The Evening Complex Establishes Repressive Chromatin Domains Via H2A.Z Deposition1 Meixuezi Tong,a,2 Kyounghee Lee,b,2 Daphne Ezer,a Sandra Cortijo,a Jaehoon Jung,a,b Varodom Charoensawan,a Mathew S. Box,a Katja E. Jaeger,a Nozomu Takahashi,c Paloma Mas,c,d,3 Philip A. Wigge,a,e,3 and Pil Joon Seo b,f,g,3,4 a Sainsbury Laboratory, University of Cambridge, Cambridge CB2 1LR, United Kingdom b Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Republic of Korea c Center for Research in Agricultural Genomics, Consortium Consejo Superior de Investigaciones Cientificas-Institute of Agrifood Research and Technology-Universitat Autònoma de Barcelona-Universidad de Barcelona, Parc de Recerca Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallés), Barcelona 08193, Spain d Consejo Superior de Investigaciones Científicas, Barcelona 08193, Spain e Leibniz-Institut für Gemüse- und Zierpflanzenbau, 14979 Großbeeren, Germany f Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea g Plant Genomics and Breeding Institute, Seoul National University, Seoul 08826, Republic of Korea ORCID IDs: 0000-0001-7183-6575 (M.T.); 0000-0003-3291-6729 (S.C.); 0000-0002-2199-4126 (V.C.); 0000-0002-4153-7328 (K.E.J.); 0000-0002-3780-8041 (P.M.); 0000-0003-4822-361X (P.A.W.); 0000-0002-5499-3138 (P.J.S.). The Evening Complex (EC) is a core component of the Arabidopsis (Arabidopsis thaliana) circadian clock, which represses target gene expression at the end of the day and integrates temperature information to coordinate environmental and endogenous signals. Here we show that the EC induces repressive chromatin structure to regulate the evening transcriptome. The EC component ELF3 directly interacts with a protein from the SWI2/SNF2-RELATED (SWR1) complex to control deposition of H2A.Z-nucleosomes at the EC target genes. SWR1 components display circadian oscillation in gene expression with a peak at dusk. In turn, SWR1 is required for the circadian clockwork, as defects in SWR1 activity alter morning-expressed genes. The EC-SWR1 complex binds to the loci of the core clock genes PSEUDO-RESPONSE REGULATOR7 (PRR7) and PRR9 and catalyzes deposition of nucleosomes containing the histone variant H2A.Z coincident with the repression of these genes at dusk. This provides a mechanism by which the circadian clock temporally establishes repressive chromatin domains to shape oscillatory gene expression around dusk. The circadian clock generates biological rhythms (Alabadí et al., 2001), while in turn the TOC1 protein with a period of ;24 h to coordinate plant growth and represses CCA1 and LHY during the night (Alabadí development with environmental cycles (Greenham et al., 2001; Gendron et al., 2012; Huang et al., 2012; and McClung, 2015). A large fraction of the Arabidopsis Pokhilko et al., 2013). Repression of CCA1 and LHY (Arabidopsis thaliana) transcriptome is circadian-regulated throughout the day also occurs by the sequential action (Staiger and Green, 2011). Circadian transcription allows of additional members of the PRR family, including the molecular anticipation of the environmental cycles, PRR9, PRR7, and PRR5 (Nakamichi et al., 2010; Salomé which improves plant fitness and adaptation (Yerushalmi et al., 2010). Additional repressors of morning gene et al., 2011). Consistent with its adaptive function, the expression include the components of the Evening circadian clock is subject to multiple layers of regulation, Complex (EC), EARLY FLOWERING3 (ELF3), ELF4, which contribute toward accurate oscillations (Seo and and LUX ARRYTHMO/PHYTOCLOCK1 (Nusinow Mas, 2014). et al., 2011; Chow et al., 2012; Herrero et al., 2012). Transcriptional regulation of circadian genes is a The EC is a core component of the circadian clock, basic framework of clock architecture (Carré and Kim, and elf3 mutants are arrhythmic under continuous light 2002; Salomé and McClung, 2004). The Arabidopsis (Hicks et al., 1996; Lu et al., 2012). By binding to the central oscillator consists of a series of sequential reg- promoters of hundreds of key regulators of circadian ulatory loops composed of genes expressed at different clock, photosynthesis, and phytohormone signaling, times during the diurnal cycle. Morning-expressed genes the EC is able to repress their expression (Ezer et al., such as CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) 2017). Because the activity of the EC is reduced at and LATE ELONGATED HYPOCOTYL (LHY) repress warmer temperatures, and environmental sensing phy- the expression of evening-expressed genes like TIMING tochromes cobind target promoters, the EC is able to OF CAB EXPRESSION1/PSEUDO-RESPONSE REGULATOR1 integrate environmental information into endogenous 612 Plant PhysiologyÒ, January 2020, Vol. 182, pp. 612–625, www.plantphysiol.org Ó 2020 American Society of Plant Biologists. All Rights Reserved. Downloaded from on February 13, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. EC-Dependent H2A.Z Deposition developmental programs (Ezer et al., 2017). However, immune responses, and temperature sensing (Noh and the molecular mechanisms by which the EC represses Amasino, 2003; Deal et al., 2005; Kumar and Wigge, gene expression are not known. 2010). Here, we report that the EC associates with the Histone variants influence chromatin structure and SWR1 complex to repress the evening transcriptome. therefore transcription. H2A.Z is the most well-conserved As a part of its biological impact, this complex shapes histone variant, enriched near transcription start sites circadian oscillations by targeting clock genes such as (TSSs; Raisner et al., 2005; Raisner and Madhani, 2006) PRR7 and PRR9 for H2A.Z deposition and gene repres- and influencing transcriptional activities of associated sion. These results indicate that diurnal H2A.Z deposition genes (Raisner et al., 2005). Effects of H2A.Z deposition provides a mechanism contributing to circadian gene are likely variable depending on chromatin context: expression in Arabidopsis. H2A.Z deposition at promoters prevents the spread of heterochromatin in yeast and is associated with transcriptional inducibility (Guillemette et al., 2005), whereas in metazoans, H2A.Z has been shown to play RESULTS a role in heterochromatin formation and maintenance ELF3 Stabilizes Nucleosome Architecture at EC (Rangasamy et al., 2003; Swaminathan et al., 2005). In Target Genes plants, H2A.Z-nucleosomes confer transcriptional com- petence (Deal et al., 2007; To and Kim, 2014), and also To understand how the EC functions, we created a appear to wrap DNA more tightly, facilitating inducible stringent list of direct EC targets (Supplemental Table gene expression (Kumar and Wigge, 2010; Coleman-Derr S1), defined as genes whose promoters are bound by and Zilberman, 2012). at least two EC proteins and which are misexpressed The Arabidopsis genome encodes putative homologs at the end of the day in elf3-1 (Ezer et al., 2017). Pre- of catalytic subunits of the Swi2/Snf2-Related (SWR1)/ viously, we have seen that EC targets show the same Swi2/Snf2-Related CBP Activator Protein complex pattern of misexpression in both elf3-1 and lux-4, responsible for H2A.Z deposition, including PHOTOPERIOD- when compared to wild type over a 24-h time course INDEPENDENT EARLY FLOWERING1 (PIE1), ACTIN- (Ezer et al., 2017). In both cases, there is minimal de- RELATED PROTEIN6 (ARP6), and SERRATED LEAVES viation from wild-type gene expression during the AND EARLY FLOWERING (SEF; March-Díaz and Reyes, day, but maximal deviation during the evening and 2009). The SWR1 complex associates extensively with night-time, coinciding with the activity of the EC chromatin and catalyzes H2A.Z exchange at genomic (Huang and Nusinow, 2016). Furthermore, the fold- levels. Consistently, SWR1-mediated chromatin re- increase in expression in elf3-1 and lux-4 is also conserved modeling is involved in diverse aspects of plant phys- between target genes (Ezer et al., 2017). This indicates that iology and development, such as the floral transition, the EC components control target gene expression pos- sibly altogether. Thus, we next asked how the EC glob- 1 This work was supported by the National Research Foundation of ally controls endogenous gene expression programs. Korea’s Basic Science Research programs (NRF-2019R1A2C2006915), Because chromatin accessibility, which is related to the Basic Research Laboratory (NRF-2017R1A4A1015620), and the gene responsiveness, is usually coordinated with tran- Rural Development Administration’s Next-Generation BioGreen 21 scriptional regulation, we investigated if EC targets have Program (PJ01314501 to P.J.S.); the Federación Española de Enferme- a distinctive nucleosome structure, and if this is per- dades Raras/Spanish Ministry of Economy and Competitiveness, the turbed in elf3-1. Micrococcal nuclease (MNase) produces Ramon Areces Foundation, the Generalitat de Catalunya (Agency for double-stranded cuts between nucleosomes, thus pro- Management of University and Research Grants), the Centres de Re- viding a simple method for obtaining information on the cerca de Catalunya Program/Generalitat de Catalunya, and the locations and arrangements of nucleosomes (Shu et al., Spanish Ministry of Economy and Competitiveness “Severo Ochoa 2013). We investigated the genome accessibility of the Program for Centers of Excellence in R&D” 2016–2019 (SEV-2015- 0533 to P.M.); and the Gatsby Foundation (GAT3273/GLB EC target loci at a range of time points using MNase to P.A.W.). digestion coupled with sequencing (MNase-seq). We 2 These authors contributed equally to this work. observed high nucleosome occupancy for EC target 3 Senior authors. genes (Fig. 1), and this increased at Zeitgeber Time 4 Author for contact: pjseo1@snu.ac.kr. (ZT)8 and ZT12 (Fig. 1, B, C, E, and F), coinciding with The author responsible for distribution of materials integral to the maximal EC activity. Notably, a marked increase in findings presented in this article in accordance with the policy de- chromatin accessibility was observed in the elf3-1 mutant scribed in the Instructions for Authors (www.plantphysiol.org) is: Pil (Fig. 1, B, C, E, and F), consistent with the EC repression Joon Seo (pjseo1@snu.ac.kr). of gene expression. The nucleosome occupancy in elf3-1 was P.A.W., P.M., and P.J.S. participated in the design of the study and most similar to wild type at ZT0 (Fig. 1, A and D), but wrote the article; M.T., K.L., and N.T. performed the molecular ex- periments; M.T. and D.E. performed the analysis of sequencing data; decreased as the day progresses, showing the greatest M.T., S.C., J.J., V.C., and M.S.B. performed large-scale time-course loss at ZT12, by which time the gene body nucleosome experiments; M.T. and K.E.J. performed ChIP-seq experiments; occupancy was barely detectable (Fig. 1, C and F). P.A.W., P.M., and P.J.S. conceived the project; all authors read and These results suggest that EC-dependent gene re- approved the final article. pression is induced in part by temporal stabilization www.plantphysiol.org/cgi/doi/10.1104/pp.19.00881 of repressive chromatin structures. Plant Physiol. Vol. 182, 2020 613 Downloaded from on February 13, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Tong et al. Figure 1. Nucleosome occupancy in EC target genes in Col-0 and elf3-1 at ZT0, 8, and 12. Average nucleosome signals (nor- malized MNase-seq reads) on 52 EC target genes (see also Supplemental Table S1) at ZT0, ZT8, and ZT12 (A–C) in Col-0 and elf3-1 grown under long day conditions. Heat map visualization of nucleosome signals over 52 EC target genes on Col-0 and elf3-1 at ZT0, ZT8, and ZT12 (D–F). Data are plotted from 1 kb upstream of TSSs to 1 kb downstream of transcription end site (TES) of EC target genes. Graphs show the results of two replicates. Components of the SWR1 Complex Are Necessary for Because warm temperature reduces the ability of the EC Function EC to associate with target promoters (Box et al., 2015; Ezer et al., 2017), we investigated how these genes re- To identify genes mediating the connection between spond to 27°C. As expected, EC targets were upregu- the EC and chromatin architecture, we surveyed the lated in wild type at 27°C particularly around evening DIURNAL dataset (http://diurnal.mocklerlab.org) for and nighttime (Fig. 3B), reflecting impaired EC func- transcripts having a similar expression pattern to the tion. This pattern was, however, strongly enhanced by EC. Transcripts encoding histone variants as well as the arp6-1 mutation (Fig. 3B). These results indicate that SWR1 components required for H2A.Z deposition were the EC and H2A.Z-nucleosomes function to repress included, and they were regulated in diurnal and cir- EC targets. cadian patterns (Supplemental Fig. S1A). To confirm these observations, we examined the circadian expres- sion of genes associated with H2A.Z exchange by reverse transcription quantitative PCR (RT-qPCR) analysis. ELF3 Physically Interacts with SEF Genes encoding the SWR1 components, including ARP6, PIE1, and SEF, displayed circadian oscillation Having established a connection between the EC- under free-running conditions with a peak around dusk dependent gene repression and H2A.Z-nucleosomes, (Fig. 2, A–C), similar to ELF3 (Fig. 2D). The transcript we sought to investigate if there might be a direct accumulation of histone variant genes, HTA8, HTA9, and physical interaction between the components involved HTA11, however, did not significantly oscillate during the in these processes. To test this possibility, we performed circadian cycle in our conditions (Supplemental Fig. S1B). yeast-two-hybrid (Y2H) assays. Constructs of compo- To determine if H2A.Z-nucleosomes are important nents of the SWR1 complex—ARP4, ARP6, SEF, SWR for EC function, we analyzed whether EC targets are COMPLEX PROTEIN2, and SWR COMPLEX PROTEIN5, misexpressed in arp6-1, which is compromised in its fused with GAL4 DNA-binding domain (BD) and ability to incorporate H2A.Z-nucleosomes (Kumar and evening-expressed clock components fused with GAL4 Wigge, 2010). Expression of EC target genes over a di- activation domain (AD)—were coexpressed in yeast urnal cycle was analyzed using RNA-seq. These genes cells. Cell growth on selective medium revealed that in this cluster showed a pattern of increased expression SEF interacts specifically with ELF3 (Fig. 4A; Supplemental at the end of day and early night in arp6-1 compared to Fig. S2). Although ELF4 is also a member of the EC (Huang wild type (Fig. 3A), a pattern consistent with reduced et al., 2016), we did not observe interactions of SEF with EC function in arp6-1. ELF4 in yeast cells (Fig. 4A). Because the PIE1 protein is a 614 Plant Physiol. Vol. 182, 2020 Downloaded from on February 13, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. EC-Dependent H2A.Z Deposition of PIE1 was able to interact with ELF3 (Supplemental Fig. S3). Together, the results indicate that PIE1, possibly along with SEF, participate in the physical interactions with ELF3. To confirm the physical interaction in vivo, we car- ried out bimolecular fluorescence complementation (BiFC) assays using Arabidopsis protoplasts. The SEF complementary DNA (cDNA) sequence was fused in- frame to the 59-end of a gene sequence encoding the N-terminal half of yellow fluorescent protein (YFP), and the ELF3 gene was fused in-frame to the 59-end of a sequence encoding the C-terminal half of YFP. The fusion constructs were then transiently coexpressed in Ara- bidopsis protoplasts. Strong yellow fluorescence was detected in the nucleus of cells coexpressing the combination of SEF-ELF3 (Fig. 4B), while coexpression with empty vectors did not show visible fluorescence (Fig. 4B). To quantify the physical interaction, split Luciferase (Luc) assay was also employed. ELF3 was fused with the amino part of Luc (NLuc), and SEF was fused with carboxyl-part of Luc (CLuc; Fig. 4C). Coex- pression of ELF3-NLuc and SEF-CLuc in Arabidopsis protoplasts resulted in 2-fold increase of Luc activity, while coexpression of controls showed background- level Luc activities (Fig. 4C). The in planta interactions of ELF3 and SEF were confirmed by coimmunopreci- pitation (Co-IP) assays using Nicotiana benthamiana cells transiently coexpressing 35S:ELF3-GFP and 35S:SEF-MYC fusion constructs. Because the full-length ELF3 protein is a large protein, it is difficult to express transiently in N. benthamiana. As an alternative, we designed frag- ments of ELF3 fused with GFP and used them to test physical interactions with SEF (Fig. 4D). Co-IP analysis revealed that the C-terminal region of ELF3 was re- sponsible for interactions with SEF in planta (Fig. 4D). These results indicate that the EC directly interacts with the SWR1 complex, and this may facilitate the direct deposition of H2A.Z-nucleosomes at EC target genes. H2A.Z-Nucleosomes Are Deposited at EC Target Loci Because EC target genes are misregulated in arp6-1 and ELF3 and SEF interact directly, we investigated if H2A.Z- nucleosomes are enriched at EC target genes. HTA11 occupancy, assayed by chromatin-immunoprecipitation Figure 2. Coexpression of the SWR1 components with EC. A to D, sequencing (ChIP-seq; Cortijo et al., 2017), showed a Circadian expression of SWR1 components and ELF3. Seedlings grown strong enrichment across the gene body of ELF3 tar- under neutral day conditions (12-h light: 12-h dark) for 2 weeks were gets (Fig. 5A). The occupancy was particularly high at transferred to continuous light conditions at ZT0. Whole seedlings were the region surrounding the TSS at the presumptive 11 harvested from ZT24 to ZT68 to analyze transcript accumulation. nucleosome, but was also markedly higher over the Transcript levels of ARP6 (A), PIE1 (B), SEF (C), and ELF3 (D) were de- gene body of ELF3 targets (Fig. 5A). By comparison, termined by RT-qPCR. Gene expression values were normalized to the eIF4A expression. Biological triplicates were averaged. Bars indicate randomly selected control genes showed H2A.Z en- the mean 6 SE. The white and gray boxes indicate the subjective day and richment around the TSS only (Fig. 5B; Supplemental night, respectively. Table S2). We then compared the genome-wide association of ELF3 with H2A.Z. Consistent with the fact that the EC catalytic core of SWR1 complex, we also examined inter- is recruited to target sites via LUX binding sites and actions between PIE1 and EC components. We had diffi- G-box motifs (Ezer et al., 2017), we observed that these culties cloning the full-length PIE1 and instead assayed motifs were strongly enriched at ELF3-binding sites domains of PIE1. Our results showed that the SANT domain (Ezer et al., 2017). Notably, the footprint bound by ELF3 Plant Physiol. Vol. 182, 2020 615 Downloaded from on February 13, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Tong et al. Figure 3. Elevated expression of EC target genes in arp6-1. Heat map visualization of log fold changes of the expression levels of EC target genes in arp6-1 at 22°C compared with Col-0 at 22°C grown under long day conditions (A) and in arp6-1 at 27°C compared with Col-0 at 22°C grown under short days (B). Values in (A) and (B) represent log2 (TPM in arp6-1 22°C/TMP in Col-0 22°C) and log2 (TPM in arp6-1 27°C/TMP in Col-0 22°C), respectively. The log fold change of the expression level of each gene was calculated by Z-score (mean 5 0, SD 5 1). The heat map was generated using the heatmap.2 function in R (v3.2.5). 616 Plant Physiol. Vol. 182, 2020 Downloaded from on February 13, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. EC-Dependent H2A.Z Deposition Figure 4. Interactions of the SWR1 complex with EC. A, Y2H assays. Y2H assays were performed with the SEF protein fused to the DNA BD of GAL4 and evening-expressed clock compo- nents fused with the transcriptional AD of GAL4 for analysis of interactions. Interactions were examined by cell growth on selective media. -LWHA in- dicates Leu, Trp, His, and Ade drop-out plates. -LW indicates Leu and Trp drop- out plates. GAL4 was used as a positive control (P). B, BiFC assays. Partial frag- ments of YFP protein were fused with SEF and ELF3, and coexpressed in Ara- bidopsis protoplasts. Reconstituted flu- orescence was examined by confocal microscopy. IDD14-RFP was used as a nucleus marker. Scale bars 5 10 mm. DIC, differential interference contrast. C, Interaction of ELF3-NLuc with SEF- CLuc. Partial fragments of Luc (NLuc and CLuc) were fused with ELF3 or SEF. The fusion constructs were coexpressed in Arabidopsis protoplasts and Luc activi- ties were measured and normalized against total protein. Three independent bio- logical replicates were averaged and statistically analyzed with Student’s t test (*P , 0.05). Bars indicate the mean 6 SE. D, Co-IPassays. A. tumefaciens cells containing 35S:ELF3N(1-345aa)-GFP, 35S:ELF3C(346-695aa)-GFP, and 35S:SEF- MYC constructs were coinfiltrated to 3-week–old N. benthamiana leaves. Epitope-tagged proteins were detected immunologically using corresponding antibodies. in the promoters of EC target genes, including PRR7, (1) genes bound by both ELF3 and LUX, but not upre- PRR9, and LUX, was devoid of H2A.Z-nucleosome signal gulated in elf3-1 and lux-4 mutants (Supplemental Table (Fig. 5C; Supplemental Fig. S4, A–C), but the adjacent S3); and (2) genes upregulated in elf3-1 and lux-4, but regions including the gene bodies were highly occupied not bound by either ELF3 or LUX (Supplemental Table with H2A.Z-nucleosomes (Fig. 5C; Supplemental Fig. S4, S4). Again, H2A.Z deposition was not diurnally regulated A–C). This antiphasing between H2A.Z occupancy and and influenced by ELF3 in both cases (Supplemental the EC was observed for all the EC targets (Fig. 5C; Figs. S5 and S6). These results indicate that the EC- Supplemental Fig. S4, A–C). H2A.Z-nucleosomes are re- SWR1 complex contributes to H2A.Z deposition at fractory to transcription (Thakar et al., 2010), suggesting the EC target loci. that the initial binding of the EC to the promoters of target genes may assist subsequent recruitment of H2A.Z- nucleosomes to repress EC target gene expression. Temporal Regulation of H2A.Z Deposition at the PRR7 We thus further determined whether H2A.Z deposi- and PRR9 Promoters Underlies Proper tion is dependent on the EC. ChIP-seq assays showed Circadian Oscillation that the H2A.Z occupancy was observed in EC-target genes particularly during night time (Fig. 5D; Supplemental Given the essential role of the EC in the circadian clock Fig. S4D), whereas enrichment of H2A.Z-nucleosomes at and the direct physical and functional interaction be- those loci was compromised in the elf3-1 mutant (Fig. 5E; tween the EC and the SWR1 complex, SWR1 might be Supplemental Fig. S4D). As a negative control, randomly also important for proper circadian clock function. We selected control genes showed no temporal H2A.Z enrichment used genetic mutants affected in the key SWR1 subunits, regardless of genetic background (Supplemental Fig. S4E). arp6-1 and sef-1, and analyzed circadian function by Further, we also obtained two additional lists of control genes: monitoring the expression of a circadian output gene, Plant Physiol. Vol. 182, 2020 617 Downloaded from on February 13, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Tong et al. Figure 5. ELF3 and H2A.Z occupancy of EC target genes. A and B, H2A.Z enrichment (normalized HTA11-FLAG ChIP-seq reads) on 52 EC target genes (A) and control genes (B) was analyzed. For the class of “control genes,” a sample of 52 genes was randomly selected to compare their H2A.Z occupancy with EC target genes (see also Supplemental Table S2). (A) and (B) are plotted from 1 kb upstream of the TSS to 1 kb downstream of the TES of the corresponding genes. C, ELF3 and H2A.Z average binding plot on 52 EC target genes. D and E, H2A.Z enrichment in wild type (D) and elf3-1 mutant (E) at various time points under long day conditions. COLD, CIRCADIAN RHYTHM, AND RNA BINDING2 exchange of H2A.Z-nucleosomes might be important for (CCR2). RT-qPCR analysis revealed that the rhythmic circadian function. amplitude of CCR2 expression was significantly damp- Next we aimed to decipher the clock factors that are ened in the two mutants (Supplemental Fig. S7). Simi- direct targets of the SWR1 complex. We thus employed larly, the circadian expression of CCR2 was also reduced pHTA11:HTA11-GFP transgenic plants and examined in hta9-1 hta11-2 double mutant (Supplemental Fig. S8), H2A.Z deposition at core clock gene promoters by ChIP in a comparable trend to that of SWR1 mutants. These assays with a GFP-specific antibody. ChIP-qPCR anal- results suggest that SWR1 activity and proper circadian ysis showed that H2A.Z deposition specifically occurs 618 Plant Physiol. Vol. 182, 2020 Downloaded from on February 13, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. EC-Dependent H2A.Z Deposition at the gene bodies of the morning-expressed genes an anti-MYC antibody showed that the SWR1 component PRR7 and PRR9 (Fig. 6A), whereas other core clock binds to the PRR7 and PRR9 loci (Fig. 6B). These results genes did not show significant diurnal enrichment of support the specific association of SEF with the morning- the H2A.Z variant (Supplemental Fig. S9). Further- expressed genes and agree with the pattern of H2A.Z de- more, H2A.Z accumulation was primarily observed position. H2A.Z-nucleosome deposition usually inhibits around dusk at the PRR7 and PRR9 loci (Fig. 6A), Pol II accessibility by stimulating closed chromatin for- which is consistent with the temporal expression mation (Kumar and Wigge, 2010). Consistent with this, Pol patterns of the ELF3 and SWR1 components (Fig. 2). II recruitment was significantly increased at PRRs in the sef- We also generated transgenic plants overexpressing SEF 1 mutant (Fig. 6C), which has lower catalytic activity of (35S:MYC-SEF), in which the core component of the SWR1 H2A.Z deposition. These results indicate that H2A.Z de- complex, SEF, is fused in-frame to six copies of MYC- position occurs around dusk at EC target loci such as coding sequence. qPCR analysis after ChIP assays with PRR7 and PRR9 to repress their expression. Figure 6. H2A.Z deposition at PRR7 and PRR9 loci by the SWR1 complex. In (A) to (C), fragmented DNA was eluted from the protein–DNA complexes and used for qPCR analysis. Enrichment was normalized relative to eIF4A. Three independent biological replicates were averaged, and the statistical significance of the measurements was determined. Bars indicate the means 6 SE. A, Accumulation of H2A.Z at clock gene loci. Two-week–old plants grown under neutral day conditions were used for ChIP analysis with anti-GFP antibody. Gene structures are presented (upper representation). Underbars represent the amplified genomic re- gions. Statistically significant differences between ZT0 and ZT12 samples are indicated by asterisks (Student’s t test, *P , 0.05, **P , 0.01). B, Binding of SEF to clock gene promoters. Two-week–old 35S:MYC-SEF transgenic plants grown under neutral day conditions were harvested at ZT12. Statistically significant differences between wild-type and 35S:MYC-SEF plants are indicated by asterisks (Student’s t test, *P , 0.05). C, Recruitment of Pol II at PRRs in sef-1. Two-week–old plants grown under neutral day conditions were harvested at ZT12 and used for ChIP analysis with an anti-N terminus of Arabidopsis Pol II antibody. qPCR was performed with a primer pair amplifying the B region of each gene promoter (see also Fig. 6A). Statistically significant differences between wild-type and sef-1 plants are indicated by asterisks (Student’s t test, *P , 0.05). Plant Physiol. Vol. 182, 2020 619 Downloaded from on February 13, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Tong et al. ELF3 Is Necessary for H2A.Z Deposition at PRRs in the compared with wild-type plants, whereas arrhyth- Control of Circadian Oscillation mic expression was observed in elf3-8 (Fig. 7F). Notably, the 35S:MYC-SEF/elf3-8 plants showed arrhythmicity The SEF-ELF3 direct interaction suggests that there similar to elf3-8 (Fig. 7F), indicating that the SWR1 may be functional coordination in the temporal regula- complex depends on the EC in the control of circa- tion of PRR7 and PRR9. To investigate this, we dian oscillation. employed pELF3::ELF3-MYC/elf3-1 and 35S:MYC-ELF3 In summary, the EC interaction with the SWR1 com- transgenic plants (Jang et al., 2015) and performed ChIP plex provides a mechanism to recruit transcriptionally assays using an anti-MYC antibody. ChIP-qPCR analy- repressive H2A.Z-nucleosomes at circadian-regulated sis confirmed that ELF3 binds to the PRR7 and PRR9 loci genes. In this way, targets may be repressed over the (Fig. 7A), and this binding occurs preferentially at dusk course of the night until the EC abundance declines (Supplemental Fig. S10). Consistent with the require- and/or strong activation signals reactivate gene ex- ment of ELF4 and LUX in a functional EC, over- pression. In the case of circadian regulation, the PRR7 expression of ELF3 still resulted in rhythmic binding. and PRR9 genes are under the temporal regulation of Chromatin binding of ELF3 is causal for H2A.Z de- H2A.Z exchange. The EC-SWR1 complex is recruited position and is independent of H2A.Z occupancy. We to PRR7 and PRR9, facilitating H2A.Z deposition to genetically crossed pELF3::ELF3-MYC transgenic plants reduce gene expression during evening and night- with arp6-1, which lacks genome-wide H2A.Z deposi- time. This indicates a role for the EC in establishing tion, and the pELF3::ELF3-MYC x arp6-1 plants were circadian waves of gene expression. used for ChIP assays using an anti-MYC antibody (Supplemental Fig. S11). As a result, ELF3 binding to PRR loci was comparable in both wild-type and arp6-1 back- grounds (Supplemental Fig. S12), indicating that ELF3 DISCUSSION binding is an active process to trigger H2A.Z depo- sition at cognate regions. EC-Dependent Coordination of Chromatin Structures To determine the possible regulation of H2A.Z depo- The ELF3, ELF4, and LUX proteins form the tripartite sition by ELF3, we examined H2A.Z occupancy by ChIP- EC complex and cooperatively regulate a variety of de- qPCR at PRR7 and PRR9 in elf3-8. As expected, H2A.Z velopmental processes. They are functionally inter- deposition was reduced at these loci in elf3-8, particularly twined in the gating of day-inducible genes (Hazen around dusk (Fig. 7B; Supplemental Fig. S13), indicating et al., 2005; Huang and Nusinow, 2016), and thus that ELF3 is both present on these promoters and facili- their genetic mutants share phenotypic alterations, tates the insertion or stability of repressive H2A.Z- such as elongated hypocotyls, early flowering, and nucleosomes (Fig. 7B). Consistently, binding of SEF was altered circadian rhythms (Hicks et al., 2001; Hazen specifically observed at dusk, and its association to the PRR7 et al., 2005; Nusinow et al., 2011). Consistently, their and PRR9 promoters occurred in an ELF3-dependent binding sites largely overlap, and the EC controls manner (Fig. 7C). target gene expression (Ezer et al., 2017). Here, we Consistent with the role of the EC and SWR1 complex show that the EC recruits repressive chromatin domains in the repression of gene expression, the EC-SWR1 to regulate the evening transcriptome. The EC interacts complex contributes to the declining phase of PRR7 with the SWR1 complex and establishes transcription- and PRR9 expression during the evening and night ally repressive H2A.Z-nucleosomes at its target genes. period, when its expression is returning to basal The high occupancy of H2A.Z-nucleosomes across the levels (Farré et al., 2005). As expected, Pol II acces- gene bodies of target genes provides an effective barrier to sibility of PRR7 and PRR9 was elevated at dusk in RNA Pol II, maintaining these genes in a repressed state. elf3-8 (Fig. 7D). In further support of these results, Notably, mutants eliminating EC activity, like elf3 expression of PRR7 and PRR9 was elevated during and lux, exhibit an arrhythmic circadian clock. In con- evening and night-time in elf3-8 and H2A.Z-deficient trast, SWR1 mutants are rhythmic but with reduced hta8 hta9 hta11 (hta.z) mutants (Fig. 7E). In addition, amplitude. Therefore, deposition of H2A.Z by the EC at consistent with the repressing functions of PRR7 and core clock genes, such as PRR7 and PRR9, is important PRR9 on CCA1 and LHY (Nakamichi et al., 2010), for correct expression amplitude. However, circadian expression of CCA1 and LHY was significantly repressed clock rhythmicity appears not to depend on this mech- during subjective morning in elf3-8 and hta.z mutants anism. Additionally, we cannot rule out the possibility (Supplemental Fig. S14). Clock-controlled PHYTO- that ELF3 may recruit additional chromatin modifiers CHROME-INTERACTING FACTOR4 expression was and/or remodelers. The EC might be an important also affected in elf3-8 and hta.z mutants, and especially, platform facilitating chromatin reconfiguration with its expression was derepressed during night period multiple epigenetic modifications. (Supplemental Fig. S14). To confirm the genetic inter- Temporal exchange of H2A.Z-nucleosomes provides actions of EC and SWR1, we crossed elf3-8 with a mechanism for diurnal gating of several physiological 35S:MYC-SEF transgenic plants (Supplemental Fig. processes. For example, a majority of stress-responsive S15). 35S:MYC-SEF transgenic plants displayed re- genes are gated primarily during day period (Seo and pressed rhythmic expression of PRR7 and PRR9 Mas, 2015), and consistently, a substantial number of 620 Plant Physiol. Vol. 182, 2020 Downloaded from on February 13, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. EC-Dependent H2A.Z Deposition Figure 7. H2A.Z exchange at PRR7 and PRR9 loci by ELF3. A, Binding of ELF3 to PRR promoters. Two-week–old pELF3::ELF3- MYC/elf3-1 seedlings grown under neutral day conditions were harvested at ZT12 and used to conduct ChIP assays. Statistically significant differences between Col-0 and pELF3::ELF3-MYC/elf3-1 plants are indicated by asterisks (Student’s t test, *P , 0.05, ***P , 0.001). B, H2A.Z deposition at clock gene promoters in elf3-8. Two-week–old plants grown under neutral day conditions were used for ChIP analysis with anti-H2A.Z antibody. Statistically significant differences between Col-0 and elf3-8 plants are indicated by asterisks (Student’s t test, *P , 0.05, ***P , 0.001). C, SEF binding to the PRR loci in elf3-8 background. Two- week–old plants grown under neutral day conditions were used for ChIP analysis with anti-MYC antibody. Statistically significant differences between ZT0 and ZT12 samples are indicated by asterisks (Student’s t test, *P , 0.05). D, Recruitment of Pol II at clock gene promoters in elf3-8. Two-week–old plants grown under neutral day conditions were harvested at ZT12 and used for ChIP analysis with an anti-N terminus of Arabidopsis Pol II antibody. Statistically significant differences between ZT0 and ZT12 samples are indicated by asterisks (Student’s t test, *P , 0.05). In (A) to (D), fragmented DNA was eluted from the protein–DNA complexes and used for qPCR analysis. Enrichment was normalized relative to eIF4A. Three independent biological replicates were averaged, and the statistical significance of the measurements was determined. Bars indicate the mean 6 SE. E, Circadian expression of PRR7 and PRR9 in elf3-8 and h2a.z. F, PRR expression in 35S:MYC-SEF/elf3-8. In (E) and (F), seedlings grown under neutral day conditions for 2 weeks were transferred to continuous light conditions at ZT0. stress-inducible genes that are responsive to drought, transcriptional activation and repression. The recruit- temperature, or pathogens are under the control of EC- ment of H2A.Z-nucleosomes provides a mechanism for dependent H2A.Z deposition to ensure their suppres- the stable repression of target clock genes over the sion during night-time (Ezer et al., 2017). In addition, course of the night, which can be robustly activated the the circadian clock relies on a series of waves of following day. Overall, the EC-SWR1 complex is a Plant Physiol. Vol. 182, 2020 621 Downloaded from on February 13, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Tong et al. global transcriptional regulator that functions in the nucleosomes may have a conserved function in the diurnal gating of many developmental and physiolog- eukaryotic circadian clock. ical processes and provides a more stable mechanism for maintaining repressive states during night-time. MATERIALS AND METHODS Chromatin-Based Regulation at the Core of the Plant Materials and Growth Conditions Circadian Clock Arabidopsis (Arabidopsis thaliana; Columbia-0 ecotype) was used for all ex- periments unless otherwise specified. Arabidopsis seeds were surface-sterilized Time-of-day–dependent accumulation of chromatin and sown on 0.7% (w/v) agar plates containing half-strengthened Murashige marks such as H3ac and H3K4me3 occurs with the and Skoog media. After 3-d stratification at 4°C, the seeds were grown in a circadian transcript abundance of clock genes including reach-in plant growth chamber (Conviron) with 170 mmol m22 s21 light in- tensity and 70% humidity under short day (8-h light/16-h dark), neutral day CCA1, LHY, TOC1, PRR7, PRR9, and LUX (Hsu et al., (12-h light/12-h dark), or long day (16-h light/8-h dark) conditions at 22°C or 2013; Voss et al., 2015). Mechanistically, H3ac stimu- 27°C, as indicated in figure legends. The arp6-1, arp6-3, elf3-1, elf3-8, hta9-1hta11-2, lates an open chromatin conformation (Song and Noh, pHTA11:HTA11-FLAG, pHTA11:HTA11-GFP, and sef-1 plants were previously 2012), whereas H3K4me3 inhibits the binding of clock reported (March-Díaz et al., 2007; Kumar and Wigge, 2010; Coleman-Derr and repressor proteins to the core clock gene promoters, Zilberman, 2012; Rosa et al., 2013; Nitschke et al., 2016). To produce transgenic plants overexpressing the SEF and ELF3 genes, a full-length cDNA was subcloned avoiding advanced circadian repressor binding (Malapeira into the binary pBA002 vector under the control of the Cauliflower mosaic virus 35S et al., 2012). The SDG2/ATXR3 histone methyltransferase promoter. Agrobacterium tumefaciens-mediated Arabidopsis transformation was contributes to the H3K4me3 accumulation and thus con- then performed. trols the timing of clock gene expression, from activation to repression (Malapeira et al., 2012). Several additional MNase-Seq and ChIP-Seq Experiments and Analysis chromatin modifiers, including HISTONE DEACETY- LASE6 (HDA6), HDA19, and JUMONJI C DOMAIN- Approximately 1 g of 9-d–old Arabidopsis seedlings was harvested at the CONTAINING PROTEIN30/JUMONJI DOMAIN time points indicated in figure legends. The harvested seedlings were ground in liquid nitrogen to fine powder. The tissue powder was fixed for 10 min with 1% CONTAINING5, are also connected with the circa- (v/v) formaldehyde (cat. no. F8775; Sigma-Aldrich) in buffer 1 (0.4 M of Suc, dian oscillation (Jones and Harmer, 2011; Lu et al., 2011; 10 mM of HEPES, 10 mM of MgCl2, 5 mM of b-mercaptoethanol, 0.1 mM of Wang et al., 2013), although the mechanisms behind phenylmethylsulfonyl fluoride (PMSF), and 13 protease inhibitor; cat. no., circadian gene regulation remain to be determined. 11836145001; Roche). The reaction was quenched by adding Gly to a final The epigenetic regulation of core clock genes relies concentration of 127 mM. The homogenate was filtered through Miracloth (EMD Millipore) twice and centrifuged to collect the pellet. The pellet was on a complex web of chromatin and clock compo- washed in buffer 2 (0.24 M of Suc, 10 mM of Tris-HCl at pH 8.0, 10 mM of MgCl2, 5 mM nents. For example, CCA1 facilitates repressive chro- of b-mercaptoethanol, 0.1 mM of PMSF, 13 protease inhibitor, and 1% [v/v] Triton) matin signatures to regulate TOC1 expression around and then spun down in buffer 3 (1.7 M of Suc, 10 mM of Tris-HCl at pH 8.0, 10 mM of dawn while HDAs contribute to the declining phase of MgCl2, 5 mM of b-mercaptoethanol, 0.1 mM of PMSF, 13 protease inhibitor, and 0.15% [v/v] Triton). The nuclei pellet was then resuspended in MNase Digestion TOC1 (Perales and Más, 2007). Another MYB-like tran- buffer (20 mM of Tris-HCl at pH 8.0, 50 mM of NaCl, 1 mM of DTT, 0.5% [v/v] NP-40, scription factor known as REVEILLE8/LHY-CCA1- 1 mM of CaCl2, 0.5 mM of PMSF, and 13 protease inhibitor) and was flash-frozen in LIKE5 favors H3 acetylation at the TOC1 promoter, most liquid nitrogen twice to break the nuclear envelope. Chromatin was digested by likely by antagonizing CCA1 function throughout the adding MNase (cat. no. N3755; Sigma-Aldrich) to a final concentration of 0.4 day (Farinas and Mas, 2011). U/mL for 12.5 min. The reaction was terminated by adding EDTA to a final concentration of 5 mM. For ChIP-seq samples, H2A.Z in transgenic line In this study, we identify a repressive chromatin state pHTA11:HTA11-FLAG was immunoprecipitated by anti-FLAG M2 magnetic that shapes the rhythmic oscillations in gene expres- beads (cat. no. M8823; Sigma-Aldrich) and then eluted with 3XFLAG peptide sion. Circadian H2A.Z deposition underlies normal (cat. no. B23112; Bimake). After reverse cross linking, the DNA was purified circadian oscillation, and PRR7 and PRR9 are pri- with solid phase reversible immobilization beads. The libraries were con- structed using TruSeq ChIP Sample Preparation Kit (cat. no. IP-202-1024; Illu- mary targets of the SWR1 complex. H2A.Z-nucleosome mina) according to the manufacturer’s instructions. The libraries were deposition occurs around dusk, when the SWR1 com- sequenced on a NextSeq 500 (Illumina) platform. The raw reads obtained from ponents are highly expressed, to suppress gene ex- the sequencing facilities were analyzed using a combination of publicly avail- pression. The interaction between the SWR1 complex able software and in-house scripts. We first assessed the quality of reads using and ELF3 provides a direct mechanism to facilitate FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/). Potential adaptor contamination and low quality trailing sequences were removed using the tool H2A.Z exchange at cognate regions, contributing to Trimmomatic (Bolger et al., 2014). Then, the reads were mapped to The Arabidopsis precise oscillations in circadian gene expression. Be- Information Resource 10 reference genome using the software BowTie2 (Langmead cause the PRR7 and PRR9 loci are also subjected to and Salzberg, 2012). Duplicates were removed with the tool Picard (https://github. H3ac and H3K4me3 modifications (Malapeira et al., com/broadinstitute/picard) and the read counts was normalized by the sample’s genome-wide reads coverage. Nucleosome positioning and occupancy were de- 2012), the higher-order combination of multiple termined using the program DANPOS (Chen et al., 2013). Nucleosome and H2A.Z chromatin modifications ultimately shapes the cir- average binding profiles and heatmaps were generated using the program deep- cadian waveforms of gene expression throughout the Tools (Ramírez et al., 2014). day–night cycle. The “randomly selected control genes” were generated using the “sample()” In mammals, rhythmical H2A.Z deposition at the function in R. The indices of the 52 genes were generated by the R code “index ← sample(33557, 52),” where “33,557” is the total number of Arabidopsis genes. promoters of CLOCK:BMAL1 targets has been ob- The corresponding Arabidopsis Genome Initiative gene names were generated served, although the underlying mechanism is not by the R code “random52 ← all_gene_names[index,].” In addition, we also known (Menet et al., 2014). This suggests that H2A.Z- obtained two additional lists of control genes with new filters: genes bound by 622 Plant Physiol. Vol. 182, 2020 Downloaded from on February 13, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. EC-Dependent H2A.Z Deposition both ELF3 and LUX, but not upregulated in elf3-1 and lux-4 mutants the N-terminal half of EYFP in the pSATN-nEYFP-C1 vector (E3081). Expres- (Supplemental Table S3); and genes upregulated in elf3-1 and lux-4, but not sion constructs were cotransformed into Arabidopsis protoplasts. Expression of bound by either ELF3 or LUX (Supplemental Table S4). The filter criteria for the the fusion constructs was monitored by fluorescence microscopy using a model former gene list are: having both ELF3 and LUX binding within 1,000 bp up- no. LSM510 confocal microscope (Carl Zeiss). stream of the gene; and the log fold changes of Transcripts Per Million (TPM) values compared with Col-0 at ZT16 are smaller than 0.5-fold in both elf3-1 and lux-4 mutants. The filter criteria for the latter gene list are: having no ELF3 or LUX ChIP Assays binding within 1,000 bp upstream of the gene; and the log fold changes of TPM The epitope-tagged transgenic plant samples were cross linked with 1% (v/v) values compared with Col-0 at ZT16 are larger than 1.5-fold in both elf3-1 and formaldehyde, ground to powder in liquid nitrogen, and then sonicated. The lux-4 mutants. sonicated chromatin complexes were bound with corresponding antibodies. Anti- MYC (cat. no. 05-724; Millipore), anti-Pol II (cat. no. sc-33754; Santa Cruz Bio- RNA-Seq Experiment and Analysis technology), anti-H2A.Z antibodies (cat. no. ab4174; Abcam), and salmon sperm DNA/protein A agarose beads (cat. no. 16-157; Millipore) were used for ChIP. Approximately 30 mg of 7-d–old Arabidopsis seedlings were harvested and DNA was purified using phenol/chloroform/isoamyl alcohol and sodium ace- their total RNA was extracted using the MagMAX-96 Total RNA Isolation kit tate (pH 5.2). The level of precipitated DNA fragments was quantified by qPCR (AM1830; Ambion) according to manufacturer’s instructions. Library prepa- using specific primer sets (Supplemental Table S6). Values were normalized ration was performed using 1 mg of high integrity total RNA (RNA integrity according to input DNA levels. Values for control plants were set to 1 after nor- number . 8) using the TruSeq Stranded mRNA library preparation kit (cat. no. malization against eIF4a for qPCR analysis. RS-122-2103; Illumina) according to the manufacturer’s instructions. The libraries were sequenced on a NextSeq 500 platform (Illumina). For bioinformatics analysis, we first assessed the quality of reads using the Split‐Luc Assays program FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/). Poten- The coding regions of SEF and ELF3 were cloned into pcFLucC or pcFLucN tial adaptor contamination and low quality trailing sequences were removed using vector. The recombinant constructs were cotransformed with into Arabidopsis the tool Trimmomatic (Bolger et al., 2014), before alignment to The Arabidopsis protoplasts by polyethylene glycol-mediated transformation. The pUBQ10::GUS Information Resource 10 transcriptome using the software TopHat (Trapnell et al., plasmid was also cotransformed as an internal control to normalize the LUC 2009). Potential optical duplicates resulting from library preparation were removed activity. using the Picard tools (https://github.com/broadinstitute/picard), and the read counts were normalized by the sample’s genome-wide reads coverage. Raw counts were determined by HTseq-count (Anders et al., 2015), and the software Cufflinks Co-IP Assays (http://cole-trapnell-lab.github.io/cufflinks/) was utilized to calculate Fragments Per Kilobase Million, which was then converted into TPM. A. tumefaciens cells containing 35S:SEF-MYC and 35S:ELF3-GFP constructs were infiltrated to 3-week–old Nicotiana benthamiana leaves. N. benthamiana leaves were homogenized in protein extraction buffer (25 mM of Tris-HCl at pH RT-qPCR Analysis 7.5, 150 mM of NaCl, 5% v/v glycerol, 0.05% w/v Nonidet P-40, 2.5 mM of EDTA, 1 mM of PMSF, and 13 complete cocktail of protease inhibitors). After Total RNA was extracted using TRI reagent (TAKARA Bio) according to the protein extraction, anti-MYC antibodies (cat. no. 05-724; Millipore) coupled to manufacturer’s recommendations. Reverse transcription was performed Protein-A sepharose beads (Sigma-Aldrich) were mixed and incubated for 4 h at using Moloney Murine Leukemia Virus reverse transcriptase (Dr. Protein) 4°C. The precipitated samples were washed at least four times with the protein with oligo(dT18) to synthesize first-strand cDNA from 2 mg of total RNA. extraction buffer and then eluted by 13 SDS-PAGE loading buffer to perform Total RNA samples were pretreated with an RNase-free DNase. cDNAs SDS-PAGE with anti-MYC (1:2,000 dilution; Millipore) or anti-GFP antibodies were diluted to 100 mL with TE buffer, and 1 mL of diluted cDNA was used (1:1,000 dilution; cat. no. sc-9996; Santa Cruz Biotechnology). for PCR amplification. RT-qPCR reactions were performed in 96-well blocks using the Step-One Plus Real-Time PCR System (Applied Biosystems). The PCR primers used are Statistical Analysis listed in Supplemental Table S5. The values for each set of primers were nor- malized relative to the EUKARYOTIC TRANSLATION INITIATION FACTOR Unless otherwise specified, quantitative data are presented as mean 6 SD and 4A1 (eIF4A) gene (At3g13920). All RT-qPCR reactions were performed with significance was assessed by the two-tailed Student’s t test. biological triplicates using total RNA samples extracted from three indepen- dent replicate samples. The comparative DDCT method was employed to eval- uate relative quantities of each amplified product in the samples. The threshold Accession Numbers cycle (CT) was automatically determined for each reaction with the analysis The raw sequencing data reported in this work have been deposited in the software set using default parameters. The specificity of the RT-qPCR reactions National Center for Biotechnology Information Gene Expression Omnibus was determined by melting curve analysis of the amplified products using the (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE109101. standard method employed by the software. Supplemental Data Y2H Assays The following supplemental materials are available. Y2H assays were performed using the BD Matchmaker system (Clontech). The pGADT7 vector was used for the GAL4 AD fusion, and the pGBKT7 vector Supplemental Figure S1. Diurnal expression of H2A.Z-related genes. was used for GAL4 BD fusion. The yeast strain AH109 harboring the LacZ and Supplemental Figure S2. Y2H assays. His reporter genes was used. PCR products were subcloned into the pGBKT7 and pGADT7 vectors. The expression constructs were cotransformed into yeast Supplemental Figure S3. Interaction of ELF3 with PIE1. AH109 cells and transformed cells were selected by growth on SD/-Leu/-Trp Supplemental Figure S4. H2A.Z occupancy on EC-target genes. medium and SD/-Leu/-Trp/-His/-Ade. Interactions between proteins were analyzed by measuring b-galactosidase activity using O -nitrophenyl- Supplemental Figure S5. H2A.Z enrichment at genes bound by both ELF3 b- D -galactopyranoside as a substrate. and LUX, but not differentially expressed in elf3-1 and lux-4 mutants. Supplemental Figure S6. H2A.Z enrichment at genes upregulated in elf3- BiFC Assays 1 and lux-4, but not bound by either ELF3 or LUX. Supplemental Figure S7. Reduced rhythmic amplitude in genetic mutants The ELF3 gene was fused in-frame to the 59 end of a gene sequence encoding of SWR1 components. the C-terminal half of EYFP in the pSATN-cEYFP-C1 vector (E3082). The SEF cDNA sequence was fused in-frame to the 59 end of a gene sequence encoding Supplemental Figure S8. Influences on circadian clock in hta9-1hta11-2. Plant Physiol. Vol. 182, 2020 623 Downloaded from on February 13, 2020 - Published by www.plantphysiol.org Copyright © 2020 American Society of Plant Biologists. All rights reserved. Tong et al. Supplemental Figure S9. Deposition of H2A.Z at clock gene promoters. coordinates environmental and endogenous signals in Arabidopsis. Nat Plants 3: 17087 Supplemental Figure S10. Binding of ELF3 to the PRR7 and PRR9 pro- Farinas B, Mas P (2011) Functional implication of the MYB transcription moters at ZT12. factor RVE8/LCL5 in the circadian control of histone acetylation. Plant J Supplemental Figure S11. Protein accumulation of ELF3 in pELF3::ELF3- 66: 318–329 MYC and pELF3::ELF3-MYCxarp6-1. Farré EM, Harmer SL, Harmon FG, Yanovsky MJ, Kay SA (2005) Over- lapping and distinct roles of PRR7 and PRR9 in the Arabidopsis circa- Supplemental Figure S12. ELF3 binding to the PRR loci in arp6- dian clock. Curr Biol 15: 47–54 1 background. Gendron JM, Pruneda-Paz JL, Doherty CJ, Gross AM, Kang SE, Kay SA Supplemental Figure S13. H2A.Z deposition in elf3-8 mutant throughout (2012) Arabidopsis circadian clock protein, TOC1, is a DNA-binding a day. transcription factor. Proc Natl Acad Sci USA 109: 3167–3172 Greenham K, McClung CR (2015) Integrating circadian dynamics with Supplemental Figure S14. Expression of CCA1, LHY, and PHYTO- physiological processes in plants. Nat Rev Genet 16: 598–610 CHROME-INTERACTING FACTOR4 in h2a.z and elf3-8 mutants. Guillemette B, Bataille AR, Gévry N, Adam M, Blanchette M, Robert F, Supplemental Figure S15. Protein accumulation of SEF in 35S:MYC-SEF Gaudreau L (2005) Variant histone H2A.Z is globally localized to the and 35S:MYC-SEFxelf3-8. promoters of inactive yeast genes and regulates nucleosome positioning. PLoS Biol 3: e384 Supplemental Table S1. EC target gene list. Hazen SP, Schultz TF, Pruneda-Paz JL, Borevitz JO, Ecker JR, Kay SA Supplemental Table S2. Fifty-two randomly selected control genes. (2005) LUX ARRHYTHMO encodes a Myb domain protein essential for circadian rhythms. Proc Natl Acad Sci USA 102: 10387–10392 Supplemental Table S3. List of genes bound by both ELF3 and LUX, but Herrero E, Kolmos E, Bujdoso N, Yuan Y, Wang M, Berns MC, Uhlworm not differentially expressed in elf3-1 and lux-4 mutants. 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