Song et al. Genome Biology (2019) 20:170 https://doi.org/10.1186/s13059-019-1777-1 RESEARCH Open Access Diurnal regulation of SDG2 and JMJ14 by circadian clock oscillators orchestrates histone modification rhythms in Arabidopsis Qingxin Song1,2,3†, Tien-Yu Huang1,2†, Helen H. Yu1,2, Atsumi Ando1,2, Paloma Mas4, Misook Ha5* and Z. Jeffrey Chen1,2,3* Abstract Background: Circadian rhythms modulate growth and development in all organisms through interlocking transcriptional-translational feedback loops. The transcriptional loop involves chromatin modifications of central circadian oscillators in mammals and plants. However, the molecular basis for rhythmic epigenetic modifications and circadian regulation is poorly understood. Results: Here we report a feedback relationship between diurnal regulation of circadian clock genes and histone modifications in Arabidopsis. On one hand, the circadian oscillators CCA1 and LHY regulate diurnal expression of genes coding for the eraser (JMJ14) directly and writer (SDG2) indirectly for H3K4me3 modification, leading to rhythmic H3K4me3 changes in target genes. On the other hand, expression of circadian oscillator genes including CCA1 and LHY is associated with H3K4me3 levels and decreased in the sdg2 mutant but increased in the jmj14 mutant. At the genome-wide level, diurnal rhythms of H3K4me3 and another histone mark H3K9ac are associated with diurnal regulation of 20–30% of the expressed genes. While the majority (86%) of H3K4me3 and H3K9ac target genes overlap, only 13% of morning-phased and 22% of evening-phased genes had both H3K4me3 and H3K9ac peaks, suggesting specific roles of different histone modifications in diurnal gene expression. Conclusions: Circadian clock genes promote diurnal regulation of SDG2 and JMJ14 expression, which in turn regulate rhythmic histone modification dynamics for the clock and its output genes. This reciprocal regulatory module between chromatin modifiers and circadian clock oscillators orchestrates diurnal gene expression that governs plant growth and development. Keywords: Diurnal, Histone modification, Arabidopsis Background plants [7, 11–14]. In mouse liver, there are time- Circadian clocks anticipate diurnal cycles to coordinate dependent patterns of transcription factor binding, physiological and developmental processes in all organisms RNA polymerase II recruitment, RNA abundance, and from cyanobacteria, flies, to humans and plants [1–3], chromatin states, which account for 22% of mRNA which are achieved through feedback transcriptional- cycling genes, suggesting a transcriptional mechanism translational regulatory modules [4–7]. In the tran- for regulating the mammal circadian network [8]. scriptional module, chromatin modifications regulate In Arabidopsis, the central transcriptional loop in- central circadian oscillators in mammals [8–10] and volves two morning-phased Myb-like transcription fac- tors, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) * Correspondence: misook.ha@gmail.com; zjchen@austin.utexas.edu and LATE ELONGATED HYPOCOTYL (LHY) [15, 16], † Qingxin Song and Tien-Yu Huang contributed equally to this work. which repress the evening-phased regulator, timing of 5 Samsung Advanced Institute of Technology, Samsung Electronics CAB expression 1 (TOC1), by directly binding to its pro- Corporation, Suwon 443-803, South Korea 1 Department of Molecular Biosciences, The University of Texas at Austin, moter [17, 18]. In turn, TOC1 indirectly promotes the Austin, TX 78712, USA expression of CCA1 and LHY by inhibiting the Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Song et al. Genome Biology (2019) 20:170 Page 2 of 12 repressive PSEUDO-RESPONSE REGULATOR (PRR) largely unknown. In Arabidopsis, five histone methyl- genes or directly represses expression of CCA1 and LHY transferases (ATX1, SDG2, SDG4, SDG25, and SDG26) by binding their promoters [19, 20]. Integrating other and three histone demethylases (JMJ14, JMJ15, and clock regulators including GI and ELF4–ELF3–LUX JMJ18) are reported to be responsible for adding and re- complex with the central CCA1/LHY/TOC1 loop pro- moving H3K4me3, respectively [28–35]. Among them, vides a classic feedback regulatory mechanism for the SDG2 and JMJ14 are associated with dramatic changes circadian clock in Arabidopsis [21, 22], which includes a of genome-wide H3K4me3 levels in their respective mu- number of core molecular oscillators that constitute tants [36–38], suggesting that these two genes may be interlocking transcriptional-translational negative feed- primary targets of the circadian clock regulation. To test back loops (see reviews in [2, 4, 7, 23, 24]). this, we examined expression patterns of SDG2 and Using candidate gene approaches, several studies have JMJ14 subjected to diurnal (24 h, 16 h light/8 h dark) indicated links between histone modifications and ex- and circadian conditions (48 h, constant light) in the pression of circadian clock genes. For example, induc- wild type (Col-0). SDG2 showed rhythmic expression tion of the morning-phased TOC1 expression wave form peaks under diurnal but not constant light conditions is related to histone acetylation, which is repressed by (Additional file 1: Figure S1a). However, JMJ14 showed CCA1 and coincident with histone deacetylase activities rhythmic expression patterns under both diurnal and and repressive chromatin status at dawn [12]. Rhythmic constant light conditions (Additional file 1: Figure S1b). expression of CCA1 and LHY and their reciprocal regu- Thus, expression patterns of SDG2 and JMJ14 are diur- lator TOC1 is associated with H3K4me3 and H3K9/14 nal and circadian, respectively. To further explore the ac levels in the translational start sites of these genes roles of clock genes in SDG2 and JMJ14 regulation, we [11, 13], but correlated negatively with H3K36me2 levels examined SDG2 and JMJ14 expression changes under di- [11]. Moreover, changes in diurnal transcript levels cor- urnal (24 h, 16 h light/8 h dark, LD) conditions in the relate with H3K9ac, H3K27ac, and H3S28p levels be- wild type (Ws) compared to the cca1 lhy double mutant tween end-of-day and end-of-night [25]. Inhibiting (Ws background) and in the CCA1 overexpression line acetylation and H3K4me3 abolishes rhythmic expression (CCA1-OX, Col-0 background) relative to Col-0. Under of circadian oscillators, while blocking H3K4me3 leads diurnal condition, the expression of SDG2 peaked in the to increased levels of clock-repressor binding, suggesting morning (ZT28, ZT0 or 24 = dawn) in the wild type and a transitional role for the H3K4me3 mark in modulating was significantly downregulated in the cca1 lhy mutant clock gene expression from activation to repression [14]. but upregulated in the CCA1-OX line (Fig. 1a). Expres- JMJD5, a histone demethylase in humans and plants, is sion of SDG2 in the WT started to increase from ZT24 co-regulated with evening-phased clock regulators and (dawn), and overexpressing CCA1 may have a “burst” ef- promotes expression of clock genes at dawn [26]. How- fect at ZT24 and accelerated the SDG2 expression peak ever, neither the basis for histone mark rhythms nor the (Fig. 1a), with a caveat of a potential genotypic effect relationship between histone modifications and circadian (Ws vs. Col). clock regulators is clearly defined. Here we report dy- In contrast to SDG2, JMJ14 expression peaked at night namic interactions between chromatin modification of (ZT40) and reached the valley after dawn (ZT28) in the circadian clock gene expression and diurnal regulation WT and was significantly upregulated in the cca1 lhy of histone modifications, which regulate diurnal gene ex- mutant and downregulated in the CCA1-OX line under pression networks in Arabidopsis. diurnal condition (Fig. 1b). The data suggest that CCA1/ LHY exerts temporal effects on positive and negative Results correlation, respectively, with SDG2 and JMJ14 expres- Diurnal regulation of histone methyltransferase and sion levels. Although both SDG2 and JMJ14 have one demethylase by clock genes CCA1-binding site (CBS) in their promoter sequences, The association of rhythmic histone modifications with SDG2 and JMJ14 were not reported as target genes of expression of individual circadian clock genes in Arabi- CCA1 according to the published dataset of chromatin dopsis thaliana [14] and with altered expression of the immunoprecipitation sequencing (ChIP-seq), which was core clock genes in Arabidopsis hybrids and allopoly- performed using CCA1p::CCA1-GFP transgenic plants ploids [27] may suggest a general role of histone modifi- at ZT2 and ZT14 [39]. To determine whether CCA1 can cations in diurnal regulation of the circadian clock and bind promoters of SDG2 and JMJ14 at different time its output genes. The diurnal rhythms are likely estab- points, we performed ChIP-qPCR using antibodies lished through diurnal transcriptional regulation of cor- against CCA1 at ZT0 when CCA expression peaks. responding genes or diurnal oscillation of histone- ChIP-qPCR showed significant enrichment of CCA1 in modifying enzymes. But the relationship between circa- the promoters of JMJ14 and TOC1 (normalized to dian clock regulators and histone-modifying factors is UBQ10), but not in the promoter of SDG2 (Fig. 1c). Song et al. Genome Biology (2019) 20:170 Page 3 of 12 a b c d e Fig. 1 The clock regulates expression of histone methyltransferase (SDG2) and demethylase (JMJ14) genes. a, b Relative expression levels (R.E.L.) of SDG2 (a) and JMJ14 (b) in the wild type (WT, Ws), cca1 lhy mutant (Ws) (upper panel), and WT (Col-0) and CCA1-OX (Col-0) (lower panel) plants under a diurnal cycle (16 h light/8 h dark). Black and white bars indicate dark (ZT16, 20, and 24) and light (ZT28, 32, 36, and 40), respectively. c ChIP-qPCR showed the fold enrichment (Y-axis) of CCA1-binding fractions in the promoters of SDG2 and JMJ14 using rosette tissues from 3-week- old A. thaliana at ZT0. The TOC1 promoter containing evening elements was used as the positive control. UBQ10 was used as a nonbinding control for normalization. Error bars in a–c indicate standard deviation of biological replicates (n = 3) with an asterisk showing the statistical significance level (P < 0.05, Student t test). d Binding profiles of H3K4me3 at the TSS ± 1.5 kb in the wild type and cca1 lhy mutant with two biological replicates. e Examples showing decreased binding of H3K4me3 to the genes AT1G21270 (upper panel) and AT3G05880 (lower panel) in cca1 lhy mutant compared with the WT (Ws) Although we could not exclude a possibility of non- mutant plants at dawn (ZT0) under diurnal conditions, specific binding of CCA1-antibodies, this result indicates when CCA1/LHY expression level peaked [41]. Compared that CCA1/LHY may directly bind to the JMJ14 pro- to the wild type, H3K4me3 levels near the transcription moter and regulates its expression, as other members start sites of genic regions were damped in the cca1 lhy (JMJ30 and JMJD5) of the gene family that are co- mutant (Fig. 1d). A total of 880 genes showed decreased regulated with evening-phased genes [26, 40], but may levels of H3K4me3 peaks (one-way ANOVA, P < 0.05) indirectly regulate SDG2 expression through other fac- (Additional file 2: Table S1), including two examples tors such as CCA1-mediated genes or in the CCA1 com- shown in Fig. 1e. A previous study has identified CCA1 plex. Alternatively, regulation of SDG2 by CCA1 could target genes by ChIP-seq using 2-week-old seedlings occur at a different time of the day. In addition, other grown under 12 h light/12 h dark for 12 days and then H3K4 methyltransferase ATX1 and H3K4 demethylase transferred to constant light for 2 days [39]. The overlap- JMJ15 were also down- and upregulated, respectively, in ping fraction is small between the CCA1 target genes (5%, the cca1 lhy mutant (Additional file 1: Figure S1c, d). 78/1433) and those of reduced H3K4me3 levels in the The data suggest that CCA1/LHY can upregulate ex- cca1 lhy mutant (P > 0.5, hypergeometric test). The results pression of H3K4me3 writers (SDG2 and ATX1) and indicate that CCA1 affects overall H3K4me3 accumula- downregulate H3K4me3 erasers (JMJ14 and JMJ15), tion levels but does not directly participate in the estab- which would result in the overall decreased distributions lishment of H3K4me3 marks for CCA1 target genes. of H3K4me3 in the cca1 lhy mutant. However, different growth conditions and tissue stages in Using ChIP-seq analysis with antibodies against these two studies may also contribute to the small over- H3K4me3, we analyzed H3K4me3 distribution patterns lapping fraction between the CCA1 target genes and genes in the rosette leaves of wild type (Ws) and cca1 lhy with reduced H3K4me3 levels in the cca1 lhy mutant. Song et al. Genome Biology (2019) 20:170 Page 4 of 12 Disrupted histone modifications lead to altered Dynamic diurnal rhythms of histone modifications in expressions of clock genes Arabidopsis To test how histone modifications affect expression of In animals and plants, histone marks like H3K4me3 and circadian clock genes, we analyzed H3K4me3 level H3K9ac are associated with active transcription and can changes of the clock genes in the sdg2 (10-day-old seed- sometimes be part of transcriptional activation [44, 45]. lings under long-day conditions) or jmj14 (3-week-old Clock-mediated diurnal expression of histone-modifying rosette leaves 16 h light/8 h dark) mutant using the pub- genes suggest rhythmic distributions of histone marks. To lished ChIP-seq datasets [42, 43]. Consistent with the examine diurnal rhythms of histone modifications, we previous report [14], disruption of SDG2 resulted in a used ChIP-seq to examine diurnal rhythms of genome- dramatic decrease of H3K4me3 accumulation levels in wide H3K4me3 levels every 3 h in a 24-h period using ros- most clock genes examined, including CCA1 and LHY ette leaves of A. thaliana grown under the long-day cycle (Fig. 2a), TOC1, PRR5, PRR7, PRR9, GI, ELF3, ELF4, and (16 h light/8 h dark). For comparative studies, we also per- LUX (Additional file 1: Figure S2). ChIP-qPCR using formed ChIP-seq experiments using another active his- rosette leaves at ZT0, ZT12, and ZT24 further con- tone mark H3K9ac. We identified a total of 15,151 firmed a significant reduction of H3K4me3 levels near H3K4me3 and 16,966 H3K9ac high-confidence peaks the 5′ region of CCA1 and LHY in the sdg2 mutant (Padj < 0.01, MACS), corresponding to 14,818 and 15,422 (Additional file 1: Figure S3a). Consequently, expression unique genes, respectively (Additional file 3: Table S2). levels of CCA1 and LHY were dramatically reduced in The distribution of H3K4me3 and H3K9ac in both even- the sdg2 mutant (Fig. 2b). In contrast to the results in ing and morning phases may suggest that these marks are the sdg2 mutant, H3K4me3 accumulation levels of also required for general transcriptional regulation [46]. CCA1, LHY, and several other clock genes were Of those peaks, 4420 (29%) H3K4me3 and 3493 (21%) slightly increased in the jmj14 mutant (Fig. 2c and H3K9ac peaks exhibited diurnal rhythms at a statistically Additional file 1: Figure S2). This moderate effect significant level (Padj < 0.01, JTK_CYCLE using Kendall’s may be related to the genetic redundancy of JMJ tau correlation and Bonferroni correction to adjust P demethylase family including six other members value) (Fig. 3a and Additional file 4: Table S3). Those os- (JMJ15, JMJ16, JMJ17, JMJ18, JMJ19, and JMJ30) in cillating H3K4me3 and H3K9ac peaks overlapped 4367 Arabidopsis [32, 34, 40]. ChIP-qPCR confirmed a sig- and 3276 genes, respectively. This distribution rhythm is nificant increase of H3K4me3 levels near the 5′ re- not likely affected by histone H3, and only 21 (0.5%) gion of CCA1 and LHY in the jmj14 mutant H3K4me3 and 32 (0.9%) H3K9ac rhythmic peaks exhib- (Additional file 1: Figure S3b), which correlated with ited histone H3 distribution rhythms at a statistically sig- increased expression levels of those clock genes nificant level (Padj < 0.01, JTK_CYCLE) (Additional file 1: (Fig. 2d). Figure S3c, d). Another study identified 657 and 1495 a b c d Fig. 2 Disrupting histone modifications affects expression of key circadian clock regulators. a ChIP-seq assays showing decreased binding of H3K4me3 to the genic regions of CCA1 (top) and LHY (bottom) in the sdg2 mutant compared with the wild type (WT). Green arrows next to the gene diagrams indicate transcriptional direction. b Relative expression levels (R.E.L.) of CCA1 (top) and LHY (bottom) in the sdg2 mutant compared with the WT at ZT0, ZT12, and ZT24 under a diurnal cycle (16 h light/8 h dark). Y-axis was broken as a result from large variation of expression levels. c ChIP-seq assays showing increased binding of H3K4me3 to the genic regions of CCA1 (top) and LHY (bottom) in the jmj14 mutant compared with the WT. d R.E.L of CCA1 (upper) and LHY (lower) in the jmj14 mutant compared with the WT under a diurnal cycle (16 h light/8 h dark). Error bars in b and d indicate standard deviation of biological replicates (n = 3) with an asterisk showing the statistical significance level (P < 0.05, Student t test) Song et al. Genome Biology (2019) 20:170 Page 5 of 12 a c b d e Fig. 3 Dynamics of diurnal histone modifications in Arabidopsis. a Heat-map view of H3K4me3 and H3K9ac binding signals every 3 h in a 24-h period (16 h light/8 h dark). The peaks are partitioned into the morning and evening clusters. The number of peaks in each cluster is indicated at the left side of the panel. b Overlap of genes with H3K4me3 and H3K9ac peaks. c Gene Ontology (GO) analysis of the genes with diurnal H3K4me3 or H3K9ac binding peaks. d Overlap between rhythmic H3K4me3 peaks and peaks with altered H3K4me3 enrichment in the sdg2 or jmj14 mutant compared with the WT. The asterisk indicates the statistical significance level for overlap with P < 0.05. e Phase distributions of the genes with rhythmic H3K4me3 or H3K9ac peaks in the morning phase (blue line) and evening phase (black line) genes with oscillating H3K4me3 and H3K9ac peaks, re- the Western blot analysis (Additional file 1: Figure S4b). spectively, using two time points (end-of-day and end-of- The ChIP-seq data further showed a diurnal rhythm of night) in the short-day condition [25]. Between two data- the total H3K4me3 levels with the peak at ZT12, whereas sets, 38% (250/657) and 28% (418/1495) of the genes with the total H3K9ac levels did not show obvious peaks during rhythmic H3K4me3 and H3K9ac peaks also showed the day and night (Additional file 1: Figure S4b). The in- oscillating H3K4me3 and H3K9ac rhythms in our consistency between diurnal H3K9ac distribution and glo- study (P < 1e−38, hypergeometric test). The difference bal H3K9ac content may be related to inconspicuous between these two studies could be related to the dif- H3K9ac distribution variation in the time points examined ferences in the time points (nine vs. two) examined in total H3K9ac levels (ZT0, 6, 12, 18, 24). Alternatively, and in the growth conditions (long-day vs short-day) non-rhythmic H3K9ac distribution may account for the used between two experiments. majority of overall H3K9ac variation because of its role in The phase distribution analysis indicated genome-wide other aspects of plant growth and development [47, 48]. binding rhythms of H3K4me3 with a major peak at The ChIP-seq data were further classified into the ZT12 and a minor peak at ZT6, whereas the major peak morning-phased (between ZT0 and ZT6) and evening- of H3K9ac binding rhythms was delayed towards ZT15 phased (between ZT9 and ZT18) peaks using k-medoids and the minor peak was moved forward to ZT3 clustering [49] (Fig. 3a and Additional file 4: Table S3). A (Additional file 1: Figure S4a). This is consistent with majority (86%, 14,029/16,391) of H3K4me3- or H3K9ac- total levels of H3K4me3 and H3K9ac as determined by enriched genes had both H3K4me3 and H3K9ac peaks, Song et al. Genome Biology (2019) 20:170 Page 6 of 12 and only a small proportion of genes was associated with expression with distinct morning-phased and evening- only one histone mark (Fig. 3b). This suggests a coordi- phased patterns [41] (Fig. 3e), indicating a strong cor- nated mechanism for H3K4me3 and H3K9ac to regulate relation between diurnal histone modifications and gene expression [45]. However, among morning-phased rhythmic gene expression. genes, only 13% (313/2335) were associated with both morning-phased H3K4me3 and H3K9ac peaks. Among Roles of histone modifications in core clock genes evening-phased genes, 696 genes (22%) were related to Further analysis of the ChIP-seq data showed rhythmic both evening-phased H3K4me3 and H3K9ac peaks patterns of either or both H3K4me3 and H3K9ac marks (Fig. 3b). The proportion of genes associated with both for all core clock genes examined, including CCA1, LHY, histone marks in morning-phased (13%) or evening- TOC1, PRR5, PRR7, GI, ELF3, and LUX (Fig. 4a–d and phased (22%) peaks was significantly lower than that Additional file 1: Figure S5 and S6). This confirms previ- enriched with either H3K4me3 or H3K9ac peaks (P < 2e ous reports about the correlation between oscillating −100, hypergeometric test). chromatin modifications and rhythmic expressions of Gene ontology (GO) analysis suggested that the morn- key clock genes, including LHY, CCA1, TOC1, PRRs, and ing- and evening-phased H3K4me3 and H3K9ac marks LUX [11–14]. Most core clock genes possessed both were enriched in genes with different gene ontology (GO) marks; the morning-expressed genes like CCA1 had groups (Fig. 3c). The morning-phased genes are enriched H3K4me3 and H3K9ac peaks at dawn (ZT0-ZT3) with photosynthetic activities and responses to a variety of (Fig. 4a), while the evening-expressed genes like TOC1 stresses, while the evening-phased genes are enriched with had H3K4me3 and H3K9ac peaks at noon (ZT12) DNA and RNA metabolism and protein ubiquitination (Fig. 4c). Expectedly, the transcript levels of CCA1 and and folding (Fig. 3c). These data implied a specificity for TOC1 correlated strongly with H3K4me3 and H3K9ac diurnal H3K4me3 and H3K9ac rhythms in the expression accumulation on a diurnal basis (Fig. 4b, d). This sug- of genes involved in different biological networks. A total gests a synchronization between circadian expression of 22% (958/4367) and 28% (1208/4367) genes with rhyth- and chromatin rhythms. mic H3K4me3 peaks in the sdg2 and jmj14 mutants, re- A possibility for expression rhythms and chromatin spectively, showed altered H3K4me3 levels (Fig. 3d), modifications is that the core circadian clock genes may which was 1.5-fold higher than those genes (12%, 1252/10, interact with chromatin-modifying enzymes to alter his- 503 and 17%, 1810/10,503) with non-rhythmic H3K4me3 tone modifications and regulate target gene expression. peaks in the mutants, respectively, displaying altered To test this, we analyzed binding targets of CCA1 and H3K4me3 levels (P < 1e−10, Fisher’s exact test). The data TOC1 from the published ChIP-seq datasets [20, 39], suggest that the genes with rhythmic histone modifica- and examined enrichment levels of H3K4me3 and tions are more sensitive to the loss of histone methyltrans- H3K9ac in the target genes. As CCA1 and LHY often re- ferase and demethylase activities than other genes. press expression of evening-phased genes by binding To examine the correlation between oscillating histone their promoters [39], the majority of CCA1 targets modifications and gene expression, we analyzed expression showed rhythmic expression patterns with an evening patterns of genes using published data of expression profil- phase. The results showed that approximately 33% and ing in Arabidopsis under diurnal (16 h light/8 h dark) and 20% of the CCA1 targets had evening-phased binding circadian (constant light) conditions [41]. For the genes patterns for H3K4me3 and H3K9ac (P < 1e−50, hyper- with both diurnal and circadian expression patterns, 14% geometric test), respectively, while fewer than 7% of (612/4377) had both rhythmic H3K4me3 and H3K9ac CCA1 targets exhibited morning-phased profiles for marks (Additional file 4: Table S3). However, the fraction H3K4me3 and H3K9ac (Fig. 4e). This finding is consist- of both rhythmic H3K4me3 and H3K9ac marks was much ent with the expression patterns of those CCA1 target lower for the genes with circadian but not diurnal expres- genes in the evening phase (Additional file 1: Figure S7), sion patterns (4%, 150/4134) or for the genes with diurnal although phased distributions of morning and evening but not circadian expression patterns (4%, 139/3121). These genes could change under long- or short-day conditions results suggest different impact of histone-modifying en- [41, 50]. In contrast, 23% and 13% of TOC1 target genes zymes in diurnal and/or circadian expression patterns. JMJ correlated with H3K4me3 and H3K9ac marks in the proteins are circadian-mediated probably through cor- morning phase (Fig. 4f) (P < 1e−5, hypergeometric test), egulation with the clock components [26, 40], while while a smaller proportion of target genes correlated SGD2 expression is indirectly regulated by CCA1 with those marks in the evening. This is consistent with (Fig. 1c and Additional file 1: Figure S1). At the TOC1 being a regulator of the morning-phased genes genome-wide level, ~ 48% (2104/4367) of the genes [19, 20] (Additional file 1: Figure S7). To further test the with oscillating H3K4me3 and ~ 47% (1552/3276) of roles of SDG2 and JMJ14 in circadian-mediated gene ex- the genes with oscillating H3K9ac displayed rhythmic pression, we randomly selected three CCA1 target genes Song et al. Genome Biology (2019) 20:170 Page 7 of 12 a b c d e f Fig. 4 Histone modifications mediate regulation of the clock genes. a, c Binding signals of H3K4me3 and H3K9ac at the genic regions of CCA1 (a) and TOC1 (c) at different time points. Each track represents the normalized ChIP-seq read coverage at a single time point. The Y-axis scales are 0– 15 for H3K4me3 and 0–10 for H3K9ac. ZT time is indicated on the left. b, d Quantifications of the ChIP-seq signals at diurnal peaks located in CCA1 (b) and TOC1 (d), respectively. Relative expression levels (R.E.L.) of CCA1 and TOC1 are shown (blue line). ACT7 was used as an internal control for gene expression. e, f Enrichment of CCA1 (e) and TOC1 (f) binding targets in the genes with rhythmic H3K4me3 or H3K9ac peaks. Error bars indicate the standard deviation of biological replicates (n = 3) with an asterisk showing the significant enrichment (P < 1e−5, hypergeometric test) and examined enrichment of H3K4me3 in genic region Discussion and conclusions of these genes in wild type and sdg2 and jmj14 mutants We provided a comprehensive genome-wide landscape of at ZT0, 12, and 24. Enrichment of H3K4me3 in all three histone modification dynamics in Arabidopsis, which has CCA1 target genes was significantly decreased in the supported diurnal regulation of histone modifications on sdg2 mutant at similar levels in the time points tested several individual circadian clock genes as previously re- but increased in the jmj14 mutant in most time points ported [11–14, 25, 26]. The current data support a model (Additional file 1: Figure S8). The effect in a few time that integrates clock rhythms with histone modifications points was moderate in the jmj14, probably due to gen- to establish rhythmic activities of gene expression, growth, etic redundancy, as observed for the clock genes (Fig. 2c and development (Fig. 5). Circadian clock genes CCA1 and Additional file 1: Figure S3). Notably, the H3K4me3 and LHY correlate indirectly with the upregulation of the levels in the sdg2 mutant are reduced at all time points histone methyltransferase gene SDG2 and directly with re- tested (Additional file 1: Figure S8a), consistent with that pression of the histone demethylase gene JMJ14 to coord- SDG2 is not directly regulated by CCA1 (Fig. 1c). JMJ14, inate histone modification levels in the downstream genes however, showed increased H3K4me3 levels with relative of the circadian output network. Coincidentally, recent rhythms (Additional file 1: Figure S8b), consistent with a studies showed REVEILLE8 interacted with NIGHT role for JMJ proteins in co-regulating with the circadian LIGHT-INDUCIBLE AND CLOCKREGULATED pro- clock and its target genes [26, 32]. These data indicate teins (LNKs) [51–53] to regulate RNA Pol II and that SDG2 and JMJ14 are involved in regulation of H3K4me3 occupancy on PRR5 and TOC1 loci to control rhythmic deposition of H3K4me3 in CCA1 target genes. their expression [54], further confirming the complex Song et al. Genome Biology (2019) 20:170 Page 8 of 12 phased genes (Fig. 3). We also noticed both H3K4me3 and H3K9ac showed lower levels at the night (ZT18-24) than during the day (Fig. 3a). One possibility is that total RNA transcription is lower at night than during the day, resulting in lower levels of H3K4me3 and H3K9ac modi- fications at night. Alternatively, activation of those genes at the night may not require both H3K4me3 and H3K9ac modifications. Other histone modifications could also be involved in evening-phased genes. Further examination of additional histone marks will provide a comprehensive view for diurnal rhythms of histone modifications and gene expression. For H3K4me3, the rhythm for regulating circadian- mediated gene expression is associated with expression of the H3K4me3 methyltransferase gene SDG2, which is expressed at significantly higher levels during the day than at night; on the other hand, the expression level of the Fig. 5 Histone modifications regulate expression of key circadian H3K4me3 demethylase gene (JMJ14) is low during the day clock regulators. A model showing feedback regulation between but high at night. This provides a basis for transcriptional histone H3K4 trimethylation (H3K4me3) and circadian clock regulation of rhythmic histone modifications. Indeed, both regulation through interactions with central clock oscillators (CCA1/ LHY) and H3K4me3 writers such as SDG2 and erasers such as JMJ14. global H3K4me3 and morning-phased H3K4me3 levels Solid lines demonstrated direct effects of positive (arrow) and peak during the day (ZT12) (Additional file 1: Figure S4), negative (stop) interactions between CCA1 and JMJ14 (red) and suggesting that H3K4me3 is largely established during the SDG2 (green); dashed lines proposed indirect effects of positive day. However, the global H3K4me3 peak is delayed 8 h, (arrow) and negative (stop) interactions between CCA1 and JMJ14 compared to the expression peak of H3K4me3 methyl- (red) and SDG2 (green). CCA1/LHY can form homodimers for autoregulation (gray lines) in positive (arrow) and negative transferase SDG2 (ZT4). This could be related to the pro- (stop) directions tein and mRNA that accumulate at different levels in a diurnal cycle. For example, CCA1 binds to the JMJ14 pro- moter at ZT0 in our ChIP-qPCR study but JMJ14 is not a interaction between circadian oscillator, histone modifica- target gene at ZT2 or ZT14 in the ChIP-seq analysis [39]. tions, and basal transcriptional machinery. It is predictable CCA1 protein peaks 3 h later than mRNA in maize [56]. that the dynamic histone modifications could change the The decrease of JMJ14 transcripts during the day period chromatin structures of genes and affect the accessibility may result in the presence of H3K4me3 for a longer time. of clock regulators and/or RNA Polymerase II, contribut- In addition, the dynamic histone modifications depend on ing to rhythmic expression of the target genes [7, 8]. On maximal activity of histone-modifying enzymes that may the other hand, histone methyltransferases and demethy- change over time. Notably, other members of H3K4me3 lases directly regulate the expression of core clock genes writers (e.g., ATX1) and erasers (e.g., JMJ15) may also through adding or removing histone modifications. This contribute to expression rhythms of circadian-mediated interactive and reciprocal regulation between circadian regulatory networks. clock oscillators and chromatin modifiers orchestrates di- How histone methyltransferases and histone demethy- urnal gene expression that controls growth and develop- lases are recruited to their target genes remains unknown. ment in plants and animals [6, 55]. One possibility is that histone methyltransferases such as A large proportion of histone modification sites exhibit ATX1 can be recruited by the transcriptional machinery rhythmic patterns, while different histone modifications through interaction with the Ser5-phosphorylated CTD of (H3K4me3 and H3K9ac) have distinct phases for their Pol II for transcription initiation and elongation [46]. Al- target genes, which is consistent with the report in ternatively, histone modifications could be established mammals [8]. The data collectively indicate a conserved through the interaction of sequence-specific transcription correlation between histone modifications and circadian factors with histone methyltransferases/demethylases to clock regulation in plants and mammals, as well as a regulate target gene expression [57, 58]. The targeting and specific distribution of different histone modifications in binding time of transcription regulators for specific se- morning-phased and evening-phased genes. This could quences could affect the time of H3K4me3 deposition. A be related to distinct peaks of H3K4me3 and H3K9ac in recent study reported that lysine-specific demethylase 1 the morning and evening phases, respectively, which (LSD1)-like histone demethylases (LDL1 and LDL2) can correspond to expression of morning- and evening- interact with CCA1/LHY to repress TOC1 expression Song et al. Genome Biology (2019) 20:170 Page 9 of 12 [59]. Moreover, LDL1 and LDL2 interact with HDA6 to PMSF, and 1x cocktail). The homogenized solution was fil- co-regulate TOC1 expression by histone demethylation tered through four layers of cheesecloth; the supernatant and deacetylation. It is probable that this type of histone was centrifuged at 3000g for 20 min at 4 °C. Pellets were re- complex can include JMJ14 and other factors to regulate suspended in 800 μl 0.4 N H2SO4 and incubated with a ro- expression of clock genes. It is equally plausible that his- tator overnight at 4 °C. After centrifugation at 16,000g at tone methyltransferases and acetyltransferases could be in 4 °C for 10 min, the supernatant was transferred into a new the same complex to carry out the opposite direction of tube by adding 264 μl trichloroacetic acid. Samples were in- circadian-mediated gene expression. It will be interesting cubated overnight at 4 °C and centrifuged again at 16,000g to further test how specific transcription factors including at 4 °C for 10 min to precipitate histone proteins. After circadian clock regulators recruit other histone methyl- washing by acetone, proteins were dissolved in water for transferases and demethylases to establish rhythmic his- Western blot analysis. The antibodies were anti-histone H3 tone modifications that exert growth and developmental (Abcam; Ab1791, 1:5000 dilution), anti-H3K4me3 (Abcam; regulation in plants and animals. Ab8580, 1:5000 dilution), and anti-H3K9Ac (Abcam; Ab10812, 1:5000 dilution). Methods Plant materials and growth conditions Chromatin immunoprecipitation (ChIP) and ChIP-seq A. thaliana ecotypes Col-0 and Ws were used in this Chromatin immunoprecipitation was performed following study. cca1 lhy mutant in Ws background and CCA1- the published protocol [61]. Briefly, 2 g of aerial rosette tis- OX transgenic lines in Col-0 background were gener- sues from ~ 3-week-old A. thaliana (Col-0) were cross- ated as previously described [27]. The mutants sdg2 linked in 1% formaldehyde, and the chromatin fractions (SALK-021008) and jmj14 (SALK_135712) were ob- were isolated and purified. Antibodies against histone H3 tained from Arabidopsis Biological Resource Center (Abcam; ab1791), H3K4me3 (Abcam; Ab8580), H3K9ac (ABRC). The primer sequences for genotyping are (Abcam; Ab10812), or CCA1 (Abiocode, R1234-3) were listed in Additional file 5: Table S4. For diurnal con- added to the chromatin extracts (with a 1:1000 dilution) ditions, plants were grown under the light/dark (L/D) and incubated overnight at 4 °C with gentle rotation. cycle of 16 h/L and 8 h/D at 22 °C. Immunoprecipitated DNA was extracted using Protein A/ G Magnetic Beads (Thermo Fisher Scientific, Waltham, RNA extraction and qRT-PCR Massachusetts). Immunoprecipitated DNA (IP DNA) and Total RNA was isolated from aerial rosette leaves of ~ 3- input DNA (without IP but with incubation of beads) were week-old plants using Plant RNA Reagent (Thermo subjected to reverse cross-linking and purified using QIA- Fisher Scientific, Waltham, Massachusetts). After diges- quick PCR Purification Kit (Qiagen, Valencia, California). tion by RNase-Free DNase (Promega, Madison, Wiscon- For ChIP-seq, libraries with three biological replicates sin), total RNA (1 μg) was used to produce first-strand were constructed from IP DNA and non-IP DNA using cDNA with the Omniscript RT Kit (Qiagen, Valencia, NEBNext® Ultra™ II DNA Library Prep Kit for Illumina California). The cDNA was used as the template for (NEB, Ipswich, Massachusetts). For ChIP-qPCR, immuno- qRT-PCR using FastStart Universal SYBR Green Master precipitated DNA was used as the template for qPCR in a LightCycler®96 System (Roche, Indianapolis, Indi- using FastStart Universal SYBR Green Master in a Light- ana). The relative expression level was quantified using Cycler®96 System (Roche, Indianapolis, Indiana). The rela- the internal control ACT7 (AT5G09810). Three bio- tive enrichment was quantified using the internal control logical replicates were performed for each experiment, AT2G26560. Three biological replicates were performed and three technical replicates were used for each bio- for each experiment with three technical replicates for logical replicate in qRT-PCR analysis. Student’s t test each biological replicate. Student’s t test was used to de- and calculation of error bars were performed to deter- termine the significance level in each comparison. mine the significance level in each comparison using three biological replicates. The primer sequences are ChIP-seq data analysis listed in Additional file 5: Table S4. ChIP-seq dataset generated in this study and the pub- lished ChIP-seq datasets in sdg2 and jmj14 mutants Histone extraction and Western blot analysis were analyzed as follows. After removing adapters, Histone extraction was performed as described previ- low-quality reads, and PCR duplicates, the filtered ously [60]. An aliquot (1 g) of aerial rosette leaves from pair-end reads were aligned to A. thaliana reference ~ 3-week-old plants were ground to powder with liquid genome (TAIR10) using Bowtie2 (Version 2.3.0) with nitrogen and then suspended in NIB buffer (250 mM su- parameters “bowtie2 --no-mixed --no-discordant” [62]. crose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM Only uniquely and concordantly mapped reads were CaCl2, 15 mM PIPES, pH 6.8, 0.8% Triton X-100, 1 mM used for further analysis. In order to adjust different Song et al. Genome Biology (2019) 20:170 Page 10 of 12 sequencing depths among different samples, the Ethics approval and consent to participate uniquely mapped reads were “down-sampled” to the Not applicable. lowest read number of samples, as previously de- scribed [56]. Enriched peaks were identified using Consent for publication Not applicable. Model-based Analysis of ChIP-Seq (MACS, version 2.1.0) with the option “macs2 callpeak -g dm -q 0.01” [63]. Peaks were used for further analysis only if they Competing interests All the authors declare that they have no competing interests. were present in two biological replicates. To make a master-peak list for all time points, the peaks ob- Author details 1 tained from each time point were merged. When dif- Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA. 2Department of Integrative Biology, The University of ferent samples were compared, the read coverage was Texas at Austin, Austin, TX 78712, USA. 3State Key Laboratory of Crop normalized to 1× sequencing depth to eliminate bias Genetics and Germplasm Enhancement, Nanjing Agricultural University, of different insertion lengths in pair-end reads. The Nanjing 210095, China. 4Center for Research in Agricultural Genomics (CRAG), Consortium CSIC-IRTA-UAB-UB, Campus UAB, Bellaterra, 08193 Barcelona, genes overlapping with peaks in the gene body or ± Spain. 5Samsung Advanced Institute of Technology, Samsung Electronics 1-kb regions were defined as peak-overlapped genes. Corporation, Suwon 443-803, South Korea. The peaks were compared between the wild type Received: 29 November 2018 Accepted: 29 July 2019 and cca1 lhy mutant using one-way ANOVA, and their significance was determined with P < 0.05. As no biological replicate was used from the ChIP-seq References datasets in sdg2 and jmj14 mutants, we used 1.5-fold 1. Bass J, Takahashi JS. Circadian integration of metabolism and energetics. changes to determine differential peaks between the Science. 2010;330(6009):1349–54. wild type and mutants. The non-parametric algo- 2. Greenham K, McClung CR. Integrating circadian dynamics with physiological processes in plants. 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