA content, active transcription and nuclear area. The cell cycle in knockdown and control cells was analyzed using flow cytometry and
EdU incorporation (Figure 4b,c and Figure 4-figure supplement 3). Of the 5 G0-stalled genes
analyzed, knockdown led to reduced EdU incorporation and G1 arrest for the same 3 genes (Ncl, Ctdp1 & Rps24) that affected RNA synthesis, while 2 (Slbp & Zc3h12A) did not show significant changes in cell cycle profile (Figure 4b,c). As these genes regulate essential processes, there is ~40% decrease in viability in for Rps24 and ~20% decrease in Ctdp1 and Ncl siRNA treated cells compared to ~10% decrease in control siRNA treated cells (Figure 4-figure supplement 4).
We directly tested the onset of quiescence in cells knocked down for G0-stalled genes, by evaluating expression of quiescence specific markers p27 (cyclin-dependent kinase inhibitor 1B) (Oki et al., 2014 ) and p130 (Rb family tumor suppressor) (Carnac et al., 2000; Litovchick et al., 2004) (Figure 4-figure supplement 5,6). Knockdown of the same 3 genes (Ctdp1, Ncl and Rps24) that showed reduced EdU incorporation, also showed a significantly increased proportion of p27+ cells, comparable to the increase observed when proliferating cells are shifted to quiescence-inducing conditions (Figure 4d). Similarly, p130 protein was induced when Ctdp1, Ncl or Rps24 were knocked down in proliferating myoblasts (Figure 4e). Taken together, our
observations suggest that repression of these G0-stalled genes is essential for quiescence, since forced reduction of their expression in proliferating cells leads to induction of markers of G0, indicative of a quiescence-like state.
G0-stalled genes contribute to self-renewal
To investigate the role of these G0 stalled genes in self-renewal, we evaluated the effect
of knockdown on colony formation after exit from quiescence. G0 cells display enhanced self- renewal compared to cycling cells, supporting the view that the quiescence program includes a self-renewal module (Collins et al., 2007; Subramaniam et al., 2013). Knockdown of the same
three genes that affected RNA synthesis and G1 blockade in proliferating cells (Ctdp1, Ncl, Rps24) also showed a decrease in colony forming ability compared with control cells (Figure 4f, Figure 4-figure supplement 7). Since equal numbers of viable cells were plated for the CFU assay, the effect of knockdown on genes that are normally induced very rapidly (30’-2 hr) would
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Gala et al, Pol II pausing in G0
be to dampen their induction upon G1 entry, accounting for the sharp decrease in colony
formation. This finding further demonstrates the importance of timely reactivation of G0-stalled
genes during the G0-G1 transition, failure of which compromises self-renewal.
We conclude that repression of key cellular processes identified by analysis of G0- specific Pol II stalling is sufficient to cause cycling cells to acquire quiescence-like features.
Consistent with the pre-existing transcriptional repression of G0-stalled genes, further repression by siRNA knockdown does not appear to affect the quiescent state itself, but failure to de-repress their expression upon exit (normally mediated by release of RNA Pol II stalling) results in attenuated cell cycle re-entry and diminished self-renewal. Collectively, our results demonstrate that Pol II undergoes a stage-specific regulation to stall on key genes that are required to poise quiescent cells for efficient cell cycle re-entry.
Aff4, a component of the Pol II super-elongation complex regulates G0-stalled genes Release of paused Pol II is mediated by promoter engagement of P-TEFb, a complex comprised of CDK9 and cyclin T. P-TEFb availability and recruitment is regulated by Hexim1, Aff4, and Brd4, with direct consequences on Pol II elongation (Adelman and Lis, 2012; Jonkers
and Lis, 2015). To determine if these key regulators of elongation affected expression of G0-
stalled genes, we used siRNA treatment (in G0) (Figure 5-figure supplement 1), and investigated
deregulation of G0-stalled genes. While, Hexim1 knockdown led to down-regulation for 4 of 5 selected (all except for Ncl) and Brd4 siRNA led to up-regulation of Ncl and Slbp, unexpectedly,
knockdown of Aff4 led to significant up-regulation of all 5 selected G0-stalled genes (Figure 5a and Figure 5-figure supplement 2-a,b). Thus, Aff4 is the Pol II elongation control factor that
commonly restrains expression of stalled IEG in G0 since its knockdown stimulated their transcription.
Since G0-stalled genes were induced by release of pausing in G1, we hypothesized that the up-regulation of these genes in Aff4 knockdown cells might prime cells for accelerated cell
cycle progression. Indeed, EdU incorporation during cell cycle reentry (6 hr and 12 hr after G0 exit) showed that only Aff4 depletion led to accelerated S phase kinetics (~22% EdU+ at 12hr compared to ~8% EdU+ control cells; re-entry kinetics of Brd4 and Hexim1 siRNA treated cells were unchanged (Figure 5 b, c & Figure 5-figure supplement 3). This accelerated exit from quiescence for Aff4 knockdown cells was observed as early as 2 hours after reactivation as
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Gala et al, Pol II pausing in G0
estimated using phosphorylation of pRB S795, a marker of the G0-G1 transition (Zarkowska and Mittnacht, 1997) (Figure 5 d, e). The role of elongation regulators on self-renewal was also assessed (Figure 5-figure supplement 4 a,b): perturbation of Aff4 and Hexim1 led to decreased CFU.
To test if Aff4 acts directly on promoters of G0 stalled genes to regulate their timely expression during exit from quiescence, we examined the occupancy of Aff4 on the promoters of
G0-stalled genes using ChIP (Figure 5 f). During quiescence, Aff4 occupancy was indeed located
at promoters of G0-stalled genes (Ncl and Rps24) but not on a G0 expressed genes (Rgs2), which is consistent with a direct role for Aff4. Further, these promoters continue to be bound by Aff4 as cells exit quiescence. Taken together, these results indicate that in quiescence, the SEC
component Aff4 unexpectedly acts as repressor of G0-stalled genes, thereby promoting stalling and restraining exit from quiescence, in spite of activating chromatin marks. Collectively, our results demonstrate that in reversibly arrested myoblasts, Pol II stalling contributes to repression of a set of genes that are not thus marked in permanently arrested myotubes, and that primed reactivation of these genes is critical for reversal of quiescence and
for the timing of G1-S progression (Figure 6).
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Gala et al, Pol II pausing in G0
Discussion In this study, we highlight the role of Pol II pausing in the maintenance and exit from the quiescent self-renewing state. While cell cycle exit is known to be associated with reduced transcriptional activity, we show here that this is not uniformly true for all mitotically inactive states: reversible arrest, which is typical of adult stem cells, elaborates global transcriptional control mechanisms distinct from terminally arrested differentiated cells. Consequently, Pol II localization and expression levels, as well as the extent of repression of global RNA content and synthesis differ quantitatively between reversibly and irreversibly arrested cells. In particular, genes regulating RNA biogenesis are among the most strongly repressed in quiescent but not differentiated cells. Strikingly, promoter-proximal polymerase pausing revealed by ChIP-seq is
enhanced in G0, genes encoding regulators of RNA biogenesis experience prominent Pol II pausing, and release of pausing on these genes is critical for cell cycle re-entry and self-renewal. Finally, we identify the SEC component Aff4 as a key regulator of quiescence-specific Pol II stalled genes, that also unexpectedly restrains cell cycle re-activation. We conclude that Pol II
stalling mediates transcriptional repression that controls quiescence, and that G0-stalled genes
contribute to competence for G1 entry and self-renewal (Figure 6). Given the similarities of gene expression between myoblasts in culture and satellite cells ex vivo, we suggest that similar regulatory mechanisms may also function in muscle stem cells.
Regulation of the RNA Pol II transcriptional machinery distinguishes reversible and irreversible arrest
Reduced transcriptional output is typical of cells whose proliferative activity has slowed, and has been documented in many systems (Bertoli et al., 2013) including quiescent adult stem cells (Freter et al., 2010). Here, we explored the differences in Pol II activity as myoblasts
withdrew into alternate arrested states –G0 or differentiation- and found several notable differences. First, while the expression of total Pol II was strongly inhibited in both MT and in
G0, the cytoplasmic localization of Pol II was increased in quiescent cells rather than predominantly nuclear as in MT. Second, the levels of active phospho-Pol II (both Ser5p
indicative of initiated Pol II and Ser2p indicative of elongating Pol II) were lower in G0 than in MT, suggesting quiescence-dependent regulation at the level of signaling to the CTD
kinases/phosphatases. Finally, consistent with the severely depressed total RNA levels in G0
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Gala et al, Pol II pausing in G0
compared to either MB or MT, the extent of active RNA synthesis (EU incorporation) was much
lower in G0 than in MB, but rapidly reversed in response to cell cycle reactivation. Importantly, active RNA synthesis differed in muscle cells ex vivo- analysis of freshly isolated skeletal myofibers showed enhanced EU incorporation in activated SC nuclei compared to the differentiated myonuclei. Together, these findings suggest that not only are quiescent cells in culture marked by specific mechanisms that control RNA biogenesis, but also that analysis of RNA synthesis may be useful in distinguishing global cellular states in vivo.
Revisiting the quiescent transcriptome using RNA-seq reveals major differences in RNA biogenesis compared to permanent arrest
Several studies have reported global gene expression profiles of quiescent cells and revealed a core quiescence signature as well as cell type-specific programs (Fukada et al., 2007; Liu et al., 2013; Hausburg et al., 2015). All studies along these lines so far have been comparative analysis using microarrays, where the experimental design forces the use of cRNA derived from equal total RNA. However, our study clearly shows that the altered RNA
metabolism in G0 myoblasts leads not only to lower RNA synthesis, but also strongly reduced total RNA content. The marked reduction in total RNA content led us to re-evaluate the quiescent transcriptome using RNA-seq: by normalizing transcript levels to cell number and not equal RNA, we reveal a revised picture of the quiescent transcriptome. A major new insight offered by this analysis is the strong repression of a much larger number of genes than previously appreciated, bringing the repression of RNA biogenesis into sharp focus. Interestingly, these pathways are not repressed in differentiated cells, consistent with the observations that Pol II regulation distinguishes two mitotically inactive states, leading to the elaboration of distinct global controls.
The transcriptome studies carried out here also show a distinct quiescence signature in
the repertoire of genes up-reglated in G0. This observation contrasts with studies of c-MYC overexpression or activation of naïve B-cells (Lovén et al., 2012; Kouzine et al., 2013), where the basal transcription of a set of genes seen at low levels in the resting phase is induced upon overexpression of c-MYC or B-cell activation: i.e. the same set is expressed to a higher extent, thereby increasing the RNA content per cell and causing transcriptional amplification. By
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Gala et al, Pol II pausing in G0
contrast, in G0 myoblasts, we find a new set of genes is induced in quiescence, and involves distinct mechanisms of global control at the level of Pol II.
Poising of the replication-dependent histone gene network in reversible arrest
A key new finding from our study concerns the differential transcriptional competence of the histone genes in arrested cells. Although replication-dependent histone mRNA expression is tightly S-phase linked, histone genes are transcribed constitutively, while mRNA processing and degradation is regulated by Slbp (Kaygun and Marzluff, 2005). We now show that while Slbp
expression is repressed in both arrested states, Pol II is retained on histone loci only in G0 and not MT. Transcriptional control of Slbp has not been previously reported, and Pol II pausing on this regulator might be the pivot for priming the entire histone gene family. Since the replication- dependent histone loci while stalled and repressed, remain primed during quiescence, and
stalling of Pol II on the Slbp gene is specific to G0, our finding provides a new node for
reversible control of replication in G0 that is silenced in MT.
Further, the expression of histone variants implicated in marking transcriptionally active or poised regions in the genome (H3f3a and H3f3b (Tang et al., 2013)) is unchanged in quiescence but repressed in MT. These variants (Yuen and Knoepfler, 2013) are translated from polyA+ transcripts and are required for normal development and growth control (Tang et al., 2013). It is tempting to speculate that expression of specific replication-independent histones is critical for quiescence, and that the condensed chromatin structure (Evertts et al., 2013) and
reduced nuclear size peculiar to G0 cells (Figure 1e, 4c) may require input of specific histone isoforms.
A new quiescence-specific regulatory node: increased global Pol II pausing
Accumulating evidence points to quiescence as a poised state where diverse regulatory mechanism at the level of chromatin (Liu 2013; Cheedipudi, 2015), mRNA mobilization (Crist,
2013), and signaling (Rodgers, 2014) work to prime G0 cells for cell cycle reactivation. Extending this concept, our study addresses the hypothesis that genes important for cycling cells are kept poised by Pol II pausing, permissive for future expression. Indeed, we identify a distinct
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Gala et al, Pol II pausing in G0
class of genes that are regulated by pausing, and allow quiescent cells to boost the activity of the core cellular machinery rapidly after activation. It is quite likely that Pol II pausing in G0 may enable the retention of open chromatin around key (inactive) promoters during quiescence, permitting their activation during cell cycle reentry. Genes exhibiting quiescence-specific
stalling are also transcriptionally repressed specifically in G0, suggesting a functional relationship between promoter-proximal pausing and low transcriptional output in this state. As this repressed gene set includes RNA metabolism and regulatory proteins, high in the hierarchy of cellular control, Pol II mediated repression of these genes would lead to a reduction in overall cellular throughput, contributing to quiescence. Further, ontologies that include Ca+ channels and neuro-muscular signaling were found to be specific for myotubes, suggesting that Pol II stalling in differentiated cells may control responses to neuronal stimulation. Overall, our finding of state-specific paused networks in myoblasts supports the emerging idea of Pol II elongation control as a major regulatory step in eukaryotic gene expression.
Quiescence-repressed genes are poised for activation in G1
An important hallmark of quiescent cells concerns their extended kinetics of S-phase
entry compared to that of a continually cycling population. This additional phase - the G0-G1 transition- has long been appreciated to integrate extracellular signaling (Pledger et al., 1978), but the regulation of its duration is still incompletely understood (Coller, 2007). Our study suggests that Pol II pausing and its reversal may play a role in determining these kinetics. Unlike
genes such as Rgs2 and Id2 that are up-regulated in quiescent cells, 95% of G0-stalled genes, are
repressed. The activation of some G0-stalled genes preconfigures changes in cellular processes that are essential for cell cycle reentry. Transcriptional activation of this gene set is accompanied by loss of Pol II stalling, increased active phosphorylation marks (Ser2-p and Ser5-p), and a
rapid burst in active RNA synthesis very early in G1. The timing of these events suggests that the increase in active Pol II marks is linked to the exit from quiescence. Taken together, we suggest that release of polymerase stalling and the burst of active transcriptional output during reversal of quiescence promotes cell cycle entry and fine-tunes the transcriptional cascade required for de-
repressing a variety of cellular processes essential for progression from G0 to G1 to S.
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Gala et al, Pol II pausing in G0
Repression of G0-stalled genes contributes to blocked proliferation and sustained cell potency
Our studies support the hypothesis that transcriptional repression of G0-stalled genes contributes to quiescence entry since direct knockdown of these genes in proliferating cells leads to arrest and compromised self-renewal. Perturbation of ribosomal biogenesis genes (Ncl and Rps24) and the Pol II recycling phosphatase (Ctdp1) led to cell cycle arrest of cycling myoblasts, consistent with the findings that these genes are anti-tumorgenic in other systems (Ugrinova et al., 2007; Badhai et al., 2009; Zhong et al., 2016). Interestingly, Ctdp1/FCP1, which controls the restoration of active Pol II during the transcription cycle, was earlier identified in a genetic screen for quiescence-regulatory genes in yeast (Sajiki et al., 2009). Further, in mammalian cells, Ctdp1 has been implicated in controlling global levels of polyA transcripts as well as in rapid induction of heat shock genes (Kobor et al., 1999; Cho et al., 2001; Mandal et al., 2002; Fuda et al., 2012), both functions ascribed to regulatory functions of Pol II mediated transcription. Thus,
stalling control of the Ctdp1 gene itself and its reduced expression in G0, may suggest a feed-
forward mechanism by which RNA Pol II activity is further depressed in G0.
The ribosomal machinery genes, Ncl and Rps24 are involved in the very first processing step that generates pre-rRNA (Ginisty et al., 1998; Choesmel et al., 2008). Rps24 mutations result in Diamond-Blackfan Anemia characterized by ribosome biogenesis defects in HSCs and erythroid progenitors (Choesmel et al., 2008; Song et al., 2014). Stalling of genes involved in
ribosomal RNA processing in G0 suggests an upstream blockade on ribosome biogenesis, and by
extension, the translational machinery. Thus, G0-specific Pol II pausing participates in keeping the major biosynthetic pathways in check to maintain the quiescent state.
The SEC component Aff4 regulates G0-stalled genes to control the timing of S-phase entry
Quiescence-specific regulation of Pol II has not been previously reported. Regulators of stalling such as DSIF and NELF are widely involved and likely to participate in all states. Therefore, we investigated specific components of the P-TEFb regulatory system complex as
good candidates for the release of stalling of specific target genes during G1 (Lu et al., 2016). Release of stalled polymerase is brought about by coordinated regulation of P-TEFb mediated by SEC, Brd4, Hexim1 (Lu et al., 2016; Puri et al., 2015). Hexim1 haplo-deficiency in mice
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Gala et al, Pol II pausing in G0
increases satellite cell activity and muscle regeneration, highlighting a role in self-renewal (Galatioto et al., 2010; Hong et al., 2012). While Hexim1 knockdown did not affect the timing of
cell cycle re-entry, an effect in quiescence cannot be ruled out and the repression of some G0- stalled genes warrants further investigation. The pause-release regulator that had the most interesting phenotype in our study was Aff4. As a SEC component, Aff4 is known to display target gene specificity (Luo et al., 2012), promoting release of paused polymerase on heat shock genes and MYC (Kühl and Rensing, 2000; Luo et al., 2012; Schnerch et al., 2012). By contrast,
we identified a new repressive role for Aff4 in G0, such that knockdown led to enhanced
expression of G0-stalled genes, and accelerated exit from quiescence. Therefore, it is tempting to speculate that Aff4 mediates differential regulation (activation and repression) of stalling on different target genes, thereby regulating cell cycle progression or arrest. Moreover, the accelerated S-phase entry in knockdown quiescent cells indicates that Aff4 guards against premature activation of quiescent cells, thereby regulating the timing of cell state transitions.
In conclusion, by combining a genome-wide analysis of Pol II occupancy with its functional transcriptional output in distinct mitotically arrested states, we uncover a repressive role for Pol II pausing in the control of RNA metabolic genes that is central to the entry into quiescence. We also define a new regulatory node where the SEC component Aff4 acts to
restrain expression of these genes in G0 to set the timing of their activation during G1 entry and affects S-phase progression. Overall, this study extends the role of Pol II pausing in regulating not only developmental lineage transitions (Scheidegger and Nechaev, 2016), but also distinct cell cycle states and in particular, sheds light on the type of arrest typical of adult stem cells.
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Gala et al, Pol II pausing in G0
Acknowledgements We thank Karen Adelman (NIH), Aseem Ansari (U. of Wisconsin), Boudewijn Burgering (Utrecht), Dasaradhi Palakodeti (InStem) and Aswin Seshasayee (NCBS) for helpful discussions, Michal Mokry (Hubrecht Inst) for input to customize the ChIP-seq analysis pipeline, Dawn Cornelison (U. of Missouri) and Judy Anderson (U. of Manitoba) for teaching us myofibre methods, Shahragim Tajbakhsh (Institut Pasteur) for providing Tg:Pax7-nGFP mouse, and Ramkumar Sambasivan (InStem), Suchitra Gopinath (THSTI), Purnima Bhargava (CCMB), Surabhi Srivastava (CCMB), and Sanjeev Galande (IISER-Pune) for critical reading of the manuscript. We gratefully acknowledge facilities at the Bangalore Life Sciences Cluster (NGGS facility for sequencing, Animal Facility and CIFF for imaging and flow cytometry at C-CAMP, inStem,/NCBS Bengaluru), and at Advanced Imaging Facility and the Animal Facility at CCMB, Hyderabad. We acknowledge graduate fellowships from Govt. of India Council for Scientific and Industrial Research (CSIR) (H.G., D.S.) , DBT (N.V.) and DAE-NCBS (A.A.). H.G. was also supported by a short-term fellowship from CSIR-CCMB. J.D. is supported by core funds to CCMB from CSIR, an Indo-Australia collaborative grant and an Indo-Danish collaborative grant from DBT. Animal work was partially supported by the National Mouse Research Resource (NaMoR) grant (BT/PR5981/MED/31/181/2012;2013-2016) from the DBT to NCBS. We gratefully acknowledge an InStem collaborative science chair fellowship to Boudewijn Burgering and support from Utrecht for exchange visits between JD and BB labs.
Author contributions
HG and JD designed research, interpreted data and wrote the paper; HG performed research,
acquired and analysed data, and wrote custom scripts for NGS analysis and quantitative image
analysis; DS, NV and AA performed research. The authors declare no conflict of interest.
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Gala et al, Pol II pausing in G0
Materials and Methods
Cell Culture. C2C12 myoblasts (MB) were obtained from H. Blau (Stanford University) and a subclone A2 (Sachidanandan et al., 2002) were used in all experiments. Myoblasts were maintained in growth medium (GM; DMEM supplemented with 20% FBS and antibiotics) and passaged at 70-80% confluency. Differentiation. Myotube (MT) differentiation was induced in proliferating cultures at 80% confluence after washing with PBS and incubation in differentiation medium (DM: DMEM with 2% horse serum), replaced daily for 5 days. Multinucleated
myotubes appear after 24 h in DM. Quiescence induction: G0 synchronization by suspension culture of myoblasts was as described (Sachidanandan et al., 2002). Briefly, subconfluent proliferating MB cultures were trypsinized and cultured as a single-cell suspension at a density of 105 cells/mL in semisolid media (DMEM containing 1.3% methyl cellulose, 20% FBS, 10
mM HEPES, and antibiotics). After 48 h, when ~98% of cells have entered G0, arrested cells
were harvested by dilution of methyl cellulose media with PBS and centrifugation. G0 cells were reactivated into the cell cycle by replating at a sub-confluent density in GM and harvested at
defined times (30` - 24hr) after activation. Upon replating, G0 cells undergo a G0–G1 transition
to enter G1 ~6 h after reattachment; S-phase peaks at 20-24 h.
Muscle fiber isolation and culture
Animal work was conducted in the NCBS/inStem Animal Care and Resource Center and in the CCMB Animal Facility. All procedures were approved by the inStem and CCMB Institutional Animal Ethics Committees following norms specified by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Govt. of India. Single muscle fibers were isolated and cultured using the Anderson lab method with some modifications (Shefer and Yablonka-Reuveni, 2005; Siegel et al., 2009). Briefly, the EDL muscle was dissected from 8-12 week Tg:Pax7-nGFP mice (Sambasivan et al., 2009). Isolated muscles were digested with Type I collagenase (Worthington) in DMEM at 37ᴼC, till single fibers dissociated. All dissociated fibers were transferred into fresh DMEM medium and triturated to release individual fibers using fire polished pasteur pipettes. Fibers were cleaned by multiple transfers through media washes and then to fiber culture media (DMEM (Gibco), 20%
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Gala et al, Pol II pausing in G0
Fetal bovine serum (Gibco), 10% Horse serum (Gibco), 2% chick embryo extract (GENTAUR), 1% penicillin streptomycin (Gibco), 5ng/ml bFGF (SIGMA)). For quantification of active RNA synthesis, 10 µM EU was added to media. Freshly isolated or cultured fibers were analyzed by immuno-staining. Single fibers were fixed in 4% PFA for 5min, washed 3X with PBS, picked and placed on charged slides (Thermo-Fisher) for immuno-staining.
Knockdown of target genes using RNAi C2C12 cells were cultured in growth medium until 80% confluent. The cells were then trypsinized and ~0.3 X 106 cells were plated on 100 mm tissue culture dishes. Approximately 12- 14hr post plating, the cells were transfected with siRNA (400 picomoles of siRNA with 40 µl lipid) using Lipofectamine RNAiMAX (Invitrogen) as per manufacturer's instructions. The cells were incubated with the RNA-Lipid complex for at least 18-24hr following which they were tested to evaluate the extent of knockdown by qRTPCR and western blot and then used for further experimental analysis. Typically, siRNA-mediated knockdown was in the range of 50- 90% at RNA level. siRNA used in this study are detailed in Table S3.
Immunofluorescence and microscopy Cells plated on cover slips or harvested from suspension cultures were washed with PBS, fixed in 2% paraformaldehyde at room temperature, and permeabilized in PBS + 0.2% TritonX100. Primary antibodies (Table S4) were diluted in blocking buffer (1XPBS, 10% HS, 0.2 % TritonX100). Secondary antibody controls were negative, no cross reactivity of secondary reagents was detected. For total RNA quantification, fixed cells were incubated with 10 µM Syto® RNASelectTM for 30`. For detection of DNA/RNA synthesis, cells were pulsed for 30` with EdU/EU, fixed with 2% PFA for 15’ at RT and labeling was detected using Click-iT® imaging kit (Invitrogen) as per manufacturer’s instructions. Samples were mounted in aqueous mounting agent with DAPI. Images were obtained using a Leica TCS SP8 confocal microscope (63X, 1.4 NA). Minimum global changes in brightness or contrast were made, and composites were assembled using Image J. The automation of intensity calculation and counts was carried out using a custom pipeline created on the Cellprofiler platform (Source Code File - Cellprofiler_processing_images, Source Code File - Cellprofiler_processing_pipeline)
27 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Gala et al, Pol II pausing in G0
(Carpenter et al., 2006; Kamentsky et al., 2011).
Cell cycle analysis using flow cytometry Adherent cells were trypsinized, washed in PBS and pelleted by centrifugation. Suspension-arrested cells were recovered from methyl-cellulose by dilution with PBS followed by centrifugation as described earlier. Cell pellets were titurated in 0.75 ml of PBS, fixed by drop wise addition into 80% ice-cold ethanol with gentle stirring. Following fixation (cells could be stored up to 7 days at -200C), cells were briefly washed with PBS and re- suspended in PBS at 1 million cells per 500 µl PBS containing 40 µM DRAQ5TM (Cat. No DR50050, Biostatus) and 10 µM Syto® RNASelectTM (cat. No S-32703, Invitrogen). Cell cycle analysis was performed on a FACS Caliber® cytometer (Becton Dickinson), DRAQ5TM (wide range of absorption / emission maxima = 697 nm) and Sytox® Green (absorption / emission maxima = ~490 / 530 nm). 10,000 cells were analyzed in each sample. CelQuest® software was used to acquire For cell cycle and EdU co-staining –Cells were pulsed with EdU and fixed as described, washed twice in PBS and resuspended in PI staining solution for 30 minutes in dark. For analysis of S- phase cells during cell cycle reentry, EdU pulsed cells were fixed in 4% paraformaldehyde for 10 min, washed and stored in PBS at 4C. For staining, cells were permeabilized and blocked in PBS having 10% FBS and 0.5% TX-100 and labeling was detected using Click-iT® imaging kit (Invitrogen) as per manufacturer’s instructions. Cells were co-stained with PI (50 µg/ml, RNaseA 250 µg/ml). Cell cycle analysis was performed on Gallios cytometer (Beckman Coulter). Kaluza software (ref) was used to acquire and analyze the data. Analysis of p27 induction: A C2C12 line expressing p27mVenus (a fusion protein consisting of mVenus and a defective mutant of p27-CDKI, (p27K(-)(Oki et al., 2014)) was generated by stable transfection. Adherent myoblasts expressing p27-mVenus (excitation/emission= 515/528), +/- knockdown of various G0-stalled genes were trypsinized and fixed in 4% paraformaldehyde for 10 minutes at room temperature. Suspension cultured cells were recovered as described above. Following fixation, cells were analyzed for mVenus expression using a Gallios cytometer (Beckman Coulter). Kaluza or FlowJo software was used to acquire and analyze the data.
Western blot analysis
Soluble lysates of adherent cultures or suspension cells (2x106) were obtained after
28 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Gala et al, Pol II pausing in G0
harvesting PBS washed cells by centrifugation, resuspended in 500µl of lysis buffer (10mM HEPES, pH 7.9, 1.5mM MgCl2, 10mM KCl, 0.5mM DTT, 1.5mM PMSF and protease inhibitor cocktail (Roche). Protein amount in lysates was estimated using Amido Black staining and quantification of absorbance at 630 nm. Proteins were resolved on 8% Acrylamide gels. and transferred to PVDF membrane. The membrane was incubated with primary antibody overnight at 4°C, then washed in 1X TBS + 0.1% Tween for 10’ each followed by incubation with secondary antibody conjugated with HRP (Horse radish peroxidase) for 1 hr. After a brief wash with TBS-T for 10’, ECL western blotting detection reagent for HRP was used for chemi- luminescent detection using gel documentation system (Vilber Lourmat). Details of antibodies and dilutions used in this study are given in Table S4.
Isolation of RNA from cultured cells and RNA sequencing library preparation Accurate determination of cell number in different states: Attached cultures or suspension cells were counted 3 times (at 2 or more dilutions) using trypan blue exclusion to generate a live cell count using a Countess® Automated Cell Counter (Invitrogen). For MT samples, being multinucleate cells, accurate counts were not possible since cells with varying number of nuclei were present; therefore nuclei count was used as cell number counts. Cells were washed twice with cold PBS, residual PBS removed and cells lysed using 1 ml RLT plus buffer containing β-mercaptoethanol (RNeasy Plus Mini Kit, Qiagen). The lysate was vortexed vigorously to shear genomic DNA and then stored at –700C until further processing. RNA was isolated from cultured cells using RNeasy Plus Mini Kit (catalog no 74134, Qiagen) as per manufacturers’ protocol, using RNeasy MinElute spin column and eluted twice in 30 µl water, quantified by NanoDrop ND-1000UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE) and checked by gel electrophoresis and q-RT PCR for marker transcripts. For RNA-seq, RNA quantification and quality check was done using Agilent RNA 6000 Pico Kit on Agilent 2100 Bioanalyzer system.
RNA sequencing libraries were prepared from duplicate MB, G0 and MT samples and by quantifying the exogenously added “spike-in mix”, we determined that each of the 6 libraries were referenced to RNA content per million cells, correcting for sampling biases generated during sequencing. 3 µl per million cells of ERCC spike-in RNA (1:10 dilution) was added to
29 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Gala et al, Pol II pausing in G0
each sample. Since the RNA-seq experiment was carried out in duplicates, ERCC Mixes were added to distinguish each replicate sets (MB, MT, G0) i.e Mix1 added for MB_1, MT_1, G0_1 (similarly for replicate2 and mix2). The spiked total cellular RNA was DNase treated to remove trace amounts of DNA (DNA-freeTM kit, AmbionTM Cat. No AM1906) and 4 µg of DNase- treated spiked RNA was further processed to remove ribosomal RNA (RiboMinusTM Eukaryotic kit V2, AmbionTM cat no A15020). The Ribominus RNA was quality checked using BioAnalyser. Finally, the purified mRNA was used for cDNA synthesis and library preparation using NEBNext® UltraTM Directional RNA Library Prep Kit for Illumina® (New England Biolabs® cat. No. E7420). The library thus prepared was quality checked on BioAnalyser and verified by q-PCR for marker transcripts prior to carrying out paired-end sequencing on Illumina HiSeq1000.
Chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation Cell harvest and sonication: Wild type C2C12 (8x106) were detached from the substratum by cell dissociation buffer (Sigma) collected in 15 cc tubes and washed with PBS. Cell pellets were re-suspended in growth medium with final concentration of 1% HCHO (Sigma). Cells were incubated for 10’ at 37°C followed by two washes with cold PBS and finally re-suspended in 1.6 ml of SDS lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris HCl pH 8.1). Lysates were incubated on ice for 20’ and spun at 12,000 rpm for 10’ at 4°C. 200 µl samples were subjected to
sonication for 60 sec on/off for 16 cycles for G0 and MB samples and 18 for MT (Bioruptor ® sonicator). The aliquots of sample were stored at -800C. Immuno-precipitation: Chromatin Immunoprecipitation (ChIP) Assay Kit (milliore catalog no 17-295) was used to carry out immunopreicitation as per manufactures protocol. Briefly 200 µl of sonicated lysates were diluted up to 2 ml by adding ChIP dilution buffer and pre-cleared with DynabeadsTM A beads (agarose beads from the kit were not used). Cleared supernatants were recovered and incubated with 7.5 µg antibody (anti RNA Pol II; N20clone/ rabbit IgG) for 16hr at 4°C on a rotary shaker. Dynabeads A/G were added to collect immune complexes. Washes (1ml for each) in a series of buffers were performed as follows: low salt immune complex wash buffer, High salt immune complex wash buffer, LiCl wash buffer and twice in TE (10 mM Tris HCl and 1 mM EDTA). All washes were done at 4°C on a rotary shaker and collected by centrifugation at 7000 rpm for 5’ each. Finally, the beads were re-suspended in 250 µl freshly prepared elution buffer (1% SDS, 30 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Gala et al, Pol II pausing in G0
0.1 M NaHCO3) and incubated at RT for 15’ each. The elution process was repeated twice and 20 µl of 5 M NaCl was added to 500µl of combined eluate for reverse cross-linking at 65°C for 16hr. Reverse cross-linked eluates were subjected to proteinase K digestion at 45°C for 1 hr. Qiagen nucleotide removal kit was used to purify DNA. Purified DNA was subjected to real- time PCR with primers as shown (Table S5). Each DNA sample was analyzed in triplicate, in at least three independent experiments.
ChIP-sequencing library preparation: 20 ng of pull-down/input DNA was used to carry out sequencing library preparation using NEBNext® DNA Library Prep kit for Illumina® (New England Biolabs®, cat. No. E6000) along with NEBNext® Multiplex oligos for Illumina® (Index Primers set 1) (New England Biolabs®, cat. No. E7335), as per manufacturer’s protocol. The library thus prepared was quality checked on BioAnalyser and qPCR prior to carrying out paired-end sequencing on Illumina HiSeq1000.
Sequencing data analysis
The ChIP-seq and RNA-seq raw data from this publication have been submitted to the GEO and can be accessed at link (http://www.ncbi.nlm.nih.gov/bioproject/343309)
RNA-seq: Sequenced libraries were aligned using Tophat to Mus musculus reference genome (mm9 build with 92 spike-in mRNA sequences added as pseudo-chromosomes). Reference annotation Version M1 (gtf file, from July 2011 freeze, NCBIM37 - Ensembl 65 from GENCODE consortium) along with ERCC transcripts was used.
For equal RNA normalization: Cufflinks was used for transcript abundance and differential expression analysis (Cuffdiff) (Trapnell et al., 2012; Goff L et al., 2013). Cummerbund was used for sub-selection of differentially expressed gene list (Figure 1 - source data 1) and for representation of expression using heatmap (Figure 1 -figure supplement 2).
For Equal cell number normalization: HTcount packages (Anders et al., 2015) was used to quantify reads per gene for each of the samples using reference annotation file. This was further normalized using DEseq2 package using custom R-scripts generated specifically for this
31 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Gala et al, Pol II pausing in G0
normalization (Source Code File - RNA_Seq_DEseq_processing). The sizeFactor is estimated for the Group B counts of ERCC spike-in RNA mix since the group B genes must not vary across all samples of both mixes. The calculated sizeFactor for each sample using Group B as reference gene set is further used to normalize other genes in the respective library including those in the other ERCC spike in mix groups (Groups A, C, D). This approach ensures that the RNA-seq libraries are scaled to reference spike-in controls, and not to the sequencing depth of the library. For each of the sub groups A, C and D, we found that the mix sets 1 and 2, cluster as distinct groups and the levels of intensity within each of the mix sets is comparable. Also, the fold differences between the mix sets 1 and 2 were observed to be as expected as per the manufacturer’s descriptions for all groups. Differentially expressed gene was then identified for each of pair wise comparisons (Figure 1 - source data 1). The heatmap of ERCC spike in mixes groupB and groupA transcripts after normalization step (Figure 1 -figure supplement 2).
Cross-comparison between normalization methods: Overlap of gene identity for differentially expressed genes identified from both normalization methods (Equal RNA and equal cell number) (>2 log2 fold change and p-value<0.05) is represented using size adjusted Venn diagrams separately for both up-regulated and down- regulated genes (Figure 1h).
Correlation between replicates and samples: Dissimilarity between the sample and replicates were computed using the normalized gene count matrix to get the sample to sample distance using Euclidean Distance Method using DEseq packages. The distance matrix is further is clustered hierarchically and heatmap is plotted (Figure 1 -figure supplement 1).
Gene ontology studies: Panther tools (Thomas et al., 2003) and gProfiler (g:Cocoa) (Reimand et al., 2011) was used for over representation analysis of ontologies for single and multiple gene
lists respectively. For GSEA, cell number normalized expression values (for MB and G0) used as expression dataset and gene sets (QSCs (437) and ASC36h (355) ASC60h (1588)) as identified from earlier published dataset (Liu et al., 2013) and enrichment analysis carried out as described in manual (http://software.broadinstitute.org/gsea/doc/GSEAUserGuideFrame.html).
ChIP-seq After quality assessment, the raw ChIP-seq reads were aligned using paired option using bowtie2
32 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Gala et al, Pol II pausing in G0
to mouse genome (NCBIM37/mm9).
RNA Pol II enrichment at TSS: Only non-overlapping gene >2500 bases were used for analysis (except for histone genes panels). Using these criteria 15170 genes were used for analysis. The read density counts around TSS (+/- 300) was enumerated for all samples. Independently, for
each cell state (MB, MT, G0), the TSS regions were sub group into greater than 90 percentile and 70-90 percentiles based on read density score sorted lowest to highest value. Thus for each cell state (both input and IP) the average distribution of read densities were plotted for the each subgroups (Figure 2 f).
Stalling index: Only non-overlapping gene >1500 bases were used for analysis (except for histone genes panels). Using these criteria 16095 genes were used for analysis. To calculate RNA Pol II stalling index, the number of ChIP-seq reads at promoter region (-500 to +300 bp from TSS) and gene body (+300 to +2500 for genes longer than 2.kb and +300 to +1500 for genes between 1.5kb to 2.5kb) of each gene was enumerated using Seqmonk. Stalling index for all genes were plotted in Figure 2g. The statistical significance of RNA Pol II stalling index was assess using the Benjamini-Hochberg method corrected Fisher's exact tests and to control the
false discovery cut off was (p-value<0.005) and Stalling index greater than 2.5 in G0 IP samples were used in Figure 3a.
Statistical analysis: R-packages were used to carryout statistical significance tests. Mann- Whitney test and Student T-test were used as mentioned in the respective figure legends. For the box plots the dark horizontal bar represents median value; the upper and lower limits of box represents 75 and 25 percentiles for each box.
33 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Gala et al, Pol II pausing in G0
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Figure Legends Figure 1. Decreased total RNA content, repressed total RNA synthesis and major down- regulation of the transcriptome in quiescent myoblasts and satellite cells
a. Total RNA in distinct cell states revealed by fluorescence imaging of MB, G0 and MT stained for DNA (DAPI) and the RNA-specific dye (SYTO® RNASelectTM-green) (scale bar 10µm). All three cellular states show cytoplasmic and nuclear staining.
b. Cellprofiler was used to quantitate integrated image intensity values per cell for the 3 cell states as well as across a time course of reactivation from quiescence from 30`-18hr (R30-R18)
(number of cells analyzed >120 for MB, MT, G0 and >60 for reactivation time points). The
boxplot shows quantification of RNA levels where G0 cells show a major reduction of median RNA content compared to MB and MT, and a rapid increase in RNA levels as cells reenter the cell cycle (dark horizontal bar represents median value; the upper and lower limits of box
represents 75 and 25 percentiles for each box). G0 compared to all other time points shows significance p-value < 2e-16 but between MB & MT p-value =0.072 (NS) (Mann-Whitney test).
c. Flow cytometric quantification of total RNA (Syto-green) shows reduced RNA content in
quiescent (G0, blue) compared to proliferative (MB, red) cells. DNA is stained with DRAQ5. Dot
plots showing ~50% reduction in RNA in G0 compared to G1 population of MB. Note that >90%
G0 cells cluster as a single population with 2N DNA content.
d. FACS analysis of cell cycle re-entry from quiescence. Profiles represent a time course of
reactivation of asynchronously proliferating myoblasts (MB) out of G0 from 30`-18hr (R30- R18). The numbers represent the % of cells in S-phase.
e. Newly synthesized RNA revealed by 5-Ethynyl Uridine (EU) incorporation in a 30` pulse:
Fluorescence images of MB, G0, R30', R2hr: EU (Green) and DNA (DAPI, blue). Scale bar is
10µm. RNA synthesis is low in G0 and rapidly enhanced as cells enter G1. Note that MB cells display more nucleolar staining, consistent with rRNA synthesis and ribosome assembly, which
are reduced in G0 and increased again at 30' and 2 hr after reactivation.
42 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Gala et al, Pol II pausing in G0
f. Quantification of EU incorporation by image analysis as in (Number of cells > 80) (a). G0 cells display low levels of active RNA synthesis compared to MB. A rapid burst of very strong transcriptional activity within 30` of cell cycle activation settles back to levels typical of
proliferating cells by 2hr into the G0-G1 transition. Significant differences were observed (G0 compared to MB, R30 and R02: p-value < 2e-10, MB / R30 p-value =2e-14; but MB / R02 comparison was not significant (Mann-Whitney test).
g. Active RNA synthesis in satellite stem cells (SC) associated with single myofibers cultured ex vivo. SC marked by Pax7 (green, arrowhead) can be distinguished from differentiated myonuclei which are Pax7 negative (MN, asterisk) within the underlying myofiber. In freshly isolated fibers, EU exposure (red) during a 30` pulse ex vivo shows incorporation only in SC and not in MN, indicating rapid activation of the SC during the isolation protocol. At 3hr and 24hr post isolation, MN also show RNA synthesis to the same level as the SC.
h. Transcriptional comparison of G0 and MB states using RNA seq shows a major repression of transcript abundance in quiescence. Quantification based on equal cell number (pink) and equal RNA (blue) normalization highlight the bias towards upregulated genes when equal RNA is used for normalization. Overlap of differentially regulated genes between equal RNA (Cufflinks analysis) and cell number normalization methods (DESeq2 package with spike-in RNA controls): size-adjusted Venn diagrams showing overlap of genes between the two normalization methods. Top panel represents up-regulated genes and bottom panel represents down-regulated genes.
i. Histone genes are strongly down-regulated in both mitotically inactive states. RNA-seq
quantification of 68 histone genes in MB, G0 and MT: heat map showing the level of expression for each gene in all the 3 states. The values plotted are row normalized log2 expression scores from cell number normalized datasets. Expression of Slbp a key regulator of replication- dependent histone genes is also repressed in both the arrested states, correlating with repressed histone transcript abundance.
43 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Gala et al, Pol II pausing in G0
Figure 2. Quiescence is associated with reduced RNA Pol II levels and promoter-proximal RNA Pol II stalling at a specific set of loci
a-c Reduction in levels of RNA Pol II in G0 and MT: a. Western blot analysis using antibodies against RNA Pol II large subunit (total) shows two distinct bands (~250kd) corresponding to hyper-phosphorylated and hypo- phosphorylated RNA
Pol II isoforms. The cell cycle arrested states G0 and MT show reduced levels of RNA Pol II. GAPDH was used as loading control. b. Expression and localization of RNA Pol II: Representative immunofluorescence images for
MB, G0 and MT stained for total Pol II (large subunit, red) and active Pol II (Ser2-p [green], upper panel) or total Pol II (red) and Ser-5p [green], lower panel). Scale bar is 10µm; number of cells analyzed > 70 for each cell state. MB and MT show nuclear localization of total and active
Pol II whereas G0 cells show cytoplasmic staining. c. Cell profiler was used to quantitate integrated image intensity values per nucleus for all three states and corresponding negative controls. The boxplot shows reduced levels of Pol II and its
active modification marks-representing both initiating (Ser5-p) and elongation (Ser2-p) in G0 -7 state. For total RNA pol II levels p-value < 2e for all pairwise comparisons. For Ser2-p, G0 and -7 G0 negative control are not significantly different; whereas p-value is <2e when compared
pairwise with MB, MT and G0 states. For Ser5-p negative control (MB and G0) are not
significantly different; whereas MB and MT p-value <0.00036 and G0 compared to MB/MT p- value < 2e-7 (Mann-Whitney test).
d, e. RNA Pol II enrichment on the TSS of the Rgs2 locus, a gene up-regulated in G0. (d) Targeted ChIP q-PCR, enrichment is represented as percentage of input using primers tiling Rgs2 gene in RNA Pol II IP (N20 pAb) and IgG control. Schematic of the Rgs2 locus is shown above the graph, depicting location of primers upstream (-530), TSS (+100), gene body-1 (+500) and gene body-2 (+1500) used for ChIP-qPCR. Values represent mean + SD n=3, p-value < 0.01.
44 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Gala et al, Pol II pausing in G0
e. ChIP-seq depicted by a genome browser snapshot for Rgs2 (locus is oriented with TSS on right). The read density is normalized per million within each of the sample and plotted at equal scale. The numbers in the brackets indicates the log2 expression score from the cell number normalized RNA-seq dataset.
f. Quiescent cells show high enrichment of RNA Pol II on a subset of genes compared to MB and MT. The cumulative density of sequencing reads plotted across +/− 300 bases relative to TSS. RNA pol II enrichment densities are represented for genes grouped based on top 3 decile values (ie 1517 genes for each of 70-80, 80-90, 90-100 percentiles group) for each state are
plotted. Note the substantial difference in peak height around TSS between G0 and MB/MT for the >90th percentile gene set but equivalence in the 70-90 percentile gene set.
g. Both mitotically inactive states (G0 and MT) show higher stalling indices compared to proliferating MB. CDF plot for stalling indices of both IP and IN samples for the three cellular states are depicted. All pairwise comparisons show p-value <2e-16 (except comparison between
Inputs G0 and MT not significant (Mann-Whitney test).
h. RNA Pol II is enriched on histone genes in G0 but not in MT. Violin plots of the read density across 68 histone genes show specific enrichment in all three IP samples over the respective input samples. With respect to the corresponding input sample, enrichment is highest in MB (p- -12 -11 value < 10e ), retained at near equivalent levels in G0 (p-value < 10e ) and significant but much lower in MT (p-value < 10e-5) (Mann-Whitney test).
Figure 3. G0-specific stalled genes are repressed in quiescence and poised for activation
a. The subset of 727 genes which showing stalling index (SI) >2.5 in G0 were selected and
cumulative distribution of stalling index is represented. These genes show G0-specific stalling since more than 60% of these genes display SI < 2.5 in other states (MB and MT).
45 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Gala et al, Pol II pausing in G0
b. Heat map representing mRNA expression as enumerated from RNA-seq data for genes
exhibiting Pol II stalling specifically in G0. Note that expression of only ~5% of these genes are
induced in G0 compared to MB while 95% of stalled genes are repressed in quiescence.
c. Normalized read density of antisense RNA flanking +/-500 bases with respect to TSS for G0 state. Equal number of genes (n=410) were used for each of three categories: (i) up-regulated in G0 vs. MB, (ii) G0 stalled genes and (iii) random selected genes as control.
d. G0-stalled genes were analyzed by PANTHER over-representation test and significantly enriched ontologies (BP – Biological process, CC – cellular component, MF – molecular function) with p-value <0.05 are shown. Notably, ontologies for RNA binding, mitochondria,
ribonucleoprotein complexes and mRNA metabolism are enriched in this set of G0-stalled genes.
e. Quantitative RT-PCR analysis of G0-stalled genes in quiescence and during cell cycle re-entry.
The expression in reactivation time points is normalized to expression in G0, and mean relative expression level is plotted with increasing time as cells exit quiescence (n=3). Fos, a canonical
IEG is transiently induced only at 30` after reactivation and rapidly repressed. G0-stalled but expressed genes Rgs2 (which is repressed during entry) and Id2 (which continues to express as
cells exit quiescence). All G0-stalled and repressed genes (Ctdp1, Ncl, Rps24, Slbp & Zc3h12A) show induction within 30’ after cell cycle activation, albeit with differential kinetics at later
points during G1-S progression.
f. Stalling index of G0-stalled genes decreases during cell cycle reactivation and is associated with expression dynamics. Calculation of stalling index by ChIP-qPCR was done using ratio of ChIP enrichment for primers targeting TSS vs. gene body (+500-1000 bps) in each time point:
G0, R30’ R2h. Both IEG and G0-stalled genes show rapid decrease in stalling index at 30`. G0- expressed genes Rgs2 and Id2 show gradual increase (Rgs2) and no change (Id2) in stalling index respectively, correlating with expression dynamics (significance calculated using Students t-test * = p-value <0.05 and $ = p-values between 0.05-0.15).
46 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Gala et al, Pol II pausing in G0
g. Western blots for total RNA Pol II and active phosphorylated forms at G0, and 30’-2hr after reactivation. Densitometric analysis shows an increased proportion of ser2-p/ser5-p modification
at R30 and R2 compared to G0, correlating with activation. Values represent mean + SD, n=3, *p-value<0.05.
Figure 4. Perturbation of G0-stalled genes compromises cell cycle and self-renewal
a. Quantification of integrated image intensity values per cell (number of cells > 75 per condition) to estimate effect of siRNA treatment on total RNA, EU incorporation and nuclear area. Decreased total RNA was observed for Ncl (2.6e-16), Ctdp1, Rps24 and Slbp (< 0.01). Decreased RNA synthesis was observed for Ncl & Ctdp1 (p <1e-7), Rps24 (0.0007) and Zc3h12A (0.0128). Nuclear area was decreased for Ctdp1, Ncl and Zc3h12A (<1 x e-7), Rps24 (0.0044). (N=3, using Mann-Whitney test, gray shaded box represents p-value < 0.05).
b. Change in cell cycle due to the knockdown of G0-stalled genes. The stacked bar plots for the
proportion of cells in each stage of the cell cycle (G0/G1, S, G2M) for cells treated with siRNA in
proliferative conditions. Increase in G0/G1 phase was observed for siRNA treatments of Ctdp1, Ncl and Rps24 compared to NTS control. The overlay cell cycle traces and statistics are shown in Figure 4-figure supplement 3.
c. Proliferation measured by EdU incorporation after knockdown: knockdown of Ctdp1, Ncl and Rps24 (* = p-value <0.05, n=3 t-test) results in cell cycle blockade- increased 2N population and decreased EdU incorporation.
d. Flow cytometry shows that %p27m-Venus positive cells increase in proliferating MB treated
with siRNA for G0-stalled genes. Untransfected C2C12 lines are used as negative control (Figure 4-figure supplement 4). Control cells (p27-mVenus stable C2C12 line) show increased
proportion of p27+ cells when shifted to quiescence-inducing conditions (G0 ~ 40%) compared to proliferating (MB ~ 10 %), confirming p27 as a marker of quiescence. Further, knockdowns of Ctdp1, Ncl, Rps24 show significantly increased p27+ cells compared to NTS control, indicating
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that forced reduction in mRNA levels of G0 stalled genes in proliferating conditions is sufficient to induce features of quiescence.
e. p130 protein level increases (estimated using western blots) in proliferating MB treated with
siRNA for G0-stalled genes. The knockdown of Ctdp1, Ncl, Rps24 shows significant increase in quiescence marker P130 and graph represents the normalized densitometry levels. (*= pvalue <0.05 , N=3 compared to NTS).
f. Knockdown of selected G0-stalled genes affects self-renewal: colony-forming ability for Ncl, Rps24 and Ctdp1 knockdown is reduced. Quantification is shown in (Figure 4 -figure supplement 1- S4b (Values represent mean + SD, p value <0.05, n=3)
Figure 5. Aff4, a regulator of RNApol II stalling regulates G0 stalled genes and the G0-G1 transition: an unexpected negative role for the SEC complex member
a. Perturbation of Aff4, a regulator of Pol II stalling leads to de-repression of G0-stalled genes.
qPCR analysis of expression of G0-stalled genes after Aff4 siRNA treatment of quiescent cells
shows significantly increased mRNA expression compared to NTS treated G0 cells (Values represent mean + SD n=3, p value <0.05).
b, c. Among regulators of pol II elongation, Aff4 uniquely affects exit from quiescence. Functional effect of knockdown of RNA Pol II regulators on the kinetics of S-phase entry from
G0: EdU incorporation was estimated at 6hr and 12hr after cell cycle activation (R6, R12). Aff4 KD at R12 shows significantly increased EdU incorporation compared to NTS treated cells. Brd4 KD and Hexim1 KD cells show no difference. (Values represent mean + SD, n=3, p-value * <0.05). Horse-shoe plots of EdU-pulsed re-entry time-course accurately estimates % of S-phase cells in Aff4 knockdown using flow cytometry for cells co-stained for DNA (x-axis) and EdU (y- axis). % of S-phase cells (inset text) is significantly higher at 12-18hr in the Aff4 knockdown, showing more rapid S phase kinetics (quantification depicted in Figure 5-figure supplement 3).
d, e. Confirming accelerated cell cycle re-entry in Aff4 knockdown cells compared to NTS control. The early marker for G0-G1 transition -phospho-Rb (S795)- is activated earlier and to 48 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
Gala et al, Pol II pausing in G0
higher levels than in control cells indicating that Aff4 knockdown speeds up reactivation. d is representative western blot for p130 and GAPDH (control gene) and e show the normalized densitometry levels of p130 for NTS/ siAff4 treated samples during the quiescence and re-entry time-course.
f. Aff4 controls the expression of G0-stalled genes during quiescence and reactivation. Aff4
occupancy on promoter of G0-stalled genes (Ncl and Rps24) is higher than that of non-stalled gene Rgs2. Aff4 remains associated with promoters during early reactivation, while stalling is reduced.
Figure 6. RNA Pol II pausing regulates the quiescent state and G0 à G1 transition.
Proliferating cells can exit G1 into either reversible arrest (quiescence/G0) or permanent arrest (differentiation). While both states of mitotic inactivity show enhanced pausing, we identify a distinct regulation of quiescence-specifically stalled genes and its role in cellular fate. Pol II pausing appears to maintain repressed transcription, and thereby contributes to reversibly arrested cellular state and self-renewal. In quiescent cells, Aff4 restrains transcription of stalled
genes and blocks cell cycle reentry. During exit from quiescence (G0àG1 transition), relief of Pol II pausing leads to coordinated transcriptional activation of poised genes and cellular processes, and reconfigures cellular physiology for timely cell cycle progression.
49 a Total RNA levels b
Total bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 20002018. The copyright holder for this preprint (which was not RNAcertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1000
DNA
Integrated Intensity Syto green 0 MB MT G0 Neg MB MT G0 R30’ R1 R2 R6 R12 R18 c d G0 MB State % S MB 30.5 R24 29.4 Total RNA R20 37.2 R16 31.0 R12 2.89 R06 1.97 G0 1.42 DNA e f Active RNA synthesis
1000
EU
500
DNA Integrated Intensity EU
0 MB G0 R30’ R02 neg MB G0 R30’ R2
g h i Color Key Freshly Primary muscle fibre culture Row Z−Score G0 vs. MB Up regulated genes −1 isolated fiber 3hr 24hr 0 1 Equal cell Equal Hist1h2br Hist1h2bm numbers Hist1h2bc RNA Hist1h2ao Hist1h1e Pax7 Hist1h4m 1263 320 Hist1h4n 177 Hist1h2bg Hist1h4i Hist1h4c Hist1h2ac Hist1h2be Hist1h3e Hist1h1d Hist1h2al Hist1h2bq Hist2h4 Hist4h4 Hist2h2ac Hist1h4d EU Hist1h2bn G0 vs. MB Down regulated genes Hist1h2ak Hist1h2bp Hist1h4h Hist1h2b Hist1h3d Hist1h3i Hist2h3b Hist1h2ae Equal Hist3h2ba Hist1h3a Equal cell Hist1h2ai RNA Hist1h2bf Hist1h2ad Merge Hist1h4a 2 numbers Hist1h3h Hist1h2an EU Hist1h2b Hist1h4j Hist2h2b
DAPI Down regulated in G0 Hist1h2bj 1562 7887 Hist1h3f Hist1h1b Hist1h4f Hist1h4k Hist1h2ab Hist1h2bk Hist1h2ag Hist1h1a Hist1h2bh Hist1h3b Hist1h2af DAPI Hist1h2ah Hist1h3c Hist1h3g
SLBP Slbp MT G0 MB Fig. 1 Decreased total RNA content, repressed total RNA synthesis and major down-regulation of the transcriptome in quiescent myoblasts and satellite cells. a MB G0 MT c
RNA Pol II 40 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under GapdhaCC-BY 4.0 International license.
b 0 DAPI 20 Total RNA pol II SER5-p 0 Integrated Intensity
10 DAPI Total RNA pol II SER2-p SER5-p Total RNA pol II 0 MB G0 MB MT G0 SER2-p negative
MB MT G0 d e Rgs2 locus (6kb) MB IP (8.45)
6 MT IP (7.94)
4 G0 IP (13.51)
MB IN 2 MT IN ChIP Enrichments 0 G0 IN MB MT G0 MB MT G0 anti-RNApol II Rabbit IgG
f g h 1.1x10e-5 9.5x10e-12 1.00 17.5 G0 MB MT 0.75 30 15.0 25 0.50 20 12.5 15 0.25
Fraction of genes G0 10 10.0 IN MB 5 IN IP 0.00 IP MT 0 Read density on Histone genes ChIP-Seq Read Density -500 0 500 -500 0 500 -500 0 500 −2.5 0.0 2.5 5.0 G0 MB MT >90 %ile 80-90 %ile 70-80 %ile Stalling Index (log2)
Fig. 2 Quiescence is associated with reduced RNA Pol II levels and promoter-proximal RNA Pol II stalling at a specific set of loci RNAseq expression level : a b G0 stalled genes c
AURKA F630043A04RIK 2610002D18RIK TTK BC030867 TOP2A FIGNL1 GEN1 CENPK 6720463M24RIK HN1L LOC100048579 F730047E07RIK MCM4 GM13237 LOC100044750 BLM PCNA STARD4 POLA2 4930422G04RIK 2610039C10RIK TIPIN TUBG1 SFRS7 GM5081 ENSMUSG00000081670 FUS ENOPH1 CSE1L HIST1H2BC SNX7 ENSMUSG00000084952 COTL1 GMDS CRYL1 PLAU ALDH1L2 ISY1 POLH G0 Stalled genes ENSMUSG00000090867 GM5778 SIP1 NOP2 GM9949 LOC100046995 MAGOH−RS1 INSIG1 LOC633860 CCDC18 SMG5 ACLY GREB1L NXT1 NCL EEF1E1 SERBP1 GM6978 COL4A4 GM7083 LRRC40 FAU CCT6A MRE11A GM10171 PLA2G16 TAF15 FUBP1 U2AF2 NUP50 NAA38 FAM98A NUDT21 G0 Up regulated genes ADK 2610034B18RIK SAE1 MLH1 WDR18 TRA2B GM8566 LOC100045506 GM4899 ZC3H4 G3BP1 NAT10 MDH2 WWC2 STRAP FASTKD3 PSMA1 CCDC15 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted JanuaryPIN1L 19, 2018. The copyright holder for this preprint (which was not XPO5 EXOSC10 CISD1 CSRP2 TXN2 AI314976 MGAT4A GM5551 GM16516 TMEM183A ABCE1 LOC674157 CDK8 TXNL1 Randomly selected genes SART1 TRMT5 RCL1 1.00 ARF2 RSL1D1 2810408A11RIK RRN3 CCDC138 TAF12 METTL13 certified by peer review) is the author/funder, who has granted bioRxiv a licenseNIPAL1 to display the preprint in perpetuity. It is made available under INTS3 SLC43A3 LGALS9 2310003L22RIK BRD8 CAPRIN1 IPO9 SMC3 UTP15 ANKRD32 LOC100046698 CTDP1 RALA PSMA2 BTBD10 ARPC5 EPT1 DHX9 MAPK6 MED22 GM11582 PSMD5 DYRK3 aCC-BY 4.0 InternationalOBFC2A license. 2810006K23RIK TBC1D7 PDCL3 CIRH1A EIF3A TMEM128 NDUFA8 SEC61A1 MARCKSL1−PS3 ANAPC1 GTF2A2 NDUFA10 HSF1 AHSA1 C7 GXYLT1 GM6440 EIF4G2 CCT4 SMARCC1 CNBP 1810055G02RIK AMDHD1 MTRR SPAG7 RPS3 NOP14 GM8546 MRPL32 FUBP3 GM9040 GM8729 SMARCA5 RPS5 TMX1 MRPL45 MRPL52 RPN1 6330409N04RIK GM2802 XRRA1 KIN HSPA5 AKAP8 OBFC1 VDAC1 APIP D4ERTD22E SURF6 MRPS23 CYP4F16 2610101N10RIK SLC7A6OS GM11878 RNASEH1 UBQLN1 RPP38 2810407C02RIK CHCHD7 THAP1 BNIP2 ENSMUSG00000090771 UBE2K REL HDLBP CPSF3 TOMM7 TTRAP ENSMUSG00000087497 SARS YWHAG DDX3X POLK HOMER1 RUFY1 BICD2 RPUSD4 RAPGEF6 NDUFB9 0.75 SMAD2 FAF1 C1D NXF1 GM12844 SUGT1 1110057K04RIK QRICH1 SLC3A2 FAM35A STRN SDHA LOC100046775 BC024814 LSM7 ENSMUSG00000083816 EAF1 PDCD2L LAMP3 DNAJC27 6530418L21RIK AIP ZFP382 BZW1 ABCB7 PLK2 4922501C03RIK C2CD2L 3.0 DCP2 KLHL22 METAP1 IMMP1L HPRT TCIRG1 LBH STIM2 RBM22 ARID5A ATM GLUD1 ENSMUSG00000087260 NUP98 ENSMUSG00000091896 KBTBD2 LIAS ID2 GM2199 BC011426 SAPS1 CCDC104 CHUK 3110040N11RIK EXOC8 CMTM6 GM7204 EIF3K IDH3G 37500 PXMP3 DDB1 ENSMUSG00000089762 GM9093 ZFP146 1.2 SGTA TNRC6A GM14045 TAF13 TMCO1 GLS ENSMUSG00000073079 SLC30A5 TBC1D19 1.5 GM4149 DTWD1 TMEM107 SF3B1 SEC31A SOCS4 MOBKL1B LOC100047518 ABHD14A ZBTB41 SLC9A1 GNAI3 ANAPC10 YAF2 GM5064 HLTF TPP2 POLDIP3 SPTLC1 PNRC2 GM6560 MAN1A2 ARHGAP31 CREB1 SDHAF2 GM9786 TGFBRAP1 SETD5 TFB2M TMEM168 6330578E17RIK ZFP9 HINT1 WSB1 MALT1 GTF2B DEGS1 AASDHPPT SMYD3 0.50 HOOK2 CSRNP2 BC031181 GM6305 1.0 6720456B07RIK GM7308 TUBD1 EIF3E 4930432K21RIK FURIN ARF3 LOC100046421 LOC635419 THAP2 ENSMUSG00000086657 R3HDM1 RLIM AA386476 2410089E03RIK ADPRH BCAP31 GM10094 TIPRL YME1L1 GOLGA1 THEM4 MAMDC2 ERAP1 DNAJC5 ZFP277 CAR13 CDC42 GRPEL2 USP47 NDUFA7 ECE1 RFT1 ENSMUSG00000078909 SAFB RBM39 PTPN2 TRIM3 DGAT2 LRRC28 FEM1C VAPA -1.5 LACTB2 ATG16L2 CNR1 FNBP4 SACM1L B230219D22RIK GM11889 RNMT NDUFAF1 ZFP830 DIRAS1 IER5 ENSMUSG00000090488 USP22 0.8 ZC3H10 RBM7 BC005537 PPM1A SEC14L1 CCDC45 RBM33 GHITM PPCS DYNC1LI1 LOC100047516 KLF6 CCDC75 SEMA4B LOC627908 WAPAL GTF3C1 TM9SF2 BBS5 ZC3H12C DNAJB4 STX12 CTR9 ARIH1 PFN4
G0 repressed ST5 TDRD7 ZFP868 ZBTB8B SAFB2 -3.0 ENSMUSG00000086350 SNAI1 SMNDC1 AI597479 FAM126A MGAT4B FBXO18 A230046K03RIK VPS35 TBCK NDUFA6 TNIP2 LOC100047849 TOB2 PRCP MOCS2 2700050L05RIK CNOT8 POFUT2 LOC100047997 0.25 C130036L24RIK MTIF2 FBXO7 LOC100048079 SECISBP2 RAB18 LOC100048622 AFF1 IFT172 RASL11B RANBP2 PGAP1 CAND2 ARL15 ZFP346 GM505 PHOSPHO2 HPS5 PRKACB ENSMUSG00000086295 HINT3 CBLL1 TBC1D13 LOC674810 CDS2 GCAP14 STAT1 MTERF ERCC5 RAB33B 2900064A13RIK D6WSU116E ZFP119 FBXO30 RBM5 G0 CSGALNACT2 ZFYVE16 SECISBP2L SLC35A5 RNH1 B230317F23RIK COG6 ZFP82 A630007B06RIK NEK9 LOC100045622 LPXN AP3B1 LOC100048049 EAPP IRX5 FRG1 TPK1 CLTC SERINC5 0.4 GM7368 USP53 RC3H2 TTC7 XPR1 VEGFA ALDH2 ARHGAP28 SLC16A1 1810022K09RIK LOC676770 TM6SF2 PTS PPME1 ELMOD3 E430025E21RIK MB CCNL1 AASDH RBM4B LOC100045775 Fraction of G0 stalled genes ENSMUSG00000087440 for groups of genes INPP5D ZSWIM3 LOC100044277 TOM1L2 SEC16A DCAF8 STAM WDR60 PLOD2 LARGE 1810020D17RIK 5330426P16RIK ZFP438 B230354K17RIK TMEM30A SPTY2D1 FAM50A ARHGAP32 ZFP760 SDF4 LOC100044642 ARNT TRIM2 FAM168A ACADM ENSMUSG00000074056 MPP5 GATSL2 TMEM49 PDE1B ZSWIM6 CCNT2 MT GM9776 ENSMUSG00000085480 RC3H1 0.00 CCNDBP1 RRAGC UBXN7 1200016B10RIK NRP2 D430042O09RIK MTCP1 Levels of Anti-sense RNA SERINC1 GNG11 ANKRA2 ERP44 WLS AKAP13 BC031441 5430411K18RIK STARD3NL ENSMUSG00000084765 BMPR2 ATF7 TNFSF13B ENSMUSG00000085882 PLEKHA4 DSEL ENSMUSG00000080741 TRAFD1 LMBRD1 CD164 ATP6V0D1 EPM2AIP1 DENND4C ZFP235 ATP6AP2 ENSMUSG00000091047 ENSMUSG00000087222 GLG1 TMEM71 GAN GM9897 NDRG3 SERINC3 −500 −250 0 250 500 MAP3K2 TMEM66 1810058I24RIK BC013529 ENSMUSG00000091337 KIDINS220 ENSMUSG00000086368 UHRF2 NBEAL1 FAM162A ERN1 UBN2 TOB1 PCMTD1 2810474O19RIK PRUNE2 −2.5 7.55.02.50.0 MCEE FAM154B ENSMUSG00000086280 GORAB 4933432B09RIK CAGE1 ENSMUSG00000087377 TCTA A530017D24RIK LAMA2 LETM2 LNPEP LOC634417 PCMTD2 PROS1 MRVI1 ENSMUSG00000091727 DNAJB9 STYXL1 ANKRD12 D0H4S114 G0 ZFAND5 CSTAD D4WSU53E CYP2D22 CTSL NFKBIA UNC13B ENSMUSG00000085287 GLA ENSMUSG00000086477 MYLIP ENSMUSG00000091711 MAP3K8 P4HA1 PSAP APH1C ARL4D KCNJ2 Stalling Index (log2) induced RGS2 Distance from TSS G0 MB MT
Gene ontology MF CC BP d e catalytic activity Normalised mRNA levels protein binding -32 -2.0 1.0 1.5 5.0 polyA RNA binding Rgs2
G0 Id2
chromosome induced Ctdp1 mitochondria Ncl organelle membrane Rps24 G0 ribonucleoprotein Slbp
complex repressed Zc3h12A nucleolus IEG Fos nucleoplasm G0 R30 R02 R06 R12 R18 R24 mRNA metabolism G0/G1 S-phase G2/M organonitrogen transition compound metabolism 0.0 5.0 10.0 G0 R30 R02 −log10(p-value) f g Ser5-p Ser2-p $ 5 G0 R30' R2h RNA Pol II
4 $ G0 R30 R02 3 3 * $
2 $ 2 referenced to G0 $
Stalling index (ChIP qPCR) Stalling index (ChIP 1 * * * ** * * 1 0 Relative intensity Fos Ctdp1 Ncl Rps24 Slbp Rgs2 Id2 0 IEG G0 repressed G0 induced Ser5-p Ser2-p
Fig. 3 G0-specific stalled genes are repressed in quiescence and poised for activation. a b G0/G1 S G2/M
100 bioRxiv preprint doi: https://doi.org/10.1101/250910; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display 80the preprint in perpetuity. It is made available under 12000 aCC-BY 4.0 International license. 60
8000 40
cell cycle phase 20 % of cells in each 4000 0
NTS si-Ncl si-Slbp si-Ctdp1 si-Rps24 Integrated Intensity RNA c si-Zc3h12A
75 3000
50 2000
25 * * * 1000 % EdU positive 0
NTS si-Ncl si-Slbp si-Ctdp1 si-Rps24 si-Zc3h12A Integrated Intensity EU
50 MB G0 6000 d * 40 * 30 4000 * 20
Nuclear Area 2000 10 0 -ve
% of p27 Venus positive cells % of p27 Venus NTS si-Ncl si-Slbp NTS si-Ncl control si-Ctdp1 si-Rps24 si-Ctdp1 si-Rps24si-Slbp si-Zc3h12A
si-Zc3h12A Untreated C2C12 p27-Venus transfected C2C12 lines
e 150 kd p130 f si-Ncl si-Zc3h12A 37 kd Gapdh *
NTS si-Ncl si-Slbp si-Ctdp1 si-Rps24 si-Zc3h12A
si-Ctdp1 si-Slbp * * * 3
2 * of p130 NTS 1 si-Rps24
Normalized levels *
0
NTS si-Ncl si-Slbp si-Ctdp1 si-Rps24 si-Zc3h12A
Fig. 4 Perturbation of G0 stalled genes compromises cell cycle and self-renewal a b NTS si-Aff4 NTS si-Aff4 si-Brd4 si-Hexim1 25 bioRxiv 6preprint doi: https://doi.org/10.1101/250910* ; this version posted January 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under * aCC-BY 4.0 International license. 20 4 * * * 15 2 10 5
relative mRNA levels relative mRNA 0
Ncl EdU incorporation (%) 0 Ctdp1 Rps24 Slbp Zc3h12A Reentry 6 hours Reentry 12 hours
c R9 hr R12 hr R18 hr R24 hr
1.17% 4.20% 40.02% 44.74% NTS1
1.17% 10.30% 63.63% 36.78% si-Aff4
d e NTS si-Aff4 4 NTS si-Aff4 G0 R2 R4 R6 R12 G0 R2 R4 R6 R12 3 Rb p795 2
Gapdh 1
Relative levels of Rb-p795 0 G0 R2 R4 R6 R12 f 3 G0 R30 R2
2
1
% ChIP enrichment - Aff4 antibody % ChIP 0 Ncl Rps24 Rgs2 Respective transcription start site Fig. 5 Aff4, a regulator of RNApol II stalling regulates G0 stalled genes & the G0-G1 transition: an unexpected negative role for the SEC complex member. G2 Decreased RNA M & RNA Pol II
Proliferation S Quiescence
G1 Differentiation
G0 Stalled genes
G0 G0àG1
Increased RNA Pol II Release of RNA Pol II pausing pausing Aff4
Repressed Transcriptional transcription activation Molecularevents
TSS TSS TSSTSS Paused but not unexpressed Paused and expressed PausedPaused but notbut unexpressednot unexpressed PausedPaused and and expressed expressed Repressed cellular processes Activation of poised cellular processes Arrested cell cycle Reactivates lineage determinants Promotes self renewal Promotes cell cycle reentry
p27+ Aff4 + p130+ pRb
Quiescence Early G1
Cellphysiology Transcriptional burst Increased active RNA Pol II
Fig. 6 RNA Pol II pausing regulates quiescent state and G0 à G1 transition. pax7 neg 0.4 pos
0.2
Median Intensity - EU incorporation 0.0 NEG 30MIN 3HR 24HR
Fig.Figure S1a 1-figure supplement 1a Time in culture SatelliteFig S1a cells associated with freshly isolated myofiber are activated rapidly during the isolation protocol. BoxplotSatellite for cells median associated intensity to of freshly EU incorporation: isolated myofiber Pax7 arepositive activated (green) rapidly satellite during cells the & isolationmyonuclei (red) forprotocol. ex vivo culturesBoxplot forat variousMedian time intensity points of (30min,EU incorporation 3hrs and 24hrspax7 positivepost isolation). (green) satellite cells & myonuclie (red) for ex vivo cultures at various time points (0hr, 3hrs and 24hrs post isolation).
Euclidean Distance Count 0 6 0 50 100 150
MB_2 MB_1 MT_1 MT_2 G0_1 G0_2 Fig.FigureFig S1b 1-figure supplement 1b CellCell numbernumber normalizationnormalization ofof thethe RNA-seqRNA-seq datasetdataset doesdoes notnot affectaffect quantificationquantification asas thethe replicatesreplicated clustercluster together.together. The levels of ERCC spike in controls was used to normalize the sequencing Indepth order (see to confirm methods) that of the the modified two replicates normalization of MB, G0method and MT employed cell states. in this In studyorder todoes confirm not affect that thethe similaritymodified betweennormalization samples method and employedreplicates, herethe Euclidean does not affectdistances the replicates between thethe samplesEuclidean was calculateddistances betweenfrom the regularizedthe samples log was transformation calculated from (Deseq the regularized package). Thelog transformation distance is color (Deseq coded andpackage). shown Thein the distance key scale is color in the coded heat map.and shown The dendogram in the key scaleshows in the the distance heat map. between The dendog - samplesram shows and the their distance replicates, between indicating samples that and the theirreplicates replicates behave indicating similarly, the while replicates the three behave states aresimilarly indeed and distinct three fromstates each are other.indeed distinct to each other. NA|ERCC−00170 NA|ERCC−00156 ERCC−00116 NA|ERCC−00154 ERCC−00108 NA|ERCC−00147 Spike−RNA GroupA ERCC−00116 ERCC−00136 NA|ERCC−00144 (Log2 fold_diff mix1/mix2= 2) ERCC−00108 NA|ERCC−00136 NA|ERCC−00170 ERCC−00092 NA|ERCC−00156Spike−RNA GroupA ERCC−00136 NA|ERCC−00131 log FPKM1 + NA|ERCC−00154(Log2 fold_diff mix1/mix2= 2) 10 ERCC−00130 ERCC−00092 NA|ERCC−00130 Color Key NA|ERCC−00123 NA|ERCC−00147 1 ERCC−00004 ERCC−00130 and Histogram NA|ERCC−00116 NA|ERCC−00144 Cufflinks : Spike−RNA GroupA ERCC−00144 Color Key 0 ERCC−00004 NA|ERCC−00108 NA|ERCC−00136and Histogram (Log2 fold_diff mix1/mix2= 2) NA|ERCC−00131 −1 ERCC−00170 ERCC−00144 NA|ERCC−00095 log10 FPKM1 + NA|ERCC−00092 NA|ERCC−00130 ERCC−00019 ERCC−00170 NA|ERCC−00123 1 Count NA|ERCC−00085 ERCC−00028 ERCC−00019 NA|ERCC−00116 Cufflinks : Spike−RNA GroupA
NA|ERCC−00062 Count 0 (Log2 fold_diff mix1/mix2= 2) ERCC−00154 ERCC−00028 NA|ERCC−00033 NA|ERCC−00108
01234 ERCC−00154 NA|ERCC−00028 NA|ERCC−00095 −1
ERCC−00085 01234 −1.5−1 −0.50 0.511.5 NA|ERCC−00092 ERCC−00085 NA|ERCC−00019 ERCC−00131 Row Z−Score NA|ERCC−00004 NA|ERCC−00085−1.5−1 −0.50 0.511.5 ERCC−00131 NA|ERCC−00062Row Z−Score ERCC−00095 ERCC−00095 NA|ERCC−00033 ERCC−00062 G0_1 MB_1 MT_1 G0_2 MB_2 MT_2 EqualERCC−00062 cell number NA|ERCC−00028 Equal RNA MT_1 G0_1 MB_1 MT_2 G0_2 MB_2 MT_1 G0_1 MB_1 MT_2 G0_2 MB_2NA|ERCC−00171 NA|ERCC−00019 normalization NA|ERCC−00158 NA|ERCC−00004 normalization NA|ERCC−00150 NA|ERCC−00148 G0_1 MB_1 MT_1 G0_2 MB_2 MT_2 NA|ERCC−00138 NA|ERCC−00126 NA|ERCC−00171 ERCC−00042 ERCC−00042 NA|ERCC−00109 NA|ERCC−00158 NA|ERCC−00104 NA|ERCC−00150 log10 FPKM + 1 ERCC−00035 ERCC−00035 NA|ERCC−00096 NA|ERCC−00148 1 ERCC−00025 ERCC−00025 NA|ERCC−00073 NA|ERCC−00138 Cufflinks : Spike−RNA GroupB ERCC−00060 NA|ERCC−00067 0 ERCC−00060 NA|ERCC−00126Spike−RNA GroupB (Log2 fold_diff mix1/mix2= 0) Spike−RNA GroupB ERCC−00171 NA|ERCC−00060 (Log2NA|ERCC−00109 fold_diff mix1/mix2= 0) −1 ERCC−00171 log10 FPKM + 1 (Log2 fold_diff mix1/mix2= 0) ERCC−00009 NA|ERCC−00053 NA|ERCC−00104Color Key ERCC−00009 Color Key NA|ERCC−00051 NA|ERCC−00096and Histogram 1 and Histogram ERCC−00096 NA|ERCC−00042 NA|ERCC−00073 Cufflinks : Spike−RNA GroupB ERCC−00096 ERCC−00126 NA|ERCC−00035 NA|ERCC−00067 0 (Log2 fold_diff mix1/mix2= 0) NA|ERCC−00034 NA|ERCC−00060 ERCC−00126 ERCC−00051 −1
NA|ERCC−00031 Count NA|ERCC−00053 ERCC−00051 ERCC−00148 NA|ERCC−00025 NA|ERCC−00051 ERCC−00148 Count ERCC−00034 NA|ERCC−00009 NA|ERCC−00042 ERCC−00034 ERCC−00053 NA|ERCC−0003501234 NA|ERCC−00034−1 01 01234 ERCC−00067 G0_1Row Z−ScoreMB_1 MT_2 ERCC−00053 NA|ERCC−00031 MT_1 G0_2 MB_2 −1 01 ERCC−00031 ERCC−00067 Row Z−Score NA|ERCC−00164 NA|ERCC−00025 ERCC−00150 NA|ERCC−00009 ERCC−00031 NA|ERCC−00162 NA|ERCC−00170NA|ERCC−00157 ERCC−00150 MB_2 MB_1 G0_1 MT_2 G0_2 MT_1 NA|ERCC−00156NA|ERCC−00145 G0_1 MB_1 MT_1 G0_2 MB_2 MT_2 MB_2 MB_1 G0_1 MT_2 G0_2 MT_1 NA|ERCC−00154NA|ERCC−00143 NA|ERCC−00147NA|ERCC−00120 NA|ERCC−00164 NA|ERCC−00144NA|ERCC−00113 NA|ERCC−00162 log10 FPKM1 + ERCC−00116 NA|ERCC−00136NA|ERCC−00111 NA|ERCC−00157 ERCC−00145 NA|ERCC−00145 NA|ERCC−00131NA|ERCC−00099 1 ERCC−00108 log10 FPKM1 + Spike−RNA GroupA ERCC−00111ERCC−00116 NA|ERCC−00130NA|ERCC−00084NA|ERCC−00170NA|ERCC−00143 Cufflinks : Spike−RNA GroupC ERCC−00136 NA|ERCC−00156NA|ERCC−00120 0 (Log2 fold_diff mix1/mix2= −0.58) (Log2 fold_diff mix1/mix2= 2) ERCC−00044ERCC−00108 NA|ERCC−00123NA|ERCC−00076 1 ERCC−00145 NA|ERCC−00154NA|ERCC−00113Spike−RNASpike−RNA Gr GroupAoupC ERCC−00092 NA|ERCC−00116NA|ERCC−00074 −1 Cufflinkslog10 FPKM 1: Spik+ e−RNA GroupA ERCC−00076ERCC−00136 (Log2 fold_diff mix1/mix2= −0.58 ) 0 ERCC−00111 NA|ERCC−00108NA|ERCC−00071NA|ERCC−00147NA|ERCC−00111(Log2 fold_diff mix1/mix2= 2) (Log2 fold_diff mix1/mix2= 2) ERCC−00130 ERCC−00092 ERCC−00074 NA|ERCC−00058NA|ERCC−00144NA|ERCC−00099 −1 1 ERCC−00044 Spike−RNAColor Key GroupC NA|ERCC−00095 Color Key ERCC−00004 and Histogram ERCC−00113ERCC−00130 NA|ERCC−00092NA|ERCC−00054NA|ERCC−00136NA|ERCC−00084and Histogram Cufflinks : Spike−RNA GroupC ERCC−00076 (Log2 fold_diff mix1/mix2= −0.58 ) NA|ERCC−00131NA|ERCC−000763 Color Key 0 (Log2 fold_diff mix1/mix2= −0.58) ERCC−00144 ERCC−00071ERCC−00004 NA|ERCC−00085NA|ERCC−00044 log10 FPKM1 + .5 and Histogram ERCC−00074 NA|ERCC−00062NA|ERCC−00040NA|ERCC−00130NA|ERCC−00074 −1 ERCC−00170 Color Key ERCC−00084ERCC−00144 NA|ERCC−00123NA|ERCC−00071 and Histogram NA|ERCC−00033NA|ERCC−00039 1 ERCC−00113 . 522
3 ERCC−00099ERCC−00170 NA|ERCC−00116 Cufflinks : Spike−RNA GroupA
ERCC−00019 Count NA|ERCC−00058 NA|ERCC−00028NA|ERCC−00016 0 ERCC−00071 Count (Log2 fold_diff mix1/mix2= 2) .5 ERCC−00157ERCC−00019 NA|ERCC−00019 NA|ERCC−00108NA|ERCC−00054
ERCC−00028 . 511 NA|ERCC−00095Count −1 ERCC−00084 ERCC−00162ERCC−00028 NA|ERCC−00004 NA|ERCC−00044 ERCC−00154 NA|ERCC−0009200 G0_1 MB_1 MT_1 G0_2 MB_2 MT_2 . 522 NA|ERCC−00040 ERCC−00099 01234 ERCC−00058ERCC−00154 −1.5 −0.50 0.511.5 Count NA|ERCC−00085NA|ERCC−00039 ERCC−00085 01234 Row Z−Score ERCC−00157 −1.5−1 −0.50 0.511.5 ERCC−00039ERCC−00085 NA|ERCC−00168 G0_1 MB_1 MT_1 G0_2 MB_2 MT_2 NA|ERCC−00062NA|ERCC−00016−1.5−1 −0.50 0.511.5 ERCC−00131 . 511 Row Z−Score NA|ERCC−00165 ERCC−00162 ERCC−00143ERCC−00131 NA|ERCC−00033Row Z−Score
00 NA|ERCC−00171NA|ERCC−00163 ERCC−00095 ERCC−00054 NA|ERCC−00028 ERCC−00058 −1.5 −0.50 0.511.5 ERCC−00095 NA|ERCC−00158NA|ERCC−00160 NA|ERCC−00019 G0_1 MB_1 MT_1 G0_2 MB_2 MT_2 ERCC−00062 Row Z−Score NA|ERCC−00150NA|ERCC−00112 ERCC−00039 ERCC−00062 G0_1 MB_1 MT_1 MT_2 MB_2 G0_2 NA|ERCC−00004 NA|ERCC−00148NA|ERCC−00079 NA|ERCC−00168 ERCC−00143 MT_1 G0_1 MB_1 MT_2 G0_2 MB_2 MT_1 G0_1 MB_1 MT_2 G0_2 MB_2 NA|ERCC−00138NA|ERCC−00078 NA|ERCC−00165 log10 FPKM + 1 ERCC−00054 NA|ERCC−00126NA|ERCC−00077 NA|ERCC−00163 G0_1 MB_1 MT_1 G0_2 MB_2 MT_2 NA|ERCC−00109 NA|ERCC−00160 NA|ERCC−00069 1 ERCC−00079 NA|ERCC−00104 log10 FPKM + 1 G0_1 MB_1 MT_1 MT_2 MB_2 G0_2 NA|ERCC−00061NA|ERCC−00171NA|ERCC−00112 Cufflinks : Spike−RNA GroupD ERCC−00165 NA|ERCC−00096 NA|ERCC−00158 NA|ERCC−00059 NA|ERCC−00079 1 0 (Log2 fold_diff mix1/mix2= −1) ERCC−00042ERCC−00078 NA|ERCC−00073 NA|ERCC−00150NA|ERCC−00078 Cufflinks : Spike−RNA GroupB ERCC−00042 NA|ERCC−00046 log FPKM + 1 NA|ERCC−00067 NA|ERCC−00148 0 −1 (Log210 fold_diff mix1/mix2= 0) ERCC−00035ERCC−00112 NA|ERCC−00043 NA|ERCC−00077 ERCC−00035 NA|ERCC−00060 NA|ERCC−00138 NA|ERCC−00069Spike−RNA GroupD −1 ERCC−00079 ERCC−00025ERCC−00022 NA|ERCC−00053NA|ERCC−00041NA|ERCC−00126 1 ERCC−00025 Fig.Figure S1c 1-figure supplement 2 NA|ERCC−00022(Log2NA|ERCC−00061 fold_diff mix1/mix2= −1) Cufflinks : Spike−RNA GroupD ERCC−00165 ERCC−00060ERCC−00003 NA|ERCC−00051 NA|ERCC−00109 0 FigFig. S1C S1b Effect of normalization methodNA|ERCC−00014 on SpikeNA|ERCC−00059Spike−RNA in GrRNAoupB group B (unchangedlog10 FPKM + 1 (Log2 fold_diff mix1/mix2= −1) ERCC−00060 Spike−RNA GroupB NA|ERCC−00042 NA|ERCC−00104 ERCC−00078 Effect of normalizationERCC−00171ERCC−00043 method on Spike in RNANA|ERCC−00046 group B (unchanged between mixes)−1 and NA|ERCC−00035NA|ERCC−00013(Log2NA|ERCC−00096 fold_diffColor Key mix1/mix2= 0) 1 ERCC−00171 (Log2 fold_diffover mix1/mix2= mixes) 0) and group A (mix1:mix2 = 4 fold).NA|ERCC−00043 HeatColor Key map showing the extent of ERCC−00112 ERCC−00009ERCC−00046 NA|ERCC−00034NA|ERCC−00003NA|ERCC−00073and Histogram Cufflinks : Spike−RNA GroupB groupColor Key A (mix1: mix2 = 4 fold). and Histogram ERCC−00009 Spike−RNA GroupD NA|ERCC−00031NA|ERCC−00002NA|ERCC−00067NA|ERCC−00041 0 (Log2 fold_diff mix1/mix2= 0) and HistogramrepresentationERCC−00096ERCC−00002 of ERCC RNA in each of the MB, G0 and MT libraries. The value ERCC−00022 NA|ERCC−00025 NA|ERCC−00060NA|ERCC−00022 ERCC−00096 (Log2Heat fold_diff mapmix1/mix2= showing −1) ERCC−00126ERCC−00059 the extent of representation of ERCC RNA in each of the MB,−1 G0 and ERCC−00003 NA|ERCC−00009 NA|ERCC−00053NA|ERCC−00014 ERCC−00126 for each RNAERCC−00051ERCC−00163 is scaled within the row and relativeNA|ERCC−00013G0_1 MB_1 intensityMT_1 G0_2 is MB_2colorMT_2 coded as NA|ERCC−00051Count
ERCC−00043 MT libraries. The value for each RNA is scaled Count within the row and relative intensity is color ERCC−00051 Color Key ERCC−00148ERCC−00160 NA|ERCC−00042NA|ERCC−00003G0_1 MB_1 MT_2 and Histogramshown on adjacent scale bar. MT_1 G0_2 MB_2 ERCC−00046 Count NA|ERCC−00035NA|ERCC−00002 ERCC−00148 coded as shownERCC−00034ERCC−00014 on adjacent scale bar. NA|ERCC−0003401234 ERCC−00002 Right panel = Equal RNA normalization:NA|ERCC−00164 Extent01234 of representation of group A and ERCC−00034 ERCC−00053ERCC−00069 NA|ERCC−00031−1 −0.50 0.51 Right panel = Equal RNA normalization: NA|ERCC−00162Extent of −1Rorepresentationw Z−Score01G0_1 MB_1 MT_1 ofG0_2 groupMB_2 MT_2A and B Spike in ERCC−00059 01234 ERCC−00067ERCC−00077 NA|ERCC−00025Row Z−Score ERCC−00053 B Spike in transcripts in left panel is similarNA|ERCC−00157 ie G0 > MT >MB for both replicates ERCC−00163 −1 01 ERCC−00031 NA|ERCC−00009
Count transcripts in left panel is similar ie G0 > MT >MB for both replicates ERCC−00067 Row Z−Score MB_1 G0_1 MT_1 MB_2 G0_2 MT_2NA|ERCC−00145 ERCC−00160 Left panel =ERCC−00150 Equal cell number normalizationNA|ERCC−00143 : Group B transcript used as ERCC−00031 Left panel = Equal cell number normalization:NA|ERCC−00120 Group BG0_1 transcriptMB_1 used as MT_2reference for ERCC−00014 MT_1 G0_2 MB_2 ERCC−00150 01234 reference for normalizationMB_2 MB_1 G0_1 andMT_2 histogramG0_2 MT_1NA|ERCC−00113 shows their value close to zero. normalization and histogram shows their value is close to zero. Group A transcriptslog10 FPKM1 + show ERCC−00069 −1 −0.50Group0.51 A transcripts show average differenceNA|ERCC−00111 NA|ERCC−00164 of 2 (log2 scale) between the MB_2 MB_1 G0_1 MT_2 G0_2 MT_1 Row Z−Score NA|ERCC−00099 NA|ERCC−00162 ERCC−00077 average difference of 2 (log2 scale) between the replicate set 1 (ERCC mix1)1 and replicate replicate set 1 (ERCC mix1) and replicateNA|ERCC−00084 setNA|ERCC−00157 2 (ERCC mix 2). Cufflinks : Spike−RNA GroupC MB_1 G0_1 MT_1 MB_2 G0_2 MT_2 NA|ERCC−00076 NA|ERCC−00145 0 (Log2 fold_diff mix1/mix2= −0.58) set 2 (ERCC mix 2). NA|ERCC−00143 ERCC−00145 NA|ERCC−00074 −1 NA|ERCC−00071 NA|ERCC−00120 ERCC−00111 NA|ERCC−00113 NA|ERCC−00058 log10 FPKM1 + ERCC−00145 ERCC−00044 NA|ERCC−00054 NA|ERCC−00111Spike−RNA GroupC ERCC−00076 (Log2NA|ERCC−00099 fold_diff mix1/mix2= −0.58 ) 1 ERCC−00111 NA|ERCC−00044 Cufflinks : Spike−RNA GroupC ERCC−00074 NA|ERCC−00040 NA|ERCC−00084 ERCC−00044 Spike−RNA GroupC NA|ERCC−00076Color Key 0 (Log2 fold_diff mix1/mix2= −0.58) ERCC−00113 NA|ERCC−00039 and Histogram ERCC−00076 (Log2 fold_diff mix1/mix2= −0.58 ) NA|ERCC−00016 NA|ERCC−000743 −1 ERCC−00071 NA|ERCC−00071.5 ERCC−00074 Color Key ERCC−00084 NA|ERCC−00058
ERCC−00113 and Histogram NA|ERCC−00054. 522 G0_1 MB_1 MT_1 G0_2 MB_2 MT_2
3 ERCC−00099 Count ERCC−00071 NA|ERCC−00044 .5 ERCC−00157 NA|ERCC−00168 NA|ERCC−00040. 511 ERCC−00084 ERCC−00162 NA|ERCC−00165
NA|ERCC−0003900
. 522 NA|ERCC−00163 ERCC−00099 ERCC−00058 NA|ERCC−00016−1.5 −0.50 0.511.5 Count NA|ERCC−00160 Row Z−Score ERCC−00157 ERCC−00039
. 511 NA|ERCC−00112 ERCC−00162 ERCC−00143 NA|ERCC−00079 G0_1 MB_1 MT_1 G0_2 MB_2 MT_2 00 ERCC−00058 ERCC−00054 NA|ERCC−00078 −1.5 −0.50 0.511.5 NA|ERCC−00168 log10 FPKM + 1 Row Z−Score NA|ERCC−00077 ERCC−00039 G0_1 MB_1 MT_1 MT_2 MB_2 G0_2 NA|ERCC−00165 NA|ERCC−00069 1 ERCC−00143 NA|ERCC−00061 NA|ERCC−00163 Cufflinks : Spike−RNA GroupD NA|ERCC−00160 0 ERCC−00054 NA|ERCC−00059 (Log2 fold_diff mix1/mix2= −1) NA|ERCC−00112 NA|ERCC−00046 −1 G0_1 MB_1 MT_1 MT_2 MB_2 G0_2 ERCC−00079 NA|ERCC−00043 NA|ERCC−00079 NA|ERCC−00078 ERCC−00165 NA|ERCC−00041 log10 FPKM + 1 NA|ERCC−00077 ERCC−00078 NA|ERCC−00022 NA|ERCC−00069 1 ERCC−00112 NA|ERCC−00014 NA|ERCC−00013 NA|ERCC−00061Spike−RNA GroupD Cufflinks : Spike−RNA GroupD ERCC−00079 ERCC−00022 NA|ERCC−00059 0 (Log2 fold_diff mix1/mix2= −1) NA|ERCC−00003 (Log2 fold_diff mix1/mix2= −1) ERCC−00165 ERCC−00003 NA|ERCC−00002 NA|ERCC−00046 −1 NA|ERCC−00043 ERCC−00078 ERCC−00043 NA|ERCC−00041Color Key ERCC−00112 ERCC−00046 NA|ERCC−00022G0_1and HistogramMB_1 MT_1 G0_2 MB_2 MT_2 Spike−RNA GroupD ERCC−00002 ERCC−00022 NA|ERCC−00014 (Log2 fold_diff mix1/mix2= −1) ERCC−00059 NA|ERCC−00013 ERCC−00003 ERCC−00163 NA|ERCC−00003 ERCC−00043 NA|ERCC−00002Count Color Key ERCC−00160 ERCC−00046 and Histogram ERCC−00014 01234 ERCC−00002 G0_1 MB_1 MT_1 G0_2 MB_2 MT_2 ERCC−00069 −1 −0.50 0.51 Row Z−Score ERCC−00059 ERCC−00077 ERCC−00163 Count MB_1 G0_1 MT_1 MB_2 G0_2 MT_2 ERCC−00160 ERCC−00014 01234 ERCC−00069 −1 −0.50 0.51 Row Z−Score ERCC−00077 MB_1 G0_1 MT_1 MB_2 G0_2 MT_2 source term name term ID n. of corrected MT_UP G0_UP Gene Ontology term p-value genes
BP actin-mediated cell contraction GO:0070252 57 3.73e-06 15
BP ion homeostasis GO:0050801 607 2.68e-07 57
BP cellular cation homeostasis GO:0030003 460 7.41e-06 45
BP extracellular matrix organization GO:0030198 195 8.83e-08 23
BP cell surface receptor signaling pathway GO:0007166 2127 2.97e-08 93
BP regulation of signaling GO:0023051 2640 1.37e-06 103
BP cell adhesion GO:0007155 1289 1.15e-09 70
BP regulation of cell communication GO:0010646 2679 3.08e-06 103
BP muscle system process GO:0003012 306 5.41e-37 75
CC sarcoplasm GO:0016528 59 3.15e-11 20
CC myofibril GO:0030016 192 8.85e-37 61
CC plasma membrane part GO:0044459 2200 1.14e-06 137 87
CC sarcolemma GO:0042383 119 3.22e-09 25 13
CC transmembrane transporter complex GO:1902495 278 2.40e-06 34
CC collagen trimer GO:0005581 85 5.08e-10 18
CC extracellular region GO:0005576 4292 9.22e-35 220213
Fig.Figure S1d 1-figure supplement 3 Gene ontology analysis of genes differentially regulated between two mitotically arrested states Fig.(up-regulatedFig S1c S1d genes). Biological process process comparison comparison for for two two mitotically mitotically arrested arrested states states (Up regulated(Up regulated genes) genes) gProf gProfilerler analysis analysiscarried out carried for genes out down-regulatedfor genes up-regulated in G0 vs inMB G0 and vs MT MB vs and MB comparisons.MT vs MB comparisons. The output isThe graphically output is graphically represented represented here using here moderate using “moderate” option for f Biologicalltering option process for Biological and cellular process and component.cellular component The terms . The enriched enriched interms each in state each arestate listed is listed on tothe the left. left. The The intensity square with of color red intensity in red squareindicates on the right signi indicatesfcance of the each significance state. The mostof each signi state.fcant The among most the significant 2 states is amonghighlighted the 2 with gene bold listsboundaries are highlighted around square with bold and correspondingboundaries around p value square is shown and next corresponding to it and shaded p value in red is square..shown next to it and shaded in red square. Yellow highlight terms are enrich specifically in G0 and green are represented from MT state. source term name term ID n. of corrected G0_DOWN MT_DOWN Gene Ontology (Molecular function) term p-value genes
MF transcription cofactor activity GO:0003712 352 1.34e-09 202167
MF RNA binding GO:0003723 1261 1.12e-189 983827
MF nuclease activity GO:0004518 126 5.59e-06 82 69
MF structural constituent of ribosome GO:0003735 201 2.18e-19 146 91
MF catalytic activity GO:0003824 3878 4.31e-66 19711559 keg Protein processing in ER KEGG:04141 166 3.09e-11 118 86 keg RNA transport KEGG:03013 165 1.36e-28 141122 keg Ribosome KEGG:03010 129 2.18e-36 123 79 keg Nucleotide excision repair KEGG:03420 44 8.19e-07 39 31 keg Aminoacyl-tRNA biosynthesis KEGG:00970 44 6.87e-12 43 32 keg Basal transcription factors KEGG:03022 44 8.19e-07 39 32 keg RNA polymerase KEGG:03020 29 3.21e-06 28 25 keg Oxidative phosphorylation KEGG:00190 134 2.84e-06 91 keg Proteasome KEGG:03050 45 3.71e-06 39 keg Metabolic pathways KEGG:01100 1281 1.00e-07 635 keg Ubiquitin mediated proteolysis KEGG:04120 137 1.47e-08 97 74 keg MicroRNAs in cancer KEGG:05206 142 9.44e-06 81 keg Cell cycle KEGG:04110 123 9.65e-18 102 90 keg Ribosome biogenesis in eukaryotes KEGG:03008 79 2.07e-21 73 70 keg mRNA surveillance pathway KEGG:03015 93 2.37e-08 71 63 keg Pyrimidine metabolism KEGG:00240 104 2.26e-11 80 73 keg Spliceosome KEGG:03040 134 7.18e-27 116108 keg DNA replication KEGG:03030 36 1.35e-09 34 33
Fig.Figure S1e 1-figure supplement 4 GeneFig.Fig. S1eS1e ontology analysis of genes differentially regulated between two mitotically arrested states (Down-regulatedGeneFig S1d ontology analysis genes). of genes differentially regulated between two mitotically arrested states Biological(Down-regulatedBiological process genes).comparison comparison for for two two mitotically mitotically arrested arrested states states (Down (Down regulated regulated genes) genes) gProf ler gProfilerBiologicalanalysis carried analysis process out carried comparisonfor genes out down-regulated for for genes two mitotically down-regulated in G0 arrestedvs MB andin G0states MT vs vs MB(Down MB andcomparisons. regulated MT vs MB genes) comparisons. YellowgProfilerThe output highlight analysis is graphically terms carried are represented out enrich for genes specifically here down-regulated using in “moderate” G0 and greenin fG0ltering vsare MB optionrepresented and for MT Molecular vs from MB Bothcomparisons. function G0 and and MTYellowKEGG states. pathways highlight .terms The enriched are enrich terms specifically in each state in G0 is listed and togreen the left.are Therepresented square with from red Both intensity G0 and MTindicates states. the signifcance of each state. The most signifcant among the 2 states is highlighted with bold boundaries around square and corresponding p value is shown next to it and shaded in red square.. af 100 13
75 59 64
50 G0vsMB Up regulated
G0vsMB Down regulated 25 percent of genes (%) 229 205 1535 0 Quiescent SC 36hrs 60hrs gene set Activated SC gene set
bg
Figure 1-figure supplement 5 Fig. S1f,g IdentifyingIdentifying similaritysimilarity between G0G0 in in cultured cultured C2C12 C2C12 myoblasts myoblasts (this (this study) study) and and freshly freshly isolated isolatedsatellite satellitecells. cells. a.f. PercentagePercentage ofof overlappingoverlapping genes genes between between differentially differentially expressed expressed genes genes in G0 in vs.G0 MBvs. MB comparisoncomparison (Up/Down(Up/Down regulated)regulated) for for each each of of gene gene sets sets QSCs QSCs (437) (437) and and ASC36h ASC36h (355) (355) ASC60hASC60h (1588)(1588) gene setset identifiedidentified in in (Liu (Liu et et al., al., 2013). 2013). Note Note that that although although Quiescent Quiescent SC SChas hasgenes genes higher higher fraction fraction of genes of genes that are that Down are Down regulated regulated in G0 vsin MBG0 vscomparisons MB comparisons suggesting suggestingQSC gene listQSC depicts gene partiallylist depicts activated partially cell activated state. ASC cell 60hrs state. overlap ASC 60hrsalmost overlap entirely almostwith G0 entirelydown regulated with G0 genesdown regulatedie ASC60 geneshrs is similarie ASC60 to MB. hrs is similar to MB. b.g. Leading Leading edgeedge analysis usingusing Gene Gene set set enrichment enrichment analysis analysis (GSEA) (GSEA) comparing comparing G0 G0vs. MBvs. : MBGeneset : Geneset used usedhere ishere 437 is gene 437 identifiedgene identified as QSCs as specificQSCs specific genes (Liu genes et al., (Liu 2013) et al., and 2013) andexpression expression dataset dataset generated generated in our in study our studywas used was as used reference. as reference. Note that Note these that genes these genespartition partition into bimodal into bimodal distribution distribution one node one enriched node enriched for both G0 for andboth MB G0 state. and MB state Sample Gate G0 2N population MB 4N population MB 2N population
Figure 1-figure supplement 65 Volume estimation of MB and G0 cell containing 2N and/or4N DNA content. FSC FSC and SSC didstribution distribution forfor gatedgated cellscells isis usedused asas indicatorindicator forfor volume.volume. G0G0 cellcell areare observed to be markedly reduced in volume compared to MB 2N or 4N population. 1.00
0.75
0.05
* 0.25 *
Relative Total RNA POL II levels POL RNA Total Relative 0.0
MB G0 MT
Figure 2-figure supplement 1 Fig. S2a ReductionReduction in in levels levels of of active active RNA RNA Pol Pol II II in in G0 G0 state: state: DensitometricDensitometric analysis analysis of of western western blot blot data data shows shows an an decreased decreased proportion proportion of of RNA RNA PolPol II II G0 G0 and and MT MT compared compared to to MB. MB. Normalized Normalized RNA RNA Pol Pol II II levels levels referenced referenced to to MB MB andand GAPDH GAPDH as as loading loading control control for for each each state. state. Uniquely mapped reads
Row Labels Replicate1 Replicate2
G0_Input 26539624 12208115
G0_Pol2 22042002 3598740
MB_Input 26925144 14305695
MB_Pol2 13024284 4113686
MT_Input 25250300 6094166 ChIP-seq experiment MT_Pol2 9295852 3106101
Aligned pairs using Tophat
Row Labels Replicate1 Replicate2
G0 49335619 43894347
MB 43618085 44637091 RNA-seq
experiment MT 41130618 40704886
Figure 2-figure supplement 2
FigureDNA gel 2-figuredepicting sonication supplement profiles 3 for all 3 states, the average sonication size is observed to be around 200 DNAbases gelpairs. depicting The table enumerated sonication the profiles sequencing for depth all 3of states libraries andof each table replicate shows used the in this sequencing study. depth. a.
b.
Figure 2-figure 2-figure supplement supplement 3 2 a. MeanMean Sequencing Sequencing coverage coverage (2 replicates) (2 replicates)at GAPDH gene at shown GAPDH gene shown forfor all all three three cellular cellular state (pullstate down (pull and down input). Errorand barinput). show Errorspread barbetween show highest spread and lowest between value observedhighest andacross lowest replicates value Individual observed replicate across track for replicatesG0 shown in panelIndividual below (b). replicate track for G0 shown in panel below (b). -2kb +1 +2kb -2kb +1 +2kb -2kb +1 +2kb
G0_IP MB_IP MT_IP
Figure 2-figure supplement 4 Fig S2b. RNA pol II read density across TSS RNA pol II read density across TSS: Intensity of RNA pol II enrichment is represented aroundIntensity TSS of (+/-2kb) RNA pol for II non enrichment overlapping is representedgenes. The dark around shade TSS represent (+/-2kb) higher for nonintensity asoverlapping observed in genes. the ChIPseq The dark data shadeset for allrepresent three cellular higher states. intensity as observed in the ChIPseq data set for all three cellular states. Cut-off for selecting G0 stalled genes
Cut off G0 IN 4000 99 percent confidence
3000 G0 IP G0 IN 2000 Frequency of genes 1000 0
−4 −2 0 2 4 6
Stalling Index
Figure 2-figure supplement 5
For calculation of stalling index the input is used as reference to set stringent cutoffs. The pulldown sample has rightward tail of the stalling index compared to Input and non overlapping region with >95percent confidence is used to identify the stalled gene. The vertical line shows the cut off value for G0 sample. $% & $% &
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Fig. 4.11 Gene Ontology analysis of Stalled genes FigureFig. S2c Genes2-figure Gene withsupplement Ontology stalling analysis6 index >2.5 of Stalled were enlisted genes for MB, G0 and MT states. gProfiler based comparison for the identified list was carried out. The Gene Ontology analysis of Stalled genes Genesoutput with stalling is graphically index >2.5represented were enlisted here using for each “moderate” MB, G0 filtering and MT option states for Genesseparately. with stalling gProfiler index based >2.5 comparisonwere enlisted for for theMB, identified G0 and MT list states was separately.carried out. gProfiler The based comparisonBiological process, for the identified Cellular listcomponent was carried & Molecularout. The output function. is graphically The enriched representedoutputterms is graphically here in eachusing represented state“moderate” is listed filtering here to using theoption left. “moderate” for TheBiological square filtering process, with option redCellular intensity for Biological componemtprocess,indicates Cellular and molecularthe component significance functions. & ofMolecular eachThe terms state. function. in The each most stateThe significant enrichedis listed to terms amongthe left.The in the each square3 state withis listed redstates intensityto the is left.highlighted indicates The square the with significance withboundaries red ofintensity each around states indicates square is The andthe enriched significancecorresponding terms in of each peach statestate. indicates valueThe most is theshown significant significance next to among it.of each the state. 3 states The most is highlighted significant among with boundaries the 3 highlighted around withsquare boundaries and corresponding around square p andvalue corresponding is shown next p value to it. is shown next to it.
hist1h4h hist1h2af hist1h3g histh2bh hist1h3f hist1h4f Hist1h3e Gene mRNA CDS TSS G0_IN
G0_IP
MB_IN
MB_IP
MT_IN
MT_IP
Hist1h2ae Hist1h2bf Hist1h3d Hist1h2bg Hist1h2ad Hist1h4d Hist1h2be Gene mRNA CDS TSS G0_IN G0_IP MB_IN MB_IP MT_IN MT_I P
FigureFig. S2d 2-figure supplement 7 RNAFig Pol2Sb II Pol2abundance abundance at histone at histone 1 cluster 1 iscluster higher is in higher G0 and in MB G0 and and not MB in andMT RNAnot Pol in II MT abundance at histone 1 cluster is higher in G0 and MB and not in MT GenomeGenome browser browser snapshot snapshot was generated was generated for the Histone for the 1 geneHistone cluster 1 gene at coordinates cluster (chrat 13:23622400-23677600)coordinatesGenome browser (chr 13:23622400-23677600) snapshot on mm9 build. was The generated top panel on showsmm9 for the build.the Histonegene The location top 1 genepanel on chromosome clustershows the at andgenecoordinates its locationorientation (onchr (red chromosome 13:23622400-23677600) indicates forward and and its blue orientation in on reverse). mm9 (red build. The indicates read The density top forward panel is normalized shows and blue theper in millionreverse).gene within location The each read onsample chromosomedensity and plotted is normalized andwith theits colororientation per code million where (red within red indicates signifies each sample forwardhigher density andand plotted blueand blue indicates lower density. The intergenic regions are removed in order to represent the density within reverse).the color Thecode read where density red signifies is normalized higher perdensity million and within blue indicates each sample lower and at plottedhigher resolution. with the Strongcolor enrichmentcode where of Polred II signifiesseen in MB higher persists density in G0 but and not blue MT, despiteindicates downdensity. regulation The intergenic of transcripts regions in both arrestedare removed states. in order to represent the density at lower density. The intergenic regions are removed in order to represent the higher resolution. Say what the samples are G0 –In, G0 IP etc. Strong enrichment density at higher resolution. of Pol II seen in MB persists in G0 but not MT, despite down regulation of transcripts in both arrested states. G0 Stalled genes Comparision of stalling index across cell states 7.5
5.0
2.5 Stalling Index
0.0
−2.5
G0 MB MT
FigureFigure 3-figure 3-figure supplement supplement 1 1 Stalling Index for G0 stalled genes for each of three cellular states. The nested box plot show the distributionStalling Index for SI for(25-75 G0 percentile stalled genes. limits) and the the nested lines trace box the plot SI showvalues theacross distribution 3 states for for each SI gene.(25-75 percentile).
SI
2.2054
2.5906
2.5985
Figure 3-figure supplement 3 Fig.Fig S3a 3 a Ctdp1CTDP1 locus: locus Genome : Genome browser browser snapshot snapshot depicting depicting RNA RNA pol IIII enrichments.enrichments. Black arrow Ctdp1 locus: Genome browser snapshot depicting RNA pol II enrichments. Black arrow indicates the orientation of gene and numbers on right hand side indicate the observed indicates the orientation of gene and numbers on right hand side indicate the observed stalling index in respective state. stalling index in respective state. SI
1.8525
0.9539
3.2074
Figure 3-figure supplement 4 Fig. S3bFig Ncl Figlocus:S3b 3 b Genome browser snapshot depicting RNA pol II enrichments. Black arrow Ncl locus: Genome browser snapshot depicting RNA pol II enrichments. Black arrow indicatesNCL locus the orientation: Genome browser of gene snapshot and numbers depicting on rightRNA polhand II enrichments. side indicate the observed indicates the orientation of gene and numbers on right hand side indicate the observed stalling index in respective state. Snord is intronic transcript in the Ncl locus. stalling index in respective state. Snord is intronic transcript in the Ncl locus. 2.6238
2.1059
3.0066
Figure 3-figure supplement 5
Genome browser snapshot for Rps24, a representative G0-stalled gene. The read density is normalized per million within each sample and plotted at equal scale. Figure 3-figure supportplement 6 Fig.Fig S3c 3 c Slbp locus: Genome browser snapshot depicting RNA pol II enrichments. Black arrow Slbp SLBP locus: locus Genome : Genome browser browser snapshot snapshot depicting depicting RNARNA polpol IIII enrichments. enrichments. Black arrow indicates the orientation of gene. indicates the orientation of gene. H3K27me3 H3K4me3
35
30
25
20
15
10
5
0 Fgf8 Kit Gas1 Yy1 CTDP1 NCL RPS24 SLBP ZC3 RGS2 TSS TSS TSS TSS TSS TSS Locus positive Locus positive G0 Stalled genes G0 for K27me3 for K4me3 Induced
Figure 3-figure supplement 7 Figure 3-figure supplement 11
G0 stalledG0 stalled genes genes (CTDP1, (CTDP1, NCL.NCL. RPS24,RPS24, SLBP, SLBP, ZC3h12a) ZC3h12a) are markedare marked by by H3K4me3H3K4me3 mark mark but but not not the the H3K27me3H3K27me3 suggesting suggesting that that they they are locus are locuswith open with openchromatin. chromatin. The Positive controls for only H3K4me3 (Gas1 and Yy1 loci) and H3K27me3 (Fgf8 and Kit loci) are shown for reference. 7
6
5 G0
4 R1
R6 3 R12 Rela%ve mRNA levels 2 R24
1
0 YPEL5 MyoD L7
Figure 3-figure supplement 8 Figure 3-figure supplement 7 RelativeRelative mRNA mRNA levels levels for for group group of of genes genes repressed repressed and and not not stalled stalled in inG0 G0 DAPI NCL DAPI RPS24
MB
MT
G0
R 30 min
R 2 hr
Fig.Figure S3d 3-figure supplement 9 Fig.Restoration S3d of expression of G0-stalled genes during cell cycle reactivation. RepresentativeRestoration of expression mergeimmunofluorescence immunofluorescence of G0-stalled images genes images for during MB, for MT,cell MB, G0,cycle MT,G0 R30min reactivation. R30min and R2hrs and R2hrsstained stained for G0 for G0 stalledRepresentative genes(red)genes (red) immunofluorescence and and DAPI DAPI (Blue). (Blue). NCL imagesNcl (left(left for column)column) MB, MT, showsshows G0, distinctR30mindistinct sub-nuclear nucleolarand R2hrs staining stainedpuncta whereasfor(nucleolar) G0 and RPS24stainingstalled genes(Right whereas (red)column) and and Rps24is DAPI cytoplasmic (Blue).(Right column) Nclstaining. (left is column) Forfound both in shows thesethe cytoplasm. distinctG0 stalled sub-nuclear For gene both MB these puncta shows G0 (nucleolar) maximumstalled amountgenesstaining MB of whereas stainingshows maximumand whereas Rps24 bothprotein (Right the expression, column)arrested is state foundwhereas (G0 in andthe the cytoplasm.MT) two showsarrested For reduced states both these(G0levels. and G0 Moreover MT) stalled show G0 hasverygenes lower reduced MB amountshows levels. maximumof Expressionstaining protein which is rapidly inducesexpression, induced rapidly whereas after after reactivation reactivation(R30minthe two arrested (R30min states andand (G0 R2hrs).R2hrs) and MT) show very reduced levels. Expression is rapidly induced after reactivation (R30min and R2hrs). NCL PAX7 DAPI
0 hrs * * *
* * *
* * *
* * * 3hrs * * * * * *
Figure 3-figure supplement 10 Fig.S3e NclFig.S3e staining in satellite stem cells (SC) associated with single myofibers cultured ex vivo. Ncl staining in satellite stem cells (SC) associated with single myofibers cultured ex vivo. SC are marked by Pax7 (arrowhead) can be distinguished from differentiated myonuclei which SC are marked by Pax7 (arrowhead) can be distinguished from differentiated myonuclei which areNCL Pax7 staining negative is observed (MN, asterisk) in satellite within stem the cells underlying (SC) associated myofiber. withAt 0hrs single and myofibers 3hrs post cultured isolation, ex are Pax7 negative (MN, asterisk) within the underlying myofiber. At 0hrs and 3hrs post SCvivo. shows SC marked induced by Ncl Pax7 levels (green, whereas arrowhead) MN shows can littlebe distinguished staining. from differentiated myonuclei isolation,which are SC Pax7 shows negative induced (MN, Ncl asterisk) levels whereas within the MN underlying shows little myofiber. staining. At 0hrs and 3 hrs post isolation, SC shows induced NCL levels whereas MN shows little staining. FigureFigure 3-figure 3-figure supplement supplement 11 10 VennVenn diagram diagram depicting depicting the overlap the overlap of G0 stalledof G0 genesstalled when genes calculated when withcalculated varying the with Promoter varying region the Promoter (+500 bases region or +250 (+500 bases bases ) or +250 bases ) mean +SEMn=3,pvalue<0.05). q-PCR (normalizedtoGAPDHwithinthetreatmentandcorrespondingNTSvalue)(Values represent Extent ofknockdownrespectivemRNA levelsaftersiRNA treatmentinMBandG0estimatedusing Figure 4-figuresupplement1 G0 statecomparedtoitsrespectiveNTScontrol. Extent ofknockdownG0stalledgenesinproliferativeMBandquiescentstate Figure 4-figuresupplement1
Relative mRNA levels 0.00 0.25 0.50 0.75 1.00
NTS
si-Ctdp1 *
si-Ncl *
si-Rps24 * * * si-Slbp
si-Zc3h12A * * MB G0 * RNA EU DAPI
NTS
si-Ctdp1
si-Ncl
si-Rps24
si-Slbp
si-Zc3h12A
Figure 4-figure supplement 2
RNA content, active RNA synthesis and nuclear area altered in proliferating MB after siRNA-mediated knockdown of candidate G0-stalled genes (NTS-non targeting control siRNA, Ctdp1, Ncl, Rps24, Slbp and Zc3h12A). After 36hr, cells were co-stained for total RNA (SYTO® RNASelectTM - green) and EU incorporation following a 30` pulse (red) (scale bar 10µm). a NTS si-Zc3h12A si-Slbp si-Rps24 si-Ncl si-Ctdp1
!!b Students T test (P-value) referenced to NTS1 NTS1 si-Ctdp1 si-Ncl si-Rps24 si-Slbp si-Zc3h12a G0/G1 - 0.058 0.015 0.016 0.734 0.436 S - 0.053 0.053 0.006 0.765 0.128 G2M - 0.520 0.036 0.045 0.997 0.580
Figure 4-figure supplement 3 a. Overlayed DNA content traces plotted for the NTS (red line) treated MB. Knockdown of Ctdp1, Ncl and Rps24 show G1 arrest (green shades), for whereas Slbp and Zc3h12A are unchanged (blue shades). The quantification of proportion of cells in each cell cycle state is represented in Figure 4 b and the significance is tabulated in b and highlighted in yellow. DEAD VIABLE 100
75
50
25 proportion of viable cells (%)
0
NTS si-Ctdp1 si-Ncl si-Rps24 si-Slbp si-Zc3h12a
Figure 4-figure supplement 4
Propidium iodide staining of live cells shows a modest decrease in cell viabity for all the siRNA treated cells maximally Rps24 Un transfected cells p27-mVenus transfected
a
Myoblast Proliferating
G0 Myoblast
b 80
* 60
40
* 20 * Relative mRNA levels : Relative mRNA 0
normalised to NTS treated cells NTS Ctdp1 Ncl Rps24 Slbp Zc312H siRNA treatments Figure 4-figure supplement 5 Figure 4-figure supplement 5 ProportionProportion of ofP27 P27 positive positive cells cellsincrease increase in G0 compared in G0 compared to MB and isto good MB indicatorand is good for quiescence indicator for state. quiescencea. Stably expressing state. P27-Venus (fl-1) plasmid in C2C12 cell line (right side panel), the proportion of P27 a.positive Stably cells expressing is estimated P27-Venusby flow cytometry. (fl-1) The plasmid un- transfected in C2C12 C2C12 cell MBline is (rightused to side set referencepanel), the gates(Leftproportion side panel). of P27 positive cells is estimated by flow cytometry. The un- transfected b. TheC2C12 bar graph MB isbelow used show to setrelative reference levels of gates(LeftmRNA of p27 side marker panel). upon knockdown with G0-stalled b.genes The compared bar graph to NTS below control show during relative proliferative levels growth of mRNA conditions. of p27 Knockdowns markers ofupon Ctdp1, knockdown Ncl, and Rps24with show G0-stalled significant genes induction compared in p27,a marker to NTS of quiescence.control during (* =pvalue proliferative <0.05 Students growth T-test, conditions. N=3) Knockdowns of Ctdp1, Ncl, and Rps24 show significant induction in p27 levels indicating onset of quiescent state in a MB G0 MT
p130
Gapdh
b
1.25 0.0063 0.0008
1
0.75
0.5
0.25 – referenced to respective Gapdh Normalized densitometry levels of p130 0 MB G0 MT
Figure 4-figure supplement 6 P130 levels as marker for quiescence state Western blots (b) for P130 and Gapdh and densitometry quantification (b) shows G0 specific induction of p130. MB and MT show significantly lower level of P130 compared to G0 and numbers indicate the pvalves, Student`s T-test. 100
75 *
50 CFU (%)
25 * *
0 NTS si-Ctdp1 si-Ncl si-Rps24 si-Slbp si-Zc3h12a
Figure 4-figure supplement 7
Colony forming assay for G0 cells treated with siRNA for G40 stalled genes represented as % CFU. Reduced self renewal as estimated by colony forming ability for Ncl, Rps24 & Ctdp1 knockdown but not for Slbp and Zc3h12A knock- down (*p-value <0.05, n=3 student`s T-test) Extent of KD in G0 for regulators of pausing 0.00 NTS si-Aff4 si-Brd4 si-Hexim -1.00
-2.00 levels A
-3.00 * *
-4.00 relative mRN Extent of knock0down- -5.00 * -6.00
Figure 5-figure supplement 1 Extent of knockdown in Aff4, Brd4 and Hexim1 in G0 state compared to NTS control. (* --> pvalue <0.05 Student`s Ttest, N=3) FIG S4 a b
100 DEAD VIABLE 100 75 75 *
50 50 CFU (%) proportion of
viable cells (%) 25 25 * *
0 0
NTS si-Ncl NTS si-Ncl si-Slbp si-Ctdp1 si-Rps24 si-Slbp si-Ctdp1 si-Rps24 si-Zc3h12A si-Zc3h12A
a. b. c d
NTS si-BRD4 NTS si-HEXIM 4 8 * * 3 6
levels * A 2 4 levels A 1 2
0 0 relative mRN
Ncl -2 Ctdp1 Slbp relative mRN Rps24 * Zc3h12A -4 * * Ncl Slbp Ctdp1 Rps24 Zc3h12A Fig. S4a Propidium iodide staining of live cells shows a modest decrease in cell viability for all the siRNA treated cells, maximally for Rps24.
Fig. S4b Colony forming assay for G0 cells treated with siRNA for G0 stalled genes,represented as % CFU. ReducedFigure self renewal 5-figure as supplement estimated 2by colony forming ability for Ncl, Rps24 and Ctdp1 knockdown but not for Slbp and Zc3h12A knockdown (*p-value <0.05, n=3 student`s T-test) Effect of perturbation of RNA Pol II regulators on expression of G0 stalled genes. Fig. S4c,dReduced self renewal as estimated by colony forming ability for Ncl, Rps24 and Effect ofCtdp1 perturbation knockdown. of RNA In Polquiescent II regulators cells (G0), on expression knockdown of ofG0 Brd4 stalled (a) genes.and Hexim1 (b) by In quiescentsiRNA cells treatment (G0), knockdown results in altered of Brd4 but (c) not and for Hexim1 Slbp and (d) Zc3h12Aby siRNA knockdown treatment results (*p-value in altered level of expression<0.05, n=3 ofstudent`s G0 stalled T-test) genes level measured of expression by qRTPCR of G0 stalled (n=3 and genes * =p measured value <0.05 by using studentsqRTPCR t-test). (n=3 and * =p value <0.05 using students t-test)
Nts si-Aff4 70
60
50
40
30
20
% EdU positive cells 10 0
(estimated by horse-shoe plots) R9 R12 R18 R24 Hours after release from quiescence
Figure 5-figure supplement 3 Effect of perturbation of RNA Pol II regulators (Aff4) on cell cycle reentry. The % of EdU positive cells quantified from imaging experiments is observed to be higher in Aff4 knockdown cells at hours R12 and R18 hours after reentry indicative of faster exit from quiescence.