LARGE-SCALE BIOLOGY ARTICLE

Integrative Analysis from the Epigenome to TranslatomeUncovers Patterns of Dominant Nuclear Regulation duringTransient Stress[OPEN]

Travis A. Lee1 and Julia Bailey-Serres2

Center for Plant Cell Biology and Botany and Plant Sciences Department, University of California, Riverside, California 92521

ORCID IDs: 0000-0002-8352-8956 (T.A.L.); 0000-0002-8568-7125 (J.B.-S.).

Gene regulation is a dynamic process involving changes ranging from the remodeling of chromatin to preferential translation.To understand integrated nuclear and cytoplasmic gene regulatory dynamics, we performed a survey spanning theepigenome to translatome of Arabidopsis (Arabidopsis thaliana) seedlings in response to hypoxia and reoxygenation. Thisincluded chromatin assays (examining histones, accessibility, RNA polymerase II [RNAPII], and transcription factor binding)and three RNA assays (nuclear, polyadenylated, and ribosome-associated). Dynamic patterns of nuclear regulationdistinguished stress-induced and growth-associated mRNAs. The rapid upregulation of hypoxia-responsive genetranscripts and their preferential translation were generally accompanied by increased chromatin accessibility, RNAPIIengagement, and reduced Histone 2A.Z association. Hypoxia promoted a progressive upregulation of heat stress transcripts,as evidenced by RNAPII binding and increased nuclear RNA, with polyadenylated RNA levels only elevated after prolongedstress or reoxygenation. Promoters of rapidly versus progressively upregulated genes were enriched for cis-elements ofethylene-responsive and heat shock factor transcription factors, respectively. Genes associated with growth, including manyencoding cytosolic ribosomal proteins, underwent distinct histone modifications, yet retained RNAPII engagement andaccumulated nuclear transcripts during the stress. Upon reaeration, progressively upregulated and growth-associated genetranscripts were rapidly mobilized to ribosomes. Thus, multilevel nuclear regulation of nucleosomes, transcript synthesis,accumulation, and translation tailor transient stress responses.

INTRODUCTION

The regulated expression of protein-coding genes in eukaryotesinvolves processes within the nucleus, including the remodeling ofchromatin, recruitment of RNA polymerase II (RNAPII), cotranscrip-tional processing, and export of mature mRNA to the cytoplasm,where it may be translated, sequestered, or degraded. These pro-cesses are modulated by conditions that necessitate changes inmetabolism and growth to maintain viability. In Arabidopsis(Arabidopsis thaliana), cellular hypoxia promotes the activation ofanaerobic metabolism to enable ATP production for basic cellularprocesses. This involves the rapid activation of transcription of49 hypoxia-responsive genes (HRGs) followed by the active trans-lation of their mRNAs, while most other transcripts are sequesteredfromtranslationcomplexesuntil reaeration (Branco-Priceetal., 2005,2008; Juntawong et al., 2014; Sorenson and Bailey-Serres, 2014).Thewell-translatedHRGmRNAs lack notable sequencesor featuresthat can explain their effective translation and avoidance of

sequestration (Branco-Price et al., 2005; Juntawong et al., 2014;SorensonandBailey-Serres,2014), leading to thehypothesis that theirpreferential translational reflects aspects of their nuclear regulation.Transcriptional activation of many HRGs involves the evolu-

tionarily conserved group VII ethylene response factor (ERFVII)transcription factors (TFs), which are required for survival underhypoxia (Xu et al., 2006; Hattori et al., 2009; Gibbs et al., 2011;Licausi et al., 2011; Yang et al., 2011; Abbas et al., 2015; Paul et al.,2016; Eysholdt-Derzsó and Sauter, 2017; Giuntoli and Perata,2018). The five Arabidopsis ERFVIIs are stabilized under hypoxiadue toattenuationof their oxygen-stimulatedproteolysisvia theArgbranch of the N-degron pathway (Gibbs et al., 2011; Licausi et al.,2011; Paul et al., 2016; White et al., 2017; Millar et al., 2019). Theseinclude three constitutively synthesized ERFVIIs (RELATED TOAPETALA2.2 [RAP2.2], RAP2.3, and RAP2.12) shown to trans-activate HRG promoters in protoplasts through a hypoxia-responsive promoter cis-element (HRPE) identified in ;50%of the HRGs (Giuntoli et al., 2014; Gasch et al., 2016; Giuntoli andPerata, 2018). Two other ERFVIIs, HYPOXIA-RESPONSIVE ERF1and 2 (HRE1/2), are upregulated by hypoxia along with ALCOHOLDEHYDROGENASE1 (ADH1) and PLANT CYSTEINE OXIDASE1/2 (PCO1/2), which facilitate anaerobic metabolism and catalyzeturnover of the ERFVIIs, respectively (Giuntoli et al., 2014, 2017;Weits et al., 2014; White et al., 2017).Transcription requires the binding of TFs to specific cis-

elements typically located near the transcription start site(TSS) to facilitate the assembly of an RNAPII initiation complex.

1 Current address: Plant Biology Laboratory, The Salk Institute forBiological Studies, La Jolla, California 92037.2 Address correspondence to serres@ucr.edu.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Julia Bailey-Serres(serres@ucr.edu).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.19.00463

The Plant Cell, Vol. 31: 2573–2595, November 2019, www.plantcell.org ã 2019 ASPB.

The depletion of nucleosomes near a TSS is controlled byATP-dependent chromatin remodelers and can be influenced byinteractions with the transcriptional apparatus. TF binding andRNAPII activity both influence and are influenced by specificmodifications and variants of histones (Gates et al., 2017; TalbertandHenikoff, 2017),which therebyserveasaproxyofgeneactivity.For example, tri-methylated Histone 3-lysine 27 (H3K27me3) andH3K4me3 are prevalent in the bodies of genes transcribed atlow and high levels, respectively (Asensi-Fabado et al., 2017;Gates et al., 2017). Acetylation of Histone H3 Lys residues(H3K9ac,H3K14ac) reduces interactionsbetweenDNAandhistonesand is associated with ongoing transcription (Gates et al., 2017),including genes actively transcribed during environmentalstress in plants (Eberharter and Becker, 2002; Kim et al., 2008,2012). A characteristic of heat-stress–activated genes of Arabi-dopsis is high levels of the Histone 2A variant H2A.Z at the firstnucleosome within the gene body, a feature proposed to reducethe energy required to commence transcriptional elongation atelevated temperatures (Kumar and Wigge, 2010; Cortijo et al.,2017; Dai et al., 2017; Sura et al., 2017; Torres and Deal, 2019).

As transcription commences, the phosphorylation of specificresidueswithin theheptad repeatsof thecarboxyl terminaldomain(CTD) of RNAPII orchestrates interactions with factors that fa-cilitate transcription-coupled histone modifications as well as thecotranscriptional 59 capping, splicing, and polyadenylation of thenascent transcript (Hajheidari et al., 2013; Milligan et al., 2016).CTD phosphorylation at Ser 2 (Ser2P) demarks active elongation(Phatnani and Greenleaf, 2006). In animals, pausing of RNAPIIdownstream of the TSS is common among genes activated byheat stress (Jonkers and Lis, 2015). Themapping of the 59 ends ofnascent transcripts by Native Elongating Transcript sequencing

indicated that elongation canbe rate-limiting shortly after initiationin Arabidopsis seedlings (Zhu et al., 2018), although this was notdiscernable when nascent transcripts were surveyed by globalnuclear run-on sequencing (Hetzel et al., 2016). Yet, both studiesfound that transcription becomes rate-limited as RNAPII pausesjust beyond the site of cleavage and polyadenylation. After initi-ation, cotranscriptional intron splicing, polyadenylation, and ex-port are regulated by environmental cues including hypoxia(Branco-Price et al., 2008;Mustroph et al., 2009; Juntawonget al.,2014; Niedojadło et al., 2016; van Veen et al., 2016; de Lorenzoet al., 2017).Tobetter understand the integration of nuclear andcytoplasmic

regulation of gene activity in response to hypoxia and reoxyge-nation,weperformedamultiomicstudyofhistonedynamics,openregions of chromatin, HRE2 binding, and RNAPII–Ser2P distri-bution in Arabidopsis. Eight chromatin readouts were comparedwith the modulation of nuclear RNA (nRNA), polyadenylated RNA(polyA RNA), and polyadenylated ribosome-associated RNA. Theintegrated analysis of these data exposed patterns of histonemodifications, dynamic regulation of chromatin accessibility, cis-element enrichment, and temporal regulation of nRNA that ac-companied dynamics in mRNA abundance and translation. Thisanalysisexposedmultiple layersofgene regulation including rapidand complete upregulation of HRGs, progressive upregulation ofheat stress-response genes, and incomplete downregulation ofgenesassociatedwithgrowth, including ribosomalproteins (RPs).Both transcriptional elongation and nuclear export appear to bepointsof regulation in response tooxygenavailability. Thedatasetproduced in this study provides a resource for evaluating epi-genetic state and RNAPII activity through translation in a modelorganism.

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RESULTS

A Multiscale Data Set for Analyzing Regulatory Dynamicsfrom Chromatin to Translation

Here, we generated 11 distinct chromatin and RNA-basedgenome-scale data sets to evaluate gene regulatory dynamicsin 7-day–old Arabidopsis seedlings grown under long-day lightconditions that were treated with normoxia stress (NS; 2 h, 2NS;9 h, 9NS), nonlethal hypoxic stress (HS; 2HS, 9HS), and reaeration(R; 2HS followed by 1 h to 2 h NS, R). All growth and treatmentconditionswere consistentwith thoseofprior analyses (Figure1A;SupplementalFigure1A;Branco-Priceet al., 2008;Mustrophet al.,

2009; Juntawong et al., 2014). Chromatin immunopurification andsequencing (ChIP-seq) was used to survey the position andabundance of H3K9ac, H3K14ac, H3K4me3, H3K27me3, andH2A.Z along genes. The capture of nuclei by Isolation of NucleiTagged in Specific Cell Types (INTACT; Deal and Henikoff, 2010;Maher et al., 2018) was coupled with Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to map re-gions of nucleosome-depleted chromatin (Buenrostro et al.,2013). We also used ChIP-seq to survey regions bound byRNAPII–Ser2P and the hypoxia-induced ERFVII HRE2. Finally, weassayed three transcriptpopulationsdefinedby themethodsusedfor purification: ribosomal RNA (rRNA)-subtracted nuclear tran-scriptomeobtainedby INTACT (Reynosoetal., 2018b),polyARNA

Figure 1. Multiscale Chromatin and RNA Gene Regulatory Analyses of Control (Normoxic) and Hypoxic Arabidopsis Seedlings.

(A) Schematic of the experiments performed. Seven-day–old seedlings were subjected to control (normoxic; 2NS, 9NS), hypoxic stress (2HS, 9HS), orreoxygenation after 2HS (R) conditions. 2NS is ZT16, and 9NS is ZT1, with 1-h extended darkness. Vertical arrows indicate time of harvest. ChIP wasperformed to evaluate genomic regions bound by Ser2P, the Histone 2 variant H2A.Z, modifiedHistone 3 (H3K4me3, H3K27me3, H3K9ac, andH3K14ac),and the ERF TF, HRE2. INTACT purified nuclei were used for the ATAC and purification of nRNA. Ribosome-associated mRNA was obtained by TRAP.(B) Individual replicate samples of nRNA, polyA RNA, and TRAP mRNA [TRAP] were compared by t-SNE.(C) to (E) Distributions of histone modifications/variants across genic regions (C) for the core HRGs (n 5 49; D) and cytosolic RPs (n 5 246; E). Readdistributions (RPKM * 1,000) are plotted from 1 kb upstream to 1 kb downstream of gene units defined by the TSS and TES.

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selected by binding to oligo(dT) beads (transcriptome), and RNAobtained by Translating Ribosome Affinity Purification (TRAP andoligo[dT] selection, translatome; Zanetti et al., 2005; Juntawonget al., 2014). Each RNA population was reproducible and distin-guishable when plotted by t-distributed stochastic neighbor em-bedding (t-SNE; Figures 1B and 1C; Supplemental Figure 1).

Stress- and Growth-Associated Genes Differ in ChromatinAccessibility, Histone, Modifications, and RNA Modulationunder Hypoxia

For the nucleosome-level evaluation, we plotted the histone dataalong each annotated protein-coding gene and compared globalaverages for each condition (Figure 1C; Supplemental Figure 2).H3modificationsassociatedwith transcription (H3K9ac,H3K14ac,and H3K4me3) and stress-activated transcription (H2A.Z) wereenriched just 39 of the TSS and tapered off at the primary siteof polyadenylation, the transcription end site (TES), whereasH3K27me3 was distributed more evenly across gene bodies.Hypoxia had little effect on thesemodifications at the global level,with the exception of a slight elevation of H3K14ac and reductionof H3K4me3 levels near the TSS.

We surveyed the dynamic changes in histone modification,abundance, and distribution for the induced and translatedHRGsand stable but poorly translated cytosolic RP genes. H3K9ac sig-nificantly increased and H2A.Z decreased on HRGs at 2HS, withlimited changes in these marks on RPs (Figures 1D and 1E;Supplemental Figures 1B and 3). H3K4me3 levels were notably re-ducedontheRPs,butnotHRGs, inresponseto2HS.Whenthestresswas extended from2h to 9 h, H3K9ac significantly increased on863and 3,646 genes and H3K14ac on 2 and 1,216 genes after 2HS and9HS, respectively (Supplemental Figure 1). A progressive increase ofH3K14ac but not H3K9ac onRPswas accompanied by little changein steady-state transcript abundance (Supplemental Figures 1C, 3A,and 3C). These data indicate that these two gene cohorts undergodistinct epigenetic regulation in response to hypoxia.

Next, we evaluated chromatin accessibility dynamics in genicregions by ATAC-seq (Figures 2A and 2B). Accessibility in 59promoter regions is associated with nucleosome depletion andthe binding of transcriptional machinery (Lu et al., 2017; Maher,2018; Sijacic et al., 2018), whereas accessibility in 39 flankingregions is associated with the formation of chromatin loops andtranscriptional termination (Ansari and Hampsey, 2005; Ozsolaket al., 2008). The ATAC-seq reads mapped almost exclusivelywithin 1,000 bp 59 of the TSS and;300 bp 39 of the major TES ofgenes under all four conditions (Figure 2C). The average ATACsignal increasedwithin the promoters of bothHRGs andRPs (1.8-and 1.4-fold, respectively) after 2HS, yet ATAC reads showedexpansion intomore59and39flanking regionson theHRGs.Peak-calling of Tn5 hypersensitive insertion sites (THSs) across thegenome identified constitutively present (8,072), non-stress–specific(22,387) and an even larger group of stress-specific THSs (25,795;Figures 2D to 2F; Supplemental Figure 2A), indicating that chromatinaccessibility is readily influenced by the environment.

To complete our survey of gene activity, we compared thedistribution of the log2 fold change (FC; 2HS/2NS) values ofchromatin, RNAPII–Ser2P, and the four RNA populations at thegenome-scale as well as for the HRGs and RPs (Figure 3A;

Supplemental Figure 1A). We found that stress-induced RNA-PII–Ser2P engagement was generally correlated with increasedH3K9ac (global R 5 0.43; HRGs R 5 0.17) and H2A.Z eviction(global R520.27), particularly for the HRGs (R520.79; Figures3B and 3C; Supplemental Figures 3B and 4A). RPs contrasted tothe HRGs, displaying only minor changes in RNAPII–Ser2P as-sociation at 2HS but increased H3K14ac and nRNA levels at 9HS(Figure 3A; Supplemental Figures 3B and 3C). These resultsfurther demonstrate that the epigenetic regulation of stress-induced and growth-associated genes is distinct under hypoxia.

Regulation at the Nuclear and Cytoplasmic Scales Can BeConcordant or Discordant

Next, we evaluated the generally presumed hypothesis thatHRGsare coordinately transcribed and translated under hypoxia. To doso, we performed a global-scale systematic analysis of theRNAPII–Ser2P, nRNA, polyARNA, andTRAPRNAdata sets usingcoregulation and cluster analyses. When we investigated thedynamicsof significantly up-anddownregulatedgenes (up-DRGsanddown-DRGs; jlog2 FCj>1; falsediscovery rate [FDR] < 0.05) inall four readouts of gene activity, we found that nRNA had thegreatest (1,722) and polyA RNA the fewest (602) up-DRGs(Figure 4A). Only 213 genes were significantly upregulated in allfour readouts. These coordinately up-DRGs included 38 of the49 HRGs. A parallel analysis found that 72% of the polyA down-DRGs were significantly downregulated in at least one other dataset, with only 10 coordinately down-DRGs (Figure 4B).Gene transcripts can be nuclear-enriched (Yang et al., 2017),

but there is limited knowledge of the coordination of differentialregulation of nRNA and polyA mRNA populations under stress.Our pairwise comparison of nRNA and polyA RNA abundance at2NSand2HS identified transcripts thatwereenriched ineithernRNAor polyA RNA (Supplemental Figure 5; Supplemental Figure 3).Under both conditions, transcripts enriched in nRNA includedmanyassociated with the cell cycle and gene silencing, whereas tran-scripts enriched in the polyA pool were associated with plastid andribosome biogenesis and function. These data reveal a complexnuclear and cytoplasmic regulatory landscape, which we furtherexploredby integrativeanalysesof thechromatinandRNAdatasets.

Rapid and Coordinate Versus Progressive Upregulation AreCharacterized by Distinct Patterns of Histone Alterations

To better identify genes with similar patterns of regulation, DRGsidentified in at least one of the four data sets (n 5 3,042) wereclustered into 16 groups and evaluated by gene ontology (GO)term enrichment (Figure 4C; Supplemental Figure 4A). Thisanalysis confirmed coordinately up-DRGs (clusters 1 to 3) anddown-DRGs (cluster 16). Genes in the remaining clusters showeddiscordant regulation in one or more readout.To better understand the regulatory variation at the chromatin,

RNAPII, and RNA levels, we plotted the average signal value foreach data type for each cluster along genic regions (Figure 4D;Supplemental Figure 6). This analysis revealed that the rapid andcomplete upregulation of cluster-1 to -3 genes at 2HS was ac-companied by enhanced chromatin accessibility, H2A.Z eviction,

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and H3K9ac and RNAPII–Ser2P elevation across the gene body.Theconcomitant rise inTRAPRNA indicated that these transcriptswere translated in proportion to their increased abundance, asshown for the HRGs by high-resolution ribosome footprint anal-ysis (Juntawong et al., 2014). Not unexpectedly, the first twoclusters were enriched for genes associated with decreasedoxygen (cluster 1, P adjusted < 6.73e222) and general stress(cluster 2, <1.06e221; Supplemental Figure 4A). Genome browserviews ofADH1 andPCO2 illustrate the coordinate upregulation ofHRGs in all four assays of gene activity (Figures 5A and 5B). Theseexamples also illustrate that individual genes undergo distinctchanges in the chromatin and transcript readouts.

Genesof clusters4 to8werenotupregulated to the sameextentfrom RNAPII engagement through translation, indicating they areregulated bymultiplemechanisms that are influenced by hypoxia.For cluster 4, the rise in nRNA levels coincided with increasedH3K9ac and RNAPII–Ser2P, which is indicative of increasedtranscription, but was not accompanied by a concomitant rise inpolyA or TRAP RNA (Figure 4C; Supplemental Figure 6). Thisincomplete upregulation could reflect nuclear retention or cyto-plasmic destabilization of these transcripts. Clusters 6 to 8 geneswere elevated only in nRNA at 2HS, suggesting these transcriptsare nascent or remain in the nucleus. Of these, cluster 8 displayedlittle change in H3K9ac but a rise in H3K14ac as well as nRNA at

Figure 2. Regions of Chromatin Accessibility Determined by ATAC-Seq Are Dynamically Regulated by HS.

(A) and (B) ATAC-seq read distribution of chromatin accessibility for protein-coding genes (A), HRGs and RPs (B).(C) Distribution of THSs on genomic features for each condition.(D) and (E) Volcano plots of the log2 FC in THSs identifiable under two conditions and the significance values of their differences. Genes indicated in pinkmeet the criteria of log2 FC value > j1j, 0.05 < P value.(F) Overlap in the number of THSs identified for each of four conditions.

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2HS and/or 9HS (Supplemental Figures 6 and 7), which is con-sistent with the regulation of themanyRPs enriched in this cluster(Figure 3; Supplemental Figures 3B and 3C). RPL37B is shown asa representative RP (Figure 5D), as its regulation by hypoxia wasfollowed previously (Sorenson and Bailey-Serres, 2014).

Oneof themost distinct gene regulatory patternswas observedfor cluster 9, which showed a dramatic increase in RNAPII–Ser2Pengagement thatwasnot accompanied by a rise in nRNAor polyARNA. This could reflect an increase in RNAPII engagement (initiation)

without the completion of transcript synthesis or elevated rates oftranscription accompaniedbyhigh cytoplasmic turnover.Comparedwith cluster-1 genes, cluster-9geneshad lowerH2A.Zat 2NS,whichwasonly slightly reducedat 2HS (Figure4D;Supplemental Figures6,7A, and 7B). The association of cluster-9 genes with H3K4me3,a mark typical of actively transcribed genes, was significantly lowerthan forcluster-1genesat2HS(SupplementalFigures6,7C,and7D).By 9HS, the promoter accessibility, H3K9ac, H3K14ac, nRNA, andpolyA RNA abundance of cluster-9 genes increased (Figure 4D;Supplemental Figure 8), indicating that they are progressively acti-vated by hypoxia. Cluster-9 genes were enriched for GO terms heat(<6.20e213)andoxidativestress(<8.23e211),whichwerealsoprevalentincluster-2(heat [7.74e214];oxidativestress[<1.13e212]).Cluster2wasdistinguishable fromcluster 9 in terms of inducedSer2P engagement,which produced nRNA and translated RNA.HEAT SHOCKPROTEIN70-4 (HSP70-4), for example, displayed a 3.4-fold increase in RNA-PII–Ser2P across the gene body region accompanied by increasednRNA and TRAP RNA levels but a delayed rise in polyA RNA abun-dance (Figure 5C). The pronounced early engagement of RNAPII–-Ser2P followedbydelayedelevationofpolyARNA levels indicates thatgenes of cluster 9 are progressively upregulated by hypoxia.

Highly Downregulated Genes Undergo DistinctStimulus-Regulated Histone Modifications

Asurveyof thedown-DRGgenes (clusters 10 to16) demonstratedstimulus-driven dynamics of chromatin, RNAPII, and RNAs withtwo dominant patterns: coordinate downregulation (cluster 16)and incomplete downregulation (clusters 10 to 15; Figure 4A).Cluster10 to14geneswereprimarilydistinguishedbyasignificantreduction in nRNA at 2HS that was generally accompanied byincreasedH2A.ZandH3K14acanddecreasedH3K9ac (Figure 4C;Supplemental Figure 6). Cluster-15 and -16 genes stood out asstrongly and coordinately reduced in RNAPII–Ser2P engagementand at least two of the three RNA readouts at 2HS (Figure 4D;Supplemental Figure 6). These clusters had strongly reducedchromatin accessibility, elevation of H2A.Z, and a sharp decline inH3K9ac, all of whichwere antithetical to the changes observed forthe continuously up-DRGs (Supplemental Figures 7A and 7C).Clusters 15 and16were enriched forGOcategories including rootdevelopment (cluster 15, <3.7e28) and regulation of transcriptionand RNA biosynthesis (cluster 16, <7.78e27). Thus, H2A.Zpresence across agenebody, limited chromatin accessibility nearthe TSS, and reduced RNAPII engagement are signatures of re-stricted transcription under hypoxia.

Prolonged Hypoxia Promotes Variations in NuclearRegulation of Growth-Associated Genes

As the seedling’s response to sublethal HS changes over time(Branco-Price et al., 2008), we also evaluated changes in chro-matin accessibility, H3K9ac, H3K14ac, nRNA, and polyA RNA at9HS relative to aerated seedlings that underwent the same lighttreatment and other manipulations (Figures 2A to 2F, 6A, and 6B;Supplemental Figure 9; Supplemental Figure 1A). As observed forshort-term hypoxia, there were differences in chromatin acces-sibility and THSs between 9HS and 9NS treatment. Additional

Figure 3. Short-Term HS Promotes Pronounced Changes in Chromatin,Transcription, andNascent Transcripts that AreNot Uniformly Reflected inTranscript Steady-State Abundance or Translation

Comparison of genome-wide data for chromatin and RNA for 2HS relativeto 2NS.(A) Combined violin and boxplot of log2 FC (2HS/2NS) of histone, RNA-PII–Ser2P, and RNA outputs. Violin plot, all genes; boxplot for the HRGsplotted in orange with ADH1 depicted as a red dot and the cytosolic RPsplotted in blue, with RPL37B depicted as a dark blue dot.(B) Comparison of log2 FC (2HS/2NS) between H2A.Z on the gene bodyand Ser2P on the gene body.(C) Comparison of log2 FC (2HS/2NS) between H3K9ac and Ser2P on thegene body.For (B) and (C), HRGs are plotted in pink and RPs in blue; Pearson’scoefficient of determination is shown for all genes.

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Figure 4. Multiscale Analysis of Hypoxia-Regulated Genes Reveals Concordant and Discordant Patterns of Gene Regulation

Number of (A) up- and (B) down-RGs in response to 2 h of HS (jlog2 FCj > 1; FDR < 0.05) identified in each of the four readouts related to RNA: Ser2P ChIP,nRNA, polyA RNA, and TRAP RNA. Arc of circle circumference indicated by the gray line represents the DRGs in each of the four readouts. Genes

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study is required to determine if the distinction between 2NS and9HS reflects diurnal regulation or an effect of a 1-h extended nightcorresponding to the 9NS time point. Of the 581 THSs observedunder both 2HS and 9HS, 30 were associated with the promoterregions of the 49 HRGs. H3K9ac and H3K14ac levels were pro-gressively yet distinctly altered for the HRG and RP cohorts(Figure 6A; Supplemental Figure 3). Over 350 genes were pro-gressively upregulated, displaying a rise in H3K9ac, nRNA, andpolyA RNA at 2HS and 9HS (clusters 1b to 2b; 38 HRGs). Theremainder were incompletely up- (clusters 4b to 8b) or down-regulated (clusters 9b to 11b), indicating that the regulatorypatterns of genes within these clusters changed as hypoxia wasprolonged. Many genes with elevated nRNA levels at 9HS wereenriched for H3K14ac near their TSS (clusters 2b to 8b; Supple-mental Figure 9), as exemplified by theRPs (enriched in clusters 5band 8b). The increase in nRNA and H3K14ac without an ac-companying rise in polyA RNA suggests that these genes aretranscriptionally active but the nascent transcript remains in thenucleus,or themature (polyA) transcript is rapidlydegraded.Othergenes demonstrated dynamic up- (cluster 6b) or downregulationbetween 2HSand9HS (clusters 8b to 11b). The downregulation at9HSwas pronounced for genes related to photosynthesis (cluster8b, <5.8e27 and<1.5e26), root development (cluster 10b, <1.7e26),and transcription (cluster 11b,<7.6e26; Supplemental Figure4B). Akey finding was that temporal regulation of nuclear and polyAtranscript abundance during hypoxia can be distinct.

A number of genes associated with phasing of the circadianclock were perturbed when hypoxia was prolonged (cluster 6b;circadian rhythm <7.9e210). Upon closer examination, we found thisincluded dampened upregulation of several morning genes and pro-longedelevationofeveninggenemRNAs,whichnormallypeaktowardthe end of the light cycle when the stress was initiated (Figure 6C).Examples of this perturbation include the morning-expressed clockregulators CIRCADIAN CLOCK ASSOCIATED1 (CCA1; cluster 16b)and LATE ELONGATED HYPOCOTYL (cluster 15b), with dampenedmRNA accumulation, and night-expressed TIMING OF CAB EX-PRESSION (TOC1) with elevated polyA RNA levels after 9HS ascomparedwith9NS(Figures6Cand6D).Thus,hypoxia imposedattheend of the light cycle delays or arrests circadian phasing.

Many Coordinately Hypoxia-Upregulated Genes Are Targetsof ERFVII Regulation

We hypothesized that the coordinately up-DRGs might be tran-scriptionally activated by the low-oxygen stabilized ERFVIIs. Motifscansfor theHRPEwithinpromoters (500bp59oftheTSS)acrossthegenome confirmed significant enrichment of this cis-motif in the

cluster1genesresolved in the2HSanalysis (Figure7A;SupplementalFigure 2), including genes with strong coordinate upregulation.TheERFVII HRE2 is encodedbyanHRG. Togain insight into the

role of HRE2, ChIP-seq was performed on 2HS seedlings over-expressing a stabilized version of this protein. Over 75% of the;1,800 promoter bound peaks identified mapped within 1 kbupstream of TSSs, with clear enrichment of binding within 500 bpupstream of the TSS (Supplemental Figure 10; SupplementalFigure2A;SupplementalFile1),asevident in thepromotersofADH1andPCO2 (Figures5Aand5B;SupplementalFigures10Cand10D).The binding of HRE2 to promoter regionswas highest for cluster 1,but also significantly enriched for genes of clusters 2 and 3 of the2HSanalysis (Figure7B).Overall, HRE2peakswere present in 28%of the 213 coordinately up-DRGs (Supplemental Figure 1).We anticipated that HRE2 might be associated with the HRPE

known to be transactivated by RAP2.12, 2.2, and 2.3 (Bui et al.,2015;Gasch et al., 2016). However, de novomotif discovery usingthe HRE2 peak regions yielded an overrepresented multimeric59-GCC-39 element (P value <1e2351; Figure 7C; SupplementalFigure2) that resembles theGCC-richEBPboxboundbyRAP2.12in an in vitro DNA affinity purification and sequencing assay(O’Malley et al., 2016) and by RAP2.3 based on ChIP-quantitative(q)PCR and electrophoretic mobility shift assays (Büttner andSingh, 1997; Gibbs et al., 2014). The HRE2/GCC motif was en-riched in cluster 1 but also was more prevalent than the HRPE inother clusters (Figure 7C). Our data strongly suggest that HRE2binds to promoter regions containing a GCC-rich EBP-like box.Yet, inover50%of the213coordinatelyup-DRGs,anHRPE isalsopresent (Supplemental Figure 1). Collectively, these observationssupport theconclusion thatmanyof thecoordinatelyup-DRGsaretranscriptionally activated by one or more ERFVII.

Hypoxic-Stress Progressively Activates Genes in Heat andOxidative Stress Networks

The regulatory variation of oxidative stress and heat responsegenes displayed by clusters 2 to 3 and 9 genes at 2HS (Figure 4C)was notable. Anoxia upregulates heat-stress associated genes(Banti et al., 2010; Loreti etal., 2005), andboth theonsetofhypoxiaand reoxygenation trigger a reactive oxygen species (ROS) burstassociatedwith signal transduction (Chang et al., 2012). Elevationof RNAPII–Ser2Pwithout a commensurate increase in polyA RNAwas characteristic of HSPs, RESPIRATORY BURST OXIDASE D,and transcriptional co/activators associated with heat and oxi-dative stress (i.e. MULTIPROTEIN BRIDGING FACTOR 1C,DEHYDRATION-RESPONSIVE2A, and ZINC FINGER PROTEIN10and 12; Supplemental Figure 11). We also noted that the transcriptlevels of several HEAT SHOCK FACTOR (HSF) transcriptional

Figure 4. (continued).

differentially regulated in two to four readoutswere tabulated (number inparentheses) anddepictedby linkswithin thecircle.Genesdifferentially regulated inonly one readout are represented by unlinked gray lines.(C) Heatmap showing similarly regulated genes based on four assays of gene activity. Partitioning around medoids clustering of 3,042 DRGs for theidentification of cluster members and centers; 16 clusters. Selected enrichedGO terms are shown on the right (P adjusted < 1.47e-09), jlog2 FCj > 1, FDR <0.05. Clusters displaying increased or decreased translational status (ribosome-associated [TRAP] relative to total abundance [polyA]) are shown.(D) Average signals (RPKM * 1,000) of various chromatin and RNA outputs for genes of three clusters plotted from 1 kb upstream of the TSS to 1 kbdownstream of the TES for the 2 h (2NS, 2HS) and 9 h (9NS, 9HS) time points. Signal scale of the graphs differ.

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activators were elevated in TRAP RNA at 2HS (HSFA2, HSFA4A,andHSFA7A). We considered that the progressive upregulation ofactivity observed for cluster-9 genes may be mediated by HSFsand associated with certain epigenomic features. Indeed, motif-enrichment analysis confirmed that HSF cis-elements (HSEs) weresignificantly enriched in promoters of cluster-9 genes (85% ofgenes) and to a lesser extent in cluster-2 and -3 genes (Figure 7D).Moreover, progressively up-DRGs with a 59 HSE motif were dis-tinguishable from the continuously upregulated HRE2-boundDRGs in four ways (Figure 7F): (1) an overall 59 bias in modifiednucleosomes, (2) more 59 localized H2A.Z with less dramatic

evictionat 2HS, (3) reducedH3K4me3and increasedH3K14ac thatpreceded the rise inH3K9ac, and (4) a delay in the increase inpolyARNAuntil 9HS. Thus, the regulatory variations for progressively up-DRGs with promoter-localized HSEs are distinct from those of thecoordinately up-DRGs bound by HRE2 under hypoxia.

Nuclear-Regulated Stress-Responsive Genes Are HighlyTranslated upon Reaeration

The discovery of genes with RNAPII–Ser2P engagement ac-companied by increased nRNAbut little to no polyA accumulation

Figure 5. Genome Browser View of Normalized Read Coverage of Histone, ATAC-seq, RNAPII–Ser2P, HRE2-ChIP, and RNAOutputs for RepresentativeGenes

(A) to (D) Samples under 2NS and 2HS conditions.(E) to (H) Samples under 9NS and 9HS conditions.(A)and (E)ADH1 (Cluster 1), (B)and (F)PCO2 (Cluster 1), (C)and (G)HSP70-4 (Cluster 2), and (D)and (H)RPL37B (Cluster 9). Themaximum readscale valueused for chromatin-based and RNA-based outputs is equivalent for all genes depicted. At the bottom, the transcription unit is shown in gray with the TSSmarkedwith an arrow.PCO2 shows a stronger decline inH2A.Z thanADH1 in response to hypoxia at 2HS (PCO2,21.15 log2 FC;ADH1,20.33 log2 FC) andprovides an example of elevated H3K4me3 levels under HS.

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Figure 6. Prolonged Hypoxia Leads to Further Variation in Epigenetic, Transcriptional, and Posttranscriptional Regulation.

(A)Combinedviolin andboxplot ofH3K9acandH3K14ac, nRNA, andpolyARNAdata. Violin plots of log2 FC (9HS/9NS) data for all genes.Boxplots aredatafor the 49HRGs plotted in orange with ADH1 depicted as a red dot and data for the cytosolic RPs plotted in blue, with RP37B depicted as a dark blue dot.(B) Heatmap showing similarly regulated genes based on two dynamics in nRNA and polyA RNA. Partitioning around medoids clustering of 3,897 dif-ferentially regulated genes; 16 clusters. Selected enriched GO terms are shown at right (P adjusted < 1.37E-06), jlog2 FCj > 1, FDR < 0.05.(C)Heatmap of chromatin and RNA readouts of select circadian-regulated genes under short and prolonged HS. Heatmap scales between chromatin andRNA-based readouts differ.(D)Brower viewsof amorning (CCA1), evening (CIR1,CIRCADIAN1), andnight (TOC1) expressedgene.RNAscale ofTOC1 is threefold lower than the scaleused for CCA1 and CIR1.

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at 2HS (cluster 6; Figure 4C) suggests that somegenes are poisedfor cytosolic upregulation upon release from oxygen deprivation.By evaluating dynamics in chromatin and RNA upon reaeration,theHRGs,RPs, andheat-responsivegenesagain showeddistinctpatterns of regulation (Figures 8A and 8B; Supplemental Figures12 and 3). The coclustering of all genes differentially expressedunder hypoxia (2HS/2NS) or reaeration (R/2HS) identified cluster

8_R as upregulated in nRNA at 2HS but not in polyA RNA until R(Figures 8A and 8B; Supplemental Figure 12A). These genes wereenriched for GO categories associated with heat (4.55e231), highlight intensity (1.92e233), protein folding (8.91e233), and ROS(9.03e225). Notably,over50%ofcluster8_RgeneshadanHSE.Theexpression patterns of these HSE-containing genes contrasted tothose of the HRGs and RPs, where rapid progression toward

Figure 7. Regulation of Hypoxic Responses by Two ERFVII and HSF TF Families

(A) to (D)Motif and peak enrichment in the promoter regions (1 kb upstream to 500 bpdownstreamof the TSS) of genes in clusters identified in Figure 4C for(A) HRPE, (B) HRE2-ChIP peaks, (C) HRE2-motif, and (D) heat shock elements (HSEs). Frequency of motif and peak occurrence was calculated as thenumber of motifs per number of genes in a cluster. Clusters with significant enrichment were identified by two-tailed Fisher’s exact test P < 0.05.(E) Genome browser view of select HRGs (PCO2; CALMODULIN-LIKE38, CML38; LOB-DOMAIN CONTAINING PROTEIN41, LBD41; ALANINE AMINOTRANSFERASE1, ALAAT1). The locations of HRE2-ChIP peaks and HRPEs are depicted as a horizontal blue box and vertical red line, respectively.(F) Average signal (RPKM * 1,000) of chromatin and RNA readouts for HRE2-bound genes with coordinate upregulation and genes with progressiveupregulation (cluster 9) containing promoter HSEs. Average signal is plotted from 1 kb upstream of the TSS to 1 kb downstream of the TES.

Dynamic Nuclear Gene Regulatory Control by Hypoxia 2583

non-stress conditions was observed in all three RNA readouts,as shown by the genome browser views of five representativegenes (Figures 8B and 8C). The expression pattern demon-strated by HSE-containing genesmay be physiologically relevant,as reaeration is associated with a rapid restoration of proteinsynthesis and a ROS burst. Together, our data demonstratefunctional nuclear segregation of a subset of newly synthesizedRNAsfromcytoplasmic-localized ribosomes in response tooxygenavailability.

DISCUSSION

Gene Activity Dynamics in Response to HS Involves theRegulation of Chromatin, Transcription, andPosttranscriptional Processes

Our genome-wide measurements of chromatin state and in-termediary steps of gene expression in seedlings exposed tosublethal HS and reaeration identified time-dependent chromatin

and transcript dynamicsnot appreciatedby routine transcriptomics.HS modulated the (1) position and degree of open chromatinnear theTSS, (2)modificationofH3 lysines, (3) eviction ofH2A.Z,(4) engagement of RNAPII-Ser2P, and (5) abundance of nuclear,polyadenylated, and ribosome-associated gene transcripts.The results illustrate clear distinctions between steady-statenuclear and polyA transcriptomes, as shown with similar methodsfor rice (Oryza sativa; Reynoso et al., 2018a). The data also identifieddynamics in chromatin accessibility and confirmed translationaldynamics in response to hypoxia and reaeration (Branco-Price et al.,2005, 2008; Mustroph et al., 2009; Juntawong et al., 2014).The monitoring of RNAPII–Ser2P engagement as a proxy for

transcriptional elongation revealed that global levels of tran-scription were not dramatically dampened by 2HS (Figure 1C),although there was considerable regulation of individual genes(Figures 1C and 4C). Remarkably, less than 10%of all DRGswerecoordinatelyup-ordownregulated fromtranscriptional elongationthrough translation at 2HS. Three patterns of discontinuous generegulation were apparent: (1) progressive upregulation, charac-terized by high RNAPII–Ser2P engagement and delayed elevation

Figure 8. Reaeration Promotes the Accumulation and Translation of Polyadenylated Heat-Responsive Transcripts that Are Nucleus-Enriched underHypoxia

(A)Combined violin and boxplot of stress-recovery dynamics of hypoxia-induced nuclear-enriched transcripts. All genes shown as a violin;HRGs shown inorange,withADH1 as a reddot;RPs shown in bluewithRPL37B as ablue dot; heat shock element-containing cluster 8_Rgenes (C8_R, fromSupplementalFigure 12) shown in purple.(B) Genome browser view (RPKM * 1,000) of stress recovery dynamics of HRGs (PCO2, ADH1), heat-responsive genes (HSP70-4, HSP20 like), anda representative RP (RPL37B).(C) Hierarchical clustering of heat shock element-containing cluster 8 R genes in response to hypoxia (2HS/2NS) and reoxygenation (R/2HS).

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of polyA RNA, as observed for heat and oxidative stress genes;(2) incomplete downregulation, characterized by maintainedRNAPII–Ser2Pengagement and increased nRNAabundancewithlimited change in polyA RNA, as observed for RPs; and (3) en-hanced or reduced translational status, measured by comparingTRAP RNA and polyA RNA abundance (Branco-Price et al., 2008;Mustroph et al., 2009; Juntawong et al., 2014). We also identifiedvariations in nuclear regulation (chromatin accessibility, histonemodification, RNAPII engagement, and nRNA abundance) thatforeshadowed changes in polyA RNA abundance and translation.Finally, the evaluationof the three transcript populations after briefreaeration demonstrated a rapid rebound to homeostasis in thenRNA of the continuously up-DRGs, discontinuously up-DRGs,and discontinuously down-DRGs. In summary, these data illuminateremarkable complexity in the transcriptional and posttranscrip-tional regulation of genes associated with stress responses andgrowth.

Coordinate Upregulation of HRGs Is Characterized byHistone Modification, Histone Variant Eviction, andIncreased Chromatin Accessibility

Hypoxia had marked effects on the position, composition, andmodificationsof specifichistonesof nucleosomes. Thegenes thatwere coordinately upregulated from transcription through ribo-someassociation included theHRGs,whicharecritical for survivalunder low-oxygenstress (Mustrophet al., 2009, 2010;Gibbset al.,2011; Giuntoli et al., 2014; Gasch et al., 2016; Giuntoli and Perata,2018). The strong and coordinate upregulation of HRGs wascharacterized by eviction of H2A.Z, a rise in gene body H3K9ac,promoter accessibility, RNAP-Ser2P engagement, and nRNA,polyA, and ribosome-associated mRNA levels (Figure 9). Thecotranscriptional neutralization of a positive charge on the Nterminus of H3K9 by histone acetyltransferases is a reliable sig-nature of active transcription (Eberharter andBecker, 2002) and iscommon for stress-activated genes (Kim et al., 2008; Zhou et al.,2010; Kim et al., 2012). In animals, H3K9ac is promoted byH3K4me3andstimulates the releaseof poisedRNAPII complexes(Jonkers and Lis, 2015). H3K4me3 generally rose on HRGs inresponse to 2HS, but was not as strongly correlated with RNA-PII–Ser2P engagement as H3K9ac (Figure 3B; SupplementalFigure 4D). The stress-activated transcription coincided witheviction ofH2A.Z fromnucleosomes thatwere broadly distributedacross the bodies ofHRGs at 2NS. This observation is consistentwith the finding that H2A.Z eviction from nucleosomes is a pre-requisite for the activation of temperature-responsive genes(Kumar andWigge, 2010; Cortijo et al., 2017; Dai et al., 2017; Suraet al., 2017; Torres and Deal, 2019).

H2A.Z deposition, eviction, and the impact on transcriptionalregulation are complex and not fully understood. Depositionrequires ACTIN-RELATED PROTEIN6, a component of the SWR1remodeling complex, and other factors in Arabidopsis (Asensi-Fabado et al., 2017; Kumar, 2018). Yet disruption of H2A.Z de-position in an actin-related protein6 mutant had limited effect onregulation of HRGs under aerated growth conditions (Torres andDeal, 2019). Other chromatin remodeling proteins or specific TFscould be important. In response to small increases in temperature,displacement of H2A.Z is facilitated by HSF1A during the activation

of the heat response network (Cortijo et al., 2017). H2A.Z evictionfrom HRGs could involve ERFVIIs. Indeed, the hypoxia-stabilizedERFVII RAP2.2 interactswith theSWI–SNF remodeler “BRAHMA”and contributes to H2A.Z eviction associated with increasedchromatin accessibility (Efroni et al., 2013; Vicente et al., 2017).Many of the HRGs displayed increased chromatin accessibility intheir proximal promoter regions and gene bodies.Moreover, 69 ofthe more than 200 coordinately up-DRGs had one or more HRPEwithin 1 kb of their TSS, and many were bound by HRE2 at 2HS(Figure 7E; Supplemental Figure 1). RAP2.12 is stabilized andlocalizes to the nucleus as external oxygen levels decline below10 kPa (Kosmacz et al., 2015). The rise in nuclear levels of ERFVIIsunder hypoxiamay coordinateRNAPII initiation, facilitatingH2A.Zeviction to promote productive elongation coupled with H3K9acetylation. The interaction of ERFVII with the ATP-dependentchromatin remodeling machinery could also contribute to theexpansion of regions of accessible chromatin to facilitate highlevels of transcriptional activation. The uniform upregulation fromtranscription to translation of the up-DRGs with an HRPE- and/orHRE2-bound promoter is reminiscent of the production oftranslationally competent mRNAs during nutrient starvation inyeast (Saccharomyces cerevisiae) that is associated with specificTFs (Zid and O’Shea, 2014). We hypothesize that transcriptionalactivationby theERFVIIs promotes the fidelity of cotranscriptionalprocessing, export, and translation of their targets under hypoxia.

Genes Associated with Heat and Oxidative Stress AreProgressively Upregulated by Hypoxia

HS distinctly activated genes associated with heat and oxidativestress, despite treatment at ambient temperature. The progressiveupregulationof cluster-9genesafter 2HS (Figure 4C;SupplementalFigure 8) became more evident when their activity was comparedat 2HS and 9HS using two chromatin and two RNA readouts(Figure7F). A significant proportionof thesegenes hadHSEswithintheir promoters (Figures 4Cand7D; Supplemental Figures6 and8).Their pattern of regulatory variation included a 59 bias in histonemarks and moderate to very high RNAPII–Ser2P engagement at2HS accompanied by elevated nRNA and TRAP RNA levels, butlimited amounts of full-length polyA RNA (Figure 8).Previously, heat-stress–responsive genes were recognized as

highly induced in Arabidopsis seedlings directly transferred toanoxia (Loreti et al., 2005; Banti et al., 2010). A brief pretreatmentwith heat stress increased the resilience to anoxia of wild-typeseedlings but not loss-of-function hsfa2 or hsf1a hsf1b mutantseedlings (Banti et al., 2008). This is enigmatic because hightemperatures do not typically precede flooding in natural envi-ronments, but the availability of HSPs could reduce cellulardamage under extreme stress conditions such as anoxia. Indeed,HSPs were plentiful in cluster 9 after 2HS. The limited synthesis ofATP-dependent chaperones after brief hypoxia due to nuclearsequestration of their transcripts could minimize demands onlimited energy reserves. However, as the stress is prolonged orupon reaeration, the rapid synthesis of chaperones and proteinsthat provide protection from ROS could aid survival. For example,theupregulationofRESPIRATORYBURSTOXIDASEDcontributesto the survival of hypoxia, submergence, and postsubmergence

Dynamic Nuclear Gene Regulatory Control by Hypoxia 2585

Figure 9. Three Patterns of Dominant Nuclear Regulation in Response to Two Durations of HS and Reaeration

(A) Coordinate upregulation from chromatin to translation of HRGs characterized by hypoxia-triggered H2A.Z eviction and both H3K4me3 and H3K9acelevation. Over half of the genes with this pattern of regulation showed HRE2 binding by ChIP. Transcript abundance and translation declines uponreoxygenation.(B) Progressive upregulation of genes associated with heat and oxidative stress responses characterized by a strong 59 bias in histone marks, hypoxia-triggered H2A.Z eviction, H3K9ac elevation, and H3K4me3 reduction. Many are known targets of HSFs. These genes are transcribed at low levels undernormoxia (nonstress conditions), with evidence of transcriptional activation in response to brief hypoxia (2 h of hypoxia), as shown by elevated RNA-PII–Ser2P binding, nRNA, and TRAP RNA. However, the elevation in polyA RNA was limited until the stress was prolonged (9 h hypoxia). Transcriptabundance increases upon reoxygenation.(C) Compartmentalized downregulation of many genes associated with growth and photosynthesis, including RP genes characterized by maintainedRNAPII–Ser2P and nRNA levels under the stress, reduced H3K9ac, and progressive acquisition of H3K14ac. Transcript abundance and translation arerestored upon reoxygenation. Cartoons illustrate histonemodifications as defined by the key. HSF transcriptional activator; Ser2P, RNAPII with the Ser2Pmodification.(D) Figure key for distinct outputs of gene regulation. Levels of assocation (high, red; low, blue) of HistoneH3modifications (H3K4me3, H3K9ac, H3K14ac)and thehistonevariantH2A.Zaredepicted. Iconsdepictingbindingby the transcription factorsHRE2andHSF,andRNAPII-Ser2P.Accumulationofnulcear,whole-cell, and ribosome associated RNA populations is also depicted.

2586 The Plant Cell

recovery (Baxter-Burrell et al., 2002; Pucciariello et al., 2012;Gonzali et al., 2015; Yeung et al., 2018).

The nucleosome dynamics of cluster-2 and -9 genes after 2HSincluded similar modifications, with notable exceptions (Figures4C and 4D; Supplemental Figures 6 and 11). Genes of bothclusters underwent a loss of H2A.Z near the TSS of the gene bodyin conjunction with increased H3K9ac, but cluster-9 genesshowed a loss of H3K4me3 proximal to their TSS. This was notevident for cluster-2orother coordinately up-DRGs.ThedecreaseinH3K4me3maybean indicationofnonproductiveRNAPII–Ser2Pengagement, as this modification is associated with RNAPIIelongation (Ding et al., 2012; Kwak and Lis, 2013). Intriguingly,therewas significant enrichment of HSEs in the promoters of bothrapidly and progressively upregulated genes (Figure 7D).

We predict that the recruitment of specific TFs together withchromatin-modifying enzymes affects histone or RNAPII CTDmodifications that influence the rate of RNAPII elongation, termi-nation, or posttranscriptional processing, thereby tuning the tem-poral production of stress-induced transcripts. The progressiveupregulation of heat and oxidative stress networks could be me-diatedby theupregulationofTFgenesencodingHSFs (i.e.HSFB2B),ZINC FINGER PROTEIN10, and others during the early hours of thestress. Notably, the level of polyA mRNA accumulation and trans-lation of these mRNAs were enhanced upon reaeration (Figure 8A).Ourdatasetsprovideanopportunity for furthermeta-analysesofheatand oxidative stress gene regulatory networks.

Genes Associated with Major Cellular Processes ContinueTo Be Transcribed During Hypoxia

Our study sheds light on the transcriptional and RNA regulation ofgenes associatedwith growth (Figure 8). Our prior comparisons ofthe transcriptome and translatome determined that hypoxia limitsthe investment of energy into the production of abundant cellularmachinery such as ribosomes. Polyadenylated mRNAs encodingRPs and other abundant proteins are maintained but poorlytranslated under HS (Branco-Price et al., 2008, 2005; Juntawonget al., 2014;SorensonandBailey-Serres, 2014). Thestress-limitedtranslation ofRPsmRNAs is associatedwith their sequestration inOLIGOURIDYLATE BINDING PROTEIN1C (UBP1C)-containingmacromolecular complexes (Sorenson and Bailey-Serres, 2014).Upon reaeration, RPmRNAs rapidly reassociate with polysomes.UBP1C is nucleocytoplasmic (Branco-Price et al., 2008, 2005;Juntawong et al., 2014; Sorenson and Bailey-Serres, 2014). Asthe immunopurification of UBP1C and associated mRNAs doesnot discriminate between nuclear and cytoplasmic UBP1C-associated mRNAs, this study raises the interesting possi-bility that the association of UBP1Cwith transcripts in the nucleuscontributes to their nuclear retention.

Here, we considered on a global scale if RP or other trans-lationally limited mRNAs are synthesized during hypoxia. OurRNAP-Ser2P and nRNA data demonstrate that genes encodingRPs, metabolic enzymes, and some components of the photo-synthetic apparatus are synthesized and retained as partially orcompletely processed transcripts in the nucleus during hypoxia(Figure 6), a situation readily reversed by reaeration (Figure 8A).The finding that housekeeping gene transcription can continue isconsistent with results of traditional nuclear run-on transcription

assays performed on fully submerged maize (Zea mays; Fennoyet al., 1998). The transcriptionally active genes showed pro-gressive increases in H3K14ac but not H3K9ac between 2HS and9HS. The limited increase in their polyA RNAs could be due tolimited cleavage and polyadenylation or increased turnover.Similar uncouplingof theH3K9acandH3K14acmarks associatedwith active transcription but limited polyA RNA accumulation wasreported for promoters deemed inactive but primed for futureactivation in pluripotent embryonic stem cells of mice (Karmodiyaet al., 2012). The incomplete downregulation of housekeepinggenes may poise cells for the observed rapid recovery uponreaeration.Our genome-scale analysis demonstrated that transcripts were

retained within the nucleus during hypoxia, which is consistentwith the results of visualization studies of specific mRNAs inArabidopsis and lupine (Niedojadło et al., 2016). mRNAs asso-ciated with the cell cycle are reportedly enriched in the nucleusunder control conditions in Arabidopsis (Yang et al., 2017). Thiswas confirmed in our comparison of the nRNA and polyARNA transcriptomes under both control and hypoxia stress(Supplemental Figure 5). These data support a scenario whereinthe nuclear compartmentalization of abundant transcripts con-tributes to energy conservation by limiting translation, a mecha-nism similar to the cytoplasmic sequestration associated with theRNA binding protein UBP1C. Nuclear retention and cytoplasmicsequestration could provide two reservoirs of transcripts that canbedeployed upon reaeration, enabling rapid restoration of cellularprocesses. Indeed, our data provide evidence that nuclear-retained transcripts increase in translation status upon rea-eration (Figure 8A).Thus, the comparative monitoring of nRNA, polyA, and TRAP

RNA populations strongly support the conclusion that the nuclearexport and subsequent accumulation and translation ofmRNAs isdynamically regulated by stress in plants. Factors such aschromatin accessibility, RNAPII elongation and termination, andhistone modification (i.e. H3K14ac) could contribute to the dy-namics in nuclear transcript abundance. Circumstantial evidenceofalteredcotranscriptional processing includeshypoxia-promotedintron retention (Juntawong et al., 2014) and alternative poly-adenylation site selection (de Lorenzo et al., 2017). Although thereis no clear evidence of a general disruption of splicing or poly-adenylation during hypoxia (Koroleva et al., 2009; Juntawonget al., 2014; de Lorenzo et al., 2017), these processes could bealtered by changes in chromatin, RNAPII elongation, or associ-ation with certain proteins. The RNA helicase eIF4AIII (Korolevaet al., 2009) and several RNA binding proteins (UBP1A/B/C andCML38) form macromolecular complexes that are present in thenucleus but are primarily cytoplasmic under hypoxia (Sorensonand Bailey-Serres, 2014; Lokdarshi et al., 2016). Further experi-mentation is required to better understand the controlled dy-namics in transcript synthesis, processing, and export duringhypoxia stress.

An ERFVII Hierarchy Drives Transcriptional Activation inResponse to Hypoxia

In this study, we performed a genome-wide analysis of the cis-element targets of an ERFVII. ChIP-seq of the hypoxia-induced

Dynamic Nuclear Gene Regulatory Control by Hypoxia 2587

ERFVII HRE2 identifiedamultimeric 59-GCC-39cis-element that isdistinct from the HRPE that is enriched in HRG promoters andactivated by ERFVIIs (Gasch et al., 2016). This is consistent withthe observation that neither HRE1 nor HRE2 interacted with a33-bppromoter fragment containing theHRPE in aYeast-1hybridassay or provided significant transactivation via the HRPE inArabidopsis protoplasts (Gasch et al., 2016). The HRE2 bindingmotif strongly resembles thedoubleGCCmotifof theEBPbox thatwas first defined for an ERF in tobacco (Nicotiana tabacum; Satoet al., 1996). Previously, RAP2.3 was shown to bind to the AB-SCISIC ACID INSENSITIVE5 promoter in a region with two EBPboxes; transactivation of the ABSCISIC ACID INSENSITIVE5promoter in protoplasts by all three constitutively expressedERFVIIs (RAP2.2/2.3/2.12) was dependent on the EBP box(Gibbs et al., 2014).

Collectively, our observations support earlier conclusions thatmembers of the two ERFVII subgroups are not functionally re-dundant (Gibbs, 2014; Gasch et al., 2016). Yet, the situation re-mains enigmatic. The HRPE contains a sequence resemblinga double 59-GCC-39. Promoter regions with high scores for theHRPE andHRE2motif coincided in 40 of the 213 coordinately up-DRGs, 13 of which were bound by HRE2 (Supplemental Figure 1;Supplemental File 1). Moreover, the Arabidopsis ADH1 promoterregion contains both of these motifs and is sufficient for hypoxicupregulation (Gaschetal., 2016). Thecurrentmodel is that the low-oxygen–mediated stabilization of the constitutively expressedRAP-typeERFVIIs is responsible for rapid transactivation ofHRGsincluding HRE2 (van Dongen and Licausi, 2015; van Dongen andLicausi, 2015Voesenek and Bailey-Serres, 2013), which is syn-thesized and stabilized under hypoxia (Gibbs et al., 2011). Wepropose that HRE2 production and binding to promoters via themultimeric GCC element (HRE2 motif) is important for the con-tinuedupregulationofHRGs, as anhre1he2doublemutant fails tomaintain the upregulation of these genes after 4 h of anoxia(Licausi et al., 2010). This process may also enable cells to cir-cumvent the inhibition of RAP2.12 activity by its interaction withthe HYPOXIA RESPONSE ATTENUATOR (Giuntoli et al., 2014).Further analyses are required to comprehend the independent oroverlapping roles of the RAP-type and HRE ERFVIIs in the tem-poral activation of transcription during hypoxia.

Mammals regulate transcriptional activity in response to hyp-oxia in a manner remarkably analogous to that of Arabidopsis(Watson et al., 2010). HRGs are activated by the two-subunitHypoxia Inducible Factor (HIF) TF complex, which includes theoxygen-destabilized HIF1A subunit (LaGory and Giaccia, 2016).Similar to the three RAP-ERFVIIs of Arabidopsis, HIF1A isa constitutively synthesized protein that is destabilized in thepresence of oxygen. A group of prolylhydroxlases catalyze theoxygen-dependent hydroxylation of specific residues of HIF1Athat triggers itsubiquitylationandproteasome-mediated turnover.In animals, the chromatin landscape is also regulated throughregional accessibility (Buck et al., 2014) and the posttranslationalmodification of histones (Chervona and Costa, 2012) in anoxygen-sensitive manner. HIF signaling itself directly facilitateshistone modification of target genes through interactions withchromatin-modifying enzymes (Watson et al., 2010); severalhistone-modifying enzymes are directly targeted by HIF tran-scriptional activation, and HIF1A expression itself is regulated by

histone acetylation (Kim et al., 2007). Moreover, specific histone-modifying enzymes function asdirect oxygen sensors through themediation of their activity based on cellular oxygen levels in an-imals and likely plants (Gibbs et al., 2018; Batie et al., 2019;Chakraborty et al., 2019), confirming the integration of oxygensensing with epigenetic regulation. The low-oxygen–stabilizedtranscriptional regulators and the coordination of their functionwith chromatin modifications in Arabidopsis is evidence of con-vergence in gene regulation in response to hypoxia in plants andanimals. This may extend beyond transcriptional regulation, ascytoplasmic sequestration andselective translationofmRNAsarealso observed in plants and metazoa (Blais et al., 2004; Branco-Price et al., 2008; Young et al., 2008; Juntawong et al., 2014). Itremains to be explored if hypoxia influences the synthesis andaccumulation of nascent transcripts associated with growthin animals, as demonstrated in this multiomic analysis ofArabidopsis.This multilevel investigation into gene regulatory processes

revealed unexpectedly complex regulation of gene activity withinthe nucleus and cytoplasm that fine-tunes cellular and physio-logical acclimation to transient hypoxia, providing a model fora response to an acute stress. A key finding is that there are rapidchanges in the epigenome in response to short-term HS thatcontinue as the stress becomes more severe. Coordinate upre-gulation from chromatin accessibility to translation was observedfor over 200 HRGs. Extreme stress-responsive and growth-associated genes showed more discontinuous upregulation ofnascent transcript production, export to the cytoplasm, and re-cruitment to ribosomes. Each of these broadly defined genecohorts showed a distinct pattern of epigenetic, nuclear, andcytoplasmic transcript regulation upon reaeration. Future inves-tigations can consider roles of specific TF and their interactionswith RNAPII, transcriptional coactivators, the chromatin land-scape, and the posttranscriptional apparatus in directing thenuanced nuclear regulation uncovered in this study.

METHODS

Genetic Material

The following Arabidopsis (Arabidopsis thaliana) genotypes were used forgenome-wide experiments: Col-0, HistonemodificationChIP-seq, RNAPIIChIP-seq; pHTA11:HTA11-3xFLAG (Cortijo et al., 2017), H2A.Z ChIP-seq;pUBQ10:NTF/pACT2:BirA (Sullivan et al., 2014), ATAC-seq, nRNA-seq,polyA mRNA-seq; p35S:HF-RPL18 (Zanetti et al., 2005), TRAP se-quencing, mRNA-seq; and HRE ChIP-seq p35S:C2A-HRE2-HA (Gibbset al., 2011).

Plant Growth and Treatment

Arabidopsis seeds were sterilized by incubation in 70% (v/v) ethanol for5min, followed by incubation in 20% (v/v) bleach and 0.01% (v/v) TWEEN-20, and washed three times in double distilled water for 5 min, in triplicate.Sterilized seeds were placed onto 13 Murashige & Skoog medium (1.03MSsalts, 0.4% [w/v]Phytagel; Sigma-Aldrich), and1%[w/v]Suc, pH5.7) in9-cm2 Petri dishes and stratified by incubation at 4°C for 3 d in completedarkness. After stratification, theplateswereplaced vertically into agrowthchamber (Percival) with a 16-h light/8-h dark cycle at ;120 mmol photo-ns$s21$m-2 (cat. no. 21770; Sylvania), at 23°C for 7 d. For HS, seedlingswere removed from the growth chamber at Zeitgeber time (ZT) 16 and

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subjected to HS by bubbling argon gas into sealed chambers in completedarkness for 2 or 9 h at 24°C. The chamber setup was as described byBranco-Price et al. (2008). Oxygen partial pressure in the chamber wasmeasured with a NeoFox Sport O2 sensor and probe (Ocean Optics).Hypoxia ([O2] < 2%) was attained within 15 min of initiation of the stress.Control samples were placed in an identical chamber that was open toambient air under the same light and temperature conditions. For reaer-ation, seedlings were identically subjected to HS for 2 h and subsequentlyplaced into the control chamber for 1h (nRNAandpolyA) or 2 h (TRAP) afterthe stress. Tissuewas rapidly harvested into liquidN2 andstored at280°C.

ChIP

Seedlings were grown on Petri dishes and treated as described in thesection "Plant Growth and Treatment". For the HRE2 ChIP experiments,seedlings were pretreated by flooding with 10 mL of 100 mM of Calpaininhibitor IV (American Peptide) and 1% (v/v) DMSO for 2 h before thehypoxia treatment. For all genotypes, after control or hypoxia treatment,seedlings were immediately cross-linked in 1% (v/v) formaldehyde nucleipurification buffer (NPB: 20 mM of 3-(N-Morpholino)propanesulfonic acidat pH 7.0, 40 mM of NaCl, 90 mM of KCl, 2 mM of EDTA, and 0.5 mM ofEGTA) in a vacuum chamber under house vacuum for 10min. The reactionwas quenched by adding 5M of Gly to reach a concentration of 125 mM,followedbyvacuum infiltration for5min.Thiswas followedby threewashesin 30 mL of double distilled water. The seedlings were blotted dry andpulverizedunder liquidN2. To isolate nuclei, 0.5-g tissuewas thawed to4°Cin 10mLofNPBcontaining0.5mMof spermidine, 0.2mMof spermine, and13PlantProtease InhibitorCocktail (cat.no.P9599;Sigma-Aldrich).Nucleiwere pelleted by centrifugation in 4°C at 1200g for 10min. The nuclei werewashed in 10mLofNPBand0.1% (v/v) TritonX-100 (NPBt) andpelletedbycentrifugation in 4°C at 1200g for 10 min in triplicate. After the final NPBtwash, the supernatant was aspirated and the nuclei pellet was re-suspended in 120 mL of NPB and lysed with the addition of 120 mL of 23nuclei lysis buffer (100mMof Tris at pH8.0, 20mMofEDTA, 2% [w/v] SDS,and23PlantProtease InhibitorCocktail) byvortexing for2minat24°C.Thechromatin was sheared into 200- to 600-bp fragments by sonication(Diagenode)with 33cyclesof 30sONand30sOFFat 4°C. Thesamplewascleared by centrifugation at 16,000g at 4°C for 2 min and the supernatantwasdilutedfivefoldwithdilutionbuffer (16.7mMofTrisatpH8.0,167mMofNaCl, 1.1% [v/v] Triton X-100, and 1.2 mM of EDTA). The entire chromatinfraction was precleared by incubation with uncoupled Protein G Dyna-beads (Thermo Fisher Scientific) for 30 min, followed by collection of thesupernatant. Three-hundred microliters of the input chromatin (1 mL forHRE2) was incubated with 3 mL of undiluted anti-H3K4me3 (cat. no.ab8580; Abcam), anti-H3K27me3 (cat. no. 07-449; EMD Millipore), anti-H3K9ac (cat. no. ab4441; Abcam), anti-H3K14ac (cat. no. ab52946; Ab-cam), anti-p-CTD RNAPII (cat. no. ab5095; Abcam), anti-FLAG (cat.no. F1804; Sigma-Aldrich; 1:100 dilution), or anti-HA (cat. no. h3663;Sigma-Aldrich; 1:333 dilution) antibodies for 4 h (overnight for HRE2) whilerockingat 4°C.ProteinGDynabeads (30mL)werewashed indilutionbuffer,added to the chromatin fraction and allowed to incubate for 2 h whilerocking at 4°C. Beads were magnetically captured and washed sequen-tially in 1mL for 5minwith four buffers: lowNaClwashbuffer (20mMof Trisat pH 8.0, 150 mM of NaCl, 0.1% [w/v] SDS, 1% [v/v] Triton X-100, and2mMof EDTA), high NaCl wash buffer (20mMof Tris at pH 8.0, 500mMofNaCl, 0.1% [w/v]SDS, 1% [v/v] TritonX-100, and2mMofEDTA), LiClwashbuffer (10 mM of Tris at pH 8.0, 250 mM of LiCl, 1% [w/v] sodium deox-ycholate, 1% [v/v] Nonidet P-40, and 1 mM of EDTA), and standard TEbuffer (10 mM of Tris at pH 8.0, and 1 mM of EDTA). The beads were thenwashed twice with 10 mM of Tris at pH 8.0, and resuspended in 25 mL oftagmentation reactionmix (10mMofTri at pH8.0, 5mMofMgCl2, and10%[v/v] dimethylformamide) containing 1 mL of Tagment DNA Enzyme (Illu-mina) and incubated at 37°C for 1 min. Beads were washed twice with low

NaCl wash buffer and then once in standard TE buffer. The chromatin waseluted from the beads by heating for 15 min at 65°C in elution buffer(100 mM of NaHCO3 and 1% [w/v] SDS) and reverse cross-linked by theaddition of 20mL of 5-MNaCl with heating at 65°C overnight. After reversecross-linking, Proteinase K (0.8 units; New England Biolabs) was addedand the sample was incubated at 55°C for 15 min. The final tagged ChIP-DNA sample was purified using MinElute columns (Qiagen) according tothe manufacturer’s instructions and eluted with 14 mL of EB.

ChIP-seq Library Preparation

Library preparation for short-read sequencing (ChIP-seq) with tagmen-tation was performed according to Schmidl et al. (2015), with minormodifications. Final library enrichment was performed in a 50-mL reactioncontaining 12 mL of ChIP DNA, 0.75-mM primers, and 25 mL 23 NEBNextPCRMasterMix. Todetermine theappropriate amplification cyclenumber,aqPCRwasperformedon1mLof tagmentedChIPDNA ina10-mL reactionvolume containing 0.15-mM primers, 13 SYBR Green (Thermo FisherScientific), and 15 mL of 23 NEBNext PCR Master Mix (New EnglandBiolabs)with the followingprogram: 72°C for 5min, 98°C for 30 s, 24cyclesof 98°C for 10 s, 63°C for 30 s, 72°C for 30 s, and a final elongation at 72°Cfor 1 min. Libraries were amplified for n cycles, where n is equal to therounded-up Cq value determined in the qPCR. Amplified libraries werepurified and size-selected using SPRI AMPure XP beads (Beckman).AMPure XP beads were added at a 0.7:1.0 bead/sample ratio, and theremaining DNA was recovered by the addition of AMPure XP beads ata 2.0:1.0 bead/sample and eluted in 13 mL of EB. Quantification of the finallibraries was performed with Quant-iT PicoGreen (Thermo Fisher Scien-tific), and library fragment distribution was evaluated with the modelno. 2100 Bioanalyzer (Agilent Technologies) using the high sensitivityDNA chip. Final libraries were multiplexed to >5-nM final concentrationand sequenced on the HiSeq 3000/4000 at the UC Davis DNATechnologies Core.

Isolation of Nuclei Tagged in Specific Cell Types

INTACT was performed according to Deal and Henikoff (2010) withmodifications. Frozen pulverized tissue (0.5 g) of pUBQ:NTF/pACT2:BirAseedlings was thawed in 10 mL of cold NPB buffer and filtered through30-mm filters (Sysmex). Nuclei were pelleted by centrifugation at 1200g for7 min at 4°C. The nuclear pellet was resuspended and washed twice inNPBt and resuspended in 1 mL of NPB. M280 Streptavidin Dynabeads(10 mL; Invitrogen) washed with 1 mL of NPB and resuspended in theoriginal volume were added to the nuclei, and the sample was allowed torock for 30 min at 4°C. The bead-nuclei mixture was diluted to 14 mL withNPBt and incubated with rocking for 30 s at 4°C. The beads were mag-netically collected, the supernatant was removed, and the beads washedtwice with NPBt and resuspended in 1 mL of NPBt. A 25-mL fraction wasremoved to quantify nuclei by adding 1 mL of 1.0 mg/mL Propidium Iodide,followed by incubation on ice for 5 min before visualization and quantifi-cation with a C-Chip Hemocytometer (Incyto) under a fluorescencemicroscope.

ATAC

Guided by Maher et al. (2018), 50,000 INTACT-purified nuclei from roottissueweremagnetically captured. Thesupernatantwasaspirated andanyremainingNPBwas removed, and the nuclei were resuspended in 50mL oftransposition mix (13 TD buffer, 2.5 mL of TDE1 transposase) and in-cubated for 30 min at 37°C with occasional mixing. The transposed DNAwas purified with MinElute PCR purification columns (Qiagen), and thepurified DNAwas eluted in 11mL of EB. For library construction, a reactionwas prepared to amplify the sample via a two-step process. The reactionwas assembled containing transposed DNA (10 mL), 5 mM of Ad1.1, and

Dynamic Nuclear Gene Regulatory Control by Hypoxia 2589

an indexingprimer (Buenrostroet al., 2013), 13NEBNextHighFidelityPCRmix (New England Biolabs), cycled in the following program: 72°C 5 min;98°C30s;3cyclesof98°C10s, 63°C30s,72°C1min; 4°Cand thesampleswere placed on ice. Then a qPCR was performed using an aliquot of theamplified librarywith the followingprogram:98°C for30s;20cyclesof98°Cfor 10 s, 63°C for 30 s, 72°C for 1 min. The original PCR was resumed forn additional cycles, where n is the cycle at which the reaction was at 1/3 ofits maximum fluorescence intensity. Amplified DNA was then purified withMinElute PCR purification columns (Qiagen) and eluted in 20 mL of EB.Amplified libraries were purified and size-selected for fragments between200 bp and 600 bp using SPRI AMPure XP beads (Beckman). AMPure XPbeadswere added at a 0.55:1.0 bead/sample ratio, and the remainingDNAwas recovered by the addition of AMPure XP beads at a 1.55:1.0 bead/sample and eluted in 13 mL of EB. Library concentration was determinedusing a NEBNext (New England Biolabs) library quantification kit. Finallibraries were multiplexed to >5 nM final concentration and sequenced onthe HiSeq 3000/4000 at the UC Davis DNA Technologies Core.

INTACT Followed by nRNA Extraction

Guided by Reynoso et al., (2018a) and (2018b), 50,000 INTACT-purifiednuclei were collectedmagnetically, and the supernatant was removed andresuspended in 20 mL of NPB. nRNA was extracted and purified usinga RNeasy Micro kit (Qiagen) and eluted in 20 mL of water. The RNA wasDnaseI-digested with 1 mL (2U/mL) of Turbo DNaseI (Thermo Fisher Sci-entific) and incubated for 30min at 37°C.DNaseIwas inactivatedbyaddingEDTA to 15 mM, and heated at 75°C for 10 min, centrifuged at 2000g for5 min, and transferred to a new tube. The RNA was purified using AMPureXP beads and eluted in 15 mL of water. The rRNA was depleted from thesample using a plant Ribo-Zero rRNA Removal Kit (Illumina) according tothe manufacturer’s instructions.

TRAP Sequencing

TRAP of mRNA-ribosome complexes was performed following the pro-cedure of Mustroph et al. (2009). Briefly, pulverized tissue (1 mL) from the35S:HF-RPL18genotypewas added to 5mLofpolysomeextractionbuffer(200mMofTris at pH9.0, 200mMofKCl, 25mMofEGTA, 35mMofMgCl2,1% [w/v] PTE, 1mMof DTT, 1mM of PMSF, 100 mg/mL of cycloheximide,and 50 mg/mL of chloramphenicol) containing 1% (v/v) detergent mix(20%[w/v]polyoxyethylene[23]lauryl ether, 20% [v/v] TritonX-100,20%[v/v]Octylphenyl-polyethylene glycol, and 20% [v/v] Polyoxyethylene sorbitanmonolaurate 20) and homogenized with a glass homogenizer on ice. Thehomogenizedmixturewasallowed tostand for 10minon iceandcentrifugedat 16,000g at 4°C for 15 min. After centrifugation, the supernatant wastransferred toanewtubeandfiltered throughone layerofMiracloth (Millipore)to produce the clarified extract.

ProteinGDynabeads (50mL; ThermoFisher Scientific)were prewashedtwicewith1.5mLofwashbuffer-T (WB-T;200mMofTris atpH9.0, 200mMof KCl, 25 mM of EGTA, 35 mM of MgCl2, 5 mM of PMSF, 50 mg/mL ofcycloheximide, and 50mg/mL of chloramphenicol) and resuspended in theoriginal volume. The beads were added to the clarified tissue extract andincubated at 4°C for 2 h with gentle rocking. The beads were collectedmagnetically, the supernatant was removed, and the beads were gentlyresuspended in 6mL ofWB-T for 5min at 4°Cwith rocking. This wash stepwas repeated two additional times, after which the beads were re-suspended in 1 mL of WB-T and transferred to a new tube and the su-pernatant was removed.

RNA was purified from the sample and the reserved clarified extract byadding 105mL of lysis/binding buffer (100mMof Tris at pH 8.0, 500mM ofLiCl, 10 mM of EDTA, 1% [w/v] SDS, 5 mM of DTT, 1.5% [v/v] Antifoam A,and 5 mL/mL 2-mercaptoethanol), followed by vortexing for 5 min. Thesamples were incubated at room temperature for 10 min, centrifuged at

13,000g for 10min, and transferred toanewtube.PolyARNAselectionwasperformed by adding 1 mL of 12.5-mM biotin-20nt-dT oligos (IntegratedDNA Technologies) to 200 mL of the TRAP sample, followed by incubationat room temperature for 10 min. In a separate tube, 20 mL of magneticstreptavidin beads (New England Biolabs) were washed with 200 mL oflysis/binding buffer. The lysate was added to the washed beads and in-cubated at room temperature for 10 min with gentle agitation. The beadswere magnetically separated and washed sequentially with wash buffer A(10 mM of Tris at pH 8.0, 150 mM of LiCl, 1 mM of EDTA, and 0.1% [w/v]SDS), wash buffer B (10mM of Tris at pH 8.0, 150mM of LiCl, and 1mM ofEDTA), and low salt buffer (20 mM of Tris at pH 8.0, 150 mM of NaCl, and1 mM of EDTA). After the washes, the pellet was resuspended in 16 mL of10mMTris at pH8.0 containing 1mMof 2-Mercaptoethanol and heated at80°C for 2 min. The beads were magnetically collected, the supernatantwas transferred to a new tube, the polyA1 RNA selection process wasrepeated, and the samples combined in a new tube before storageat 280°C.

Nuclear, PolyA, and TRAP-RNA Isolation and Library Construction

TotalRNAwasextracted from50mgof frozenpulverized tissueusingTrizol(Life Sciences), treated withDNaseI, purified using AMPure XP beads, andeluted in 50 mL of water and polyA selected. PolyA RNA selection wasperformed using Oligo (dT)25 Dynabeads by two rounds of magneticcapture (Townsley et al., 2015). TRAP of mRNA-ribosome complexes wasperformed following the procedure of Mustroph et al. (2009). TRAP RNAextraction (Townsley et al., 2015) was performedwithminormodifications,and polyA RNA was selected as described by Townsley et al (2015). RNA-seq library preparation for nRNA, polyA, and TRAP RNA was performedaccording to Kumar et al. (2012).

Bioinformatic Analyses

All libraries were sequenced in excess of 5million reads per sample. For allhigh-throughput outputs, short reads were trimmed using FASTX-Toolkit(Hannon Lab) to remove barcodes, and short and low-quality reads werefiltered before alignment to the TAIR10 genome with the programsBowTie2/TopHat2 (Langmead and Salzberg, 2012; Kim et al., 2013). Readquality reports were generated using the software “FastQC” (BabrahamBioinformatics). For all data setsexcludingATAC-seq andHRE2ChIP-seq,counting of aligned reads was performed on entire transcripts (Lawrenceet al., 2013) using the latest Araport11 annotation (201606; Cheng et al.,2017), and reads per kilobase of transcript per million mapped reads(RPKM) values were calculated. Differentially expressed genes were de-terminedby “edgeR,” jFCj>1,FDR<0.01. TRAPdatawere not normalizedto total cellular polysome levels as in one of our prior analyses (Branco-Price et al., 2008, 2005).

Data Visualization and File Generation

“Bigwig” software files for Integrated Genome Viewer (Thorvaldsdottiret al., 2012) visualization were generated from aligned “bam” files andnormalized by RPKM values using the “deepTools” command “bamCo-verage” with the “-normalizeUsingRPKM” specification, with the Arabi-dopsisBlacklistedChromatin regions (SupplementalFigure5;SupplementalFile 2) masked. Within the Integrated Genome Viewer, all chromatin-basedoutputs were normalized to the same scale and all RNA outputs werenormalized to a separate scale.

Peak Calling

For ChIP-seq and ATAC-seq data sets, peak calling was performed usingindependent replicates in combination as input for the program “HOMER”using the “findPeaks” command with the specification “-gsize 1.118e8”

2590 The Plant Cell

(Heinz et al., 2010). For downstream analyses, peaks identified from eachtime point comparison were combined and counting was performed onindividual replicates. Peaks were annotated using the HOMER command“annotatePeaks.pl.” Differential regulation of peaks was performed usingthe software “edgeR.”

Motif Discovery

HRE2 peaks identified by HOMER command “findPeaks” were used asinput for the HOMER command “findMotifsGenome.pl.” Thematrix for thetop HRE2 motif (P < 1e-69) was used as input the MEME command“ceqlogo” to generate the motif logo.

DNA Affinity Purification and Sequencing Motif Analysis

Sequences of promoter regions spanning 1 kb upstream to 500 bpdownstream of the TSS were extracted for genes within each cluster. Thepromoter sequenceswere compared against each TF, per TF family, usingmotifmatrices identifiedbyO’Malley et al. (2016). Thenumber of significantmotifs identifiedwithin the promoters of each cluster were quantitated andnormalized to the number of genes within each cluster.

t-SNE

For t-SNE dimensionality reduction, a principal component analysis wasperformed using RPKM values for each replicated data set and time point.The values for principal components 1 to 10 were then used as input fort-SNE dimensionality reduction, and two-dimensional output was plottedwith the software “ggplot2.”

Wilcoxon Signed-Rank Test

AWilcoxon signed rank sum test was performed by comparing the RPKMvalues of one genomic output for each genewithin a cluster to the genes ofanother cluster.Eachclusterwascompared individually toall otherclustersto determine significance, P < 0.05.

Fisher’s Exact Test

Fisher’s exact test was performed by comparing the number of motifs/peaks associated with genes within a cluster to the total number of motifs/peaks identified for all DRGs.

Bioinformatic Tools

Analyses were performed with “Bioconductor R” packages, particularlythe Next Generation Sequencing analysis software of “systemPipeR”(Girke, 2014). Programs used within Bioconductor included: BiocParallel(Morgan et al., 2019a), BatchJobs (Bischl et al., 2015), GenomicFeatures(Lawrence et al., 2013), GenomicRanges (Lawrence et al., 2013), edgeR(McCarthy et al., 2012), gplots (Warnes et al., 2009), ggplot2 (Wickham,2009), RColorBrewer (Neuwirth, 2011), Dplyr (Wickham et al., 2015),biomaRt (Durinck et al., 2009), ChIPseeker (Yu et al., 2015), rtracklayer(Lawrence et al., 2009), Rtsne (Krijthe, 2015), Pheatmap (Kolde, 2012),e1071 (Meyer et al., 2015), cluster (Maechler et al., 2019), ShortRead(Morgan et al., 2009), clusterProfiler (Yu et al., 2012), and Rsamtools(Morgan et al., 2019b). The publicly available UNIX/Python/Perl packagesused included TopHat (Kim et al., 2013), FASTX-Toolkit (Gordon andHannon, 2010), FastQC (Andrews et al., 2010), MEME (Bailey et al., 2009),HOMER (Heinz et al., 2010), deepTools (Ramírez et al., 2016), CIRCOS(Krzywinski et al., 2009), BEDtools (Quinlan and Hall, 2010), and SAMtools(Li, 2011).

Statistical Analysis

Statistical analysisofclustermotif enrichmentwasperformedby two-tailedFisher’s exact test comparingmotif frequency for individual genes of eachcluster; P < 0.05. Statistical significance of cluster values was determinedfor each genomic output using aWilcoxon signed rank test for values of allgenes within each cluster; P < 0.05.

Accession Numbers

The Arabidopsis genes examined in this study include: ADH1, AT1G77120;PCO2, AT5G39890; HSP70-4, AT3G12580; RPL37B, AT1G52300; LHY1,AT1G01060; CCA1, AT2G46830; PRR9, AT2G46790; PRR7, AT5G02810;PRR5, AT5G24470; RV8, AT3G09600; LUX, AT3G46640; CIR1, AT5G37260;ELF3, AT2G25930; ELF4, AT2G40080; LWD1, AT1G12910; ZTL, AT5G57350;GI, AT1G22770; LNK1, AT5G64170; LNK2, AT3G54500; TOC1, AT5G61380;CML38, AT1G76650; LBD41, AT3G02550; ALAAT1, AT1G17290; HSP20 like,AT1G53540.

The data sets generated and analyzed in this study are available inthe National Center for Biotechnology Information Gene ExpressionOmnibus repository, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc5GSE122804.

Supplemental Data

Supplemental Figure 1. Expanded overview of the multiscale chro-matin and RNA gene regulatory analyses.

Supplemental Figure 2. Average signal plots and heat maps for theglobal distribution of reads of chromatin and RNA data.

Supplemental Figure 3. Multilevel evaluation of histone and generegulation activity of the HRGs and RPs.

Supplemental Figure 4. Evidence of cooperative association betweenH2A.Z eviction and H3K9ac and the relationship between histone H3modification and RNAPII engagement.

Supplemental Figure 5. Genome-Wide comparison of nRNA to polyARNA demonstrates nuclear and cytoplasmic enrichment of transcriptsunder two conditions.

Supplemental Figure 6. Histones, RNAPII–Ser2P, and RNA levels ofgenes that are coregulated in response to HS.

Supplemental Figure 7. Quantitative analysis of dynamics in histonemodifications of coregulated gene clusters after 2 h of HS.

Supplemental Figure 8. Progressive regulation of variations in geneactivity in response to hypoxia.

Supplemental Figure 9. Histone and gene activity comparisons ofgenes coregulated after 9 h of HS.

Supplemental Figure 10. Genome-Wide binding dynamics of HRE2determined by ChIP-seq.

Supplemental Figure 11. Genome browser view of normalized readcoverage of histone, ATAC-seq, RNAPII–Ser2P, HRE2-ChIP, and RNAoutputs for representative genes of cluster 9.

Supplemental Figure 12. Reoxygenation after hypoxia promotesglobal changes in chromatin accessibility and RNA populations.

Supplemental Figure 13. Regulation of variations in gene activity inresponse to hypoxia and reoxygenation.

Supplemental Data Set 1. Survey of histones, RNAPII, HRE2, andthree RNA subpopulations in response to two durations of HS.

Supplemental Data Set 2. Tabulation of ATAC and HRE2-ChIP peakson chromatin.

Dynamic Nuclear Gene Regulatory Control by Hypoxia 2591

Supplemental Data Set 3. nRNA enrichment and GO analysis.

Supplemental Data Set 4. GO analysis for clusters identified from twodurations of HS and reoxygenation.

Supplemental Data Set 5. List of arabidopsis blacklisted chromatin(ABC).

Supplemental File 1. BED File of HRE2 ChIP-seq peaks.

Supplemental File 2. BED File of arabidopsis blacklisted chromatin(ABC).

ACKNOWLEDGMENTS

We thank Mauricio Reynoso, Roger Deal, Marko Bajic, Seung-Cho Lee,Maureen Hummel, Lauren Dedow, Sonja Winte, Jérémie Bazin, ThomasGirke, DawnNagel, and Thomas Eulgem for helpful discussions. This workwas supported by the U.S. National Science Foundation (grants IOS-1121626, IOS-1238243, and MCB-1716913) and the U.S. Departmentof Agriculture, National Institute of Food and Agriculture - Agricultureand Food Research Initiative (grant 2011-04015 to J.B.-S.).

AUTHOR CONTRIBUTIONS

T.A.L. and J.B.-S. designed research; T.A.L. performed research; T.A.L.and J.B.-S. analyzed data; T.A.L. and J.B.-S. wrote the article.

Received June 20, 2019; revised August 21, 2019; accepted September12, 2019; published September 13, 2019.

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DOI 10.1105/tpc.19.00463; originally published online September 13, 2019; 2019;31;2573-2595Plant Cell

Travis A. Lee and Julia Bailey-SerresRegulation during Transient Stress

Integrative Analysis from the Epigenome to Translatome Uncovers Patterns of Dominant Nuclear

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