Ordered Changes in Histone Modifications at the Core of The

Ordered changes in histone modiﬁcations at the core of the Arabidopsis circadian clock

Jordi Malapeira1, Lucie Crhak Khaitova1, and Paloma Mas2

Molecular Genetics Department, Center for Research in Agricultural Genomics (CRAG), Consortium Consejo Superior de Investigaciones Cientíﬁcas–Institut de Recerca i Tecnologia Agroalimentaries–Universitat Autònoma de Barcelona–Universitat de Barcelona, Campus Universitat Autònoma de Barcelona, 08193 Barcelona, Spain

Edited* by Steve A. Kay, University of California at San Diego, La Jolla, CA, and approved November 15, 2012 (received for review October 1, 2012) Circadian clock function in Arabidopsis thaliana relies on a com- RELATED 3 (SDG2/ATXR3) was proposed to play a major role plex network of reciprocal regulations among oscillator components. in H3K4 trimethylation (H3K4me3) in Arabidopsis (16, 17). Loss Here, we demonstrate that chromatin remodeling is a prevalent of SDG2/ATXR3 function results in pleiotropic phenotypes, as well regulatory mechanism at the core of the clock. The peak-to-trough as a global decrease of H3K4me3 accumulation and altered ex- circadian oscillation is paralleled by the sequential accumulation of pression of a large number of genes. H3 acetylation (H3K56ac, K9ac), H3K4 trimethylation (H3K4me3), A precise regulation of gene expression is not only essential and H3K4me2. Inhibition of acetylation and H3K4me3 abolishes os- for plant responses to environmental stresses and developmental cillator gene expression, indicating that both marks are essential for transitions but also for proper function of the circadian clock. gene activation. Mechanistically, blocking H3K4me3 leads to in- The circadian clockwork allows plants to anticipate environ- creased clock-repressor binding, suggesting that H3K4me3 functions mental changes and adapt their activity to the most appropriate as a transition mark modulating the progression from activation to time of day (18). In Arabidopsis thaliana, the core of the oscillator repression. The histone methyltransferase SET DOMAIN GROUP 2/ is composed of morning- and evening-expressed components that ARABIDOPSIS TRITHORAX RELATED 3 (SDG2/ATXR3) might contrib- regulate their expression in a highly complex network of inter- ute directly or indirectly to this regulation because oscillator gene connections (19). The transcription factors CCA1 (CIRCADIAN expression, H3K4me3 accumulation, and repressor binding are al- CLOCK ASSOCIATED 1) (20), LHY (LATE ELONGATED

tered in plants misexpressing SDG2/ATXR3. Despite divergences in HYPOCOTYL) (21), and the PSEUDO-RESPONSE REGU- PLANT BIOLOGY oscillator components, a chromatin-dependent mechanism of clock LATOR (PRR) proteins (PRR9, -7, and -5) (22) have peak gene activation appears to be common to both plant and mammal phases of expression during the day, whereas TOC1 (TIMING circadian systems. OF CAB EXPRESSION 1 or PRR1) (23, 24), GI (GIGANTEA) (25, 26), and the members of the Evening Complex (EC) (27), all histone acetylation | histone methylation | circadian rhythms have evening peak-phase oscillations. The transcriptional repressing activity of TOC1 has been recently added to the myriad plant he functional properties of chromatin are modulated by var- clock repressors (28, 29), opening the question about the compo- Tious mechanisms, including, among others, posttranslational nents and mechanisms responsible for activation (30). modifications of histones, incorporation of histone variants, and The large fraction of genes regulated by the circadian clock DNA methylation (1, 2). Among histone covalent modifications, suggests that transcriptional control occurs by higher-order acetylation at specific lysine residues of the N-terminal histone changes in chromatin structure. Indeed, histone modifications tails appears to make DNA more accessible to the transcription are coupled with the generation of rhythms at the core of the machinery, which has been correlated with active transcription oscillator. Specifically, rhythmic changes in the pattern of H3 (3). Histone methylation, on the other hand, may facilitate the acetylation at TOC1 promoter correlate with TOC1 circadian recruitment of chromatin remodeling factors that can either ac- expression (31). Two MYB transcription factors antagonistically tivate or repress transcription, depending on the particular resi- contribute to this regulation. At dawn, CCA1 represses TOC1 due that is methylated and the degree of methylation (4). The expression by promoting histone deacetylation, whereas REV- sequential or combinatorial composition of these histone mod- EILLE 8/LHY-CCA1-LIKE 5 acts as an activator and facilitates ifications facilitates the switch between permissive and repressive histone acetylation (31, 32). A recent report has extended this states of chromatin that ultimately modulate the genome activity analysis showing that CCA1 and LHY are also subjected to in eukaryotes (5). rhythmic changes in H3 acetylation and methylation (33). Histone Epigenetic regulation mediated by histone modifications is demethylation might also be connected with the Arabidopsis cir- also well conserved in plants (6), with a key role in the control of cadian clock because JUMONJI DOMAIN CONTAINING 5/30 developmental transitions and plant responses to stress (7). (JMD5/JMD30) loss-of-function and JMD5/JMD30-overexpressing Among others, processes such as flowering time, transposon re- plants affect circadian period by the clock (34, 35). pression, light signaling, and abiotic stress responses are modu- Here, we have investigated the spatiotemporal distribution of lated by posttranslational modifications of histones on specific daily chromatin transitions and show their functional roles at the residues (e.g., refs. 8–12). The genomic distribution and com- core of the oscillator. The main activating histone marks, H3 binatorial association of different histone marks in Arabidopsis acetylation and H3K4me3, are enriched around the 5′ end of the appear to define various chromatin states that can be correlated with transcriptionally active or inactive genes (13). More than two-thirds of the Arabidopsis genes contain at least Author contributions: P.M. designed research; J.M., L.C.K., and P.M. performed research; one type of H3K4me (14). The enzymes responsible for meth- J.M., L.C.K., and P.M. analyzed data; and P.M. wrote the paper. ylation are histone lysine methyltransferases (HKMTases), which The authors declare no conflict of interest. usually contain a conserved SET domain that harbors the enzy- *This Direct Submission article had a prearranged editor. matic activity (15). The SET domain proteins in Arabidopsis 1J.M. and L.C.K. contributed equally to this work. belong to evolutionarily conserved classes with different spe- 2To whom correspondence should be addressed. E-mail: [email protected]. fi ci cities and different outcomes on chromatin structure (15). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. The SET DOMAIN GROUP 2/ARABIDOPSIS TRITHORAX 1073/pnas.1217022110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1217022110 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 oscillator genes and regulate the timing of the oscillator gene compared with ZT15. In contrast, the pattern of these histone expression. Despite their similar activating function, acetylation marks clearly accumulated at ZT15 in the evening-expressed and trimethylation oscillate with a different phase, which sug- oscillator genes (TOC1 and LUX ARRYTHMO)(Figs. S1 and gests a degree of specificity in their respective clock-related roles. S2). The timing of these histone marks suggest that the temporal Reduction of H3K4me3 is concomitant with increased clock- modulation of H3K56ac and H3K4me3 is in tune with the cir- repressor binding, suggesting that H3K4me3 might impede an cadian gene expression. These results are in agreement with the advanced phase of repressor activity, thus facilitating the proper previously described connection of H3ac and H3K4me3 with transition from activation to repression. Molecularly, the use of gene activation (13, 33). The phase differences in the rhythms of plants misexpressing SDG2/ATXR3 reveals that this histone histone marks among different genes suggest that chromatin methyltransferase might contribute to H3K4me3 at the core of remodeling is an intrinsic mechanism at the core of the clock. fi the clock. Collectively, our studies indicate that the precise timing Genome-wide analyses have de ned H3K27 methylation as a and combinatorial accumulation of histone marks are essential major mechanism for silencing of a large number of plant genes for proper transcriptional regulation at the core of the clock. (7). The histone mark H3K9me3 is also known to be associated with 40% of Arabidopsis genes, with no overlap with H3K27me3 Results and Discussion (36). We, therefore, examined whether these histone marks could Specific Distribution of Histone Marks Along the Genomic Structure of also contribute to circadian expression. We used WT plants syn- the Oscillator Genes. To gain further insights into the connection chronized under LD cycles and performed ChIP assays with of chromatin remodeling at the core of the clock, we performed H3K27me3, H3K27me2, and H3K9me3 antibodies. Our results showed that the ChIP signal was, overall, very low and close to ChIP assays at Zeitgeber Time 3 and 15 (ZT3 and ZT15) in wild- background for each antibody, with no evident or reproducible type (WT) plants grown under 12-h light/12-h dark (LD) cycles. enrichment at a particular time or genomic region, compared Analyses were initiated with antibodies to H3K56 acetylated with negative controls processed without antibodies (Fig. S3). (H3K56ac) and H3K4 trimethylated (H3K4me3), and different The results indicate that H3K27me3, H3K27me2, and H3K9me3 regions were amplified along the genomic structure of the main fi fi do not signi cantly contribute to the regulation of circadian gene oscillator genes. We found speci c enrichment of H3K56ac and expression at the core of the clock. H3K4me3 around the 5′ end of both sets of core genes (Fig. S1), fi fi whereas no signi cant ampli cation was obtained in samples Rhythms in Histone Marks at the Core of the Arabidopsis Oscillator. similarly processed but omitting the antibody in the ChIP assay We next performed a time course analysis of H3K56ac and (Figs. S1 and S2). The results are consistent with a recent publi- H3K4me3 accumulation over a 24-h diurnal cycle. We found that cation showing the distribution of H3K4me3 and H3ac on TOC1, for the morning and the evening set of core genes, both marks CCA1, and LHY loci (33). The decrease in 5′ to 3′ direction along followed a robust oscillation that resembled the rhythmic ex- the oscillator genes suggest that both types of modifications pression of the respective transcripts (Fig. 1). The phase of might dissociate from the elongating RNA–polymerase II dur- H3K56ac matched that of the mRNA accumulation, with the ing RNA synthesis. peak of H3K56ac temporally coinciding with the peak of mRNA The morning-expressed oscillator genes (LHY, PRR9, CCA1, (Fig. 1 A–F). The rhythmic acetylation of H3K9 was very similar and PRR7) exhibited increased H3K56ac and H3K4me3 at ZT3 to that observed for H3K56 (Fig. S4), which is consistent with

Fig. 1. Rhythmic oscillation of H3K56Ac and H3K4Me3 and its association with oscillator gene expression. ChIP assays of H3K56Ac (A–F) and H3K4me3 (G–L) were performed in WT plants sampled every 4 h over a 24-h LD cycle. Primers encompassing the peak of H3K56Ac and H3K4me3 for each gene (Fig. S1) were used to amplify LHY (A and G), CCA1 (B and H), PRR9 (C and I), PRR7 (D and J), TOC1 (E and K), and LUX (F and L). Values are represented as means ± SEM. Enrichment was normalized relative to the control At5g55840. Expression proﬁling for each gene is overlapped in each graph (red line). Gene expression was analyzed in plants grown under LD cycles. mRNA abundance was normalized to IPP2 (isopentenyl pyrophosphate:dimethyl-allyl pyrophosphate isomerase) expression. Values are represented as means ± SEM. Data were normalized relative to the maximum value to facilitate comparisons between the ChIP and the expression assays and among the different genes at the various time points.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1217022110 Malapeira et al. Downloaded by guest on September 30, 2021 previous findings focused on the TOC1 promoter (31). The phase a dynamic and complex pattern of histone marks with the se- of H3K4me3, on the other hand, was delayed, with a peak about quential enrichment H3ac→H3K4me3→H3K4me2 that tempo- 4 h later, compared with the peak of H3K56ac accumulation (Fig. rally correlates with the peak-to-trough rhythmic expression. 1 G–L and Fig. S5). Slower decay of H3K4me3 was also observed H3ac and H3K4me3 coexist during gene activation, whereas during the declining phase of H3K56ac and mRNA oscillation. H3K4me3 and H3K4me2 share high or moderate abundance These results suggest that the rhythms in H3ac and H3K4me3 during the declining phase and trough. might facilitate the transcriptional activation of the morning- and evening-expressed oscillator genes. The oscillations in histone Pharmacological Inhibition of Histone Marks Alters Oscillator Gene marks might rhythmically change the chromatin structure, con- Expression. To explore whether the rhythms in histone marks ferring differential accessibility to the transcriptional machinery are associated with transcriptional regulation, we examined and regulators controlling the expression of the genes at a specific luminescence from seedlings expressing the CCA1 and TOC1 time of the day. The differential pattern of accumulation and the promoter fused to the luciferase (CCA1::LUC, TOC1::LUC)in differences in peak phases also open the possibility of specific the absence or in the presence of specific inhibitors. The roles for acetylation and K4me3. rhythms in seedlings treated with nicotinamide (NAM) [shown Histone demethylation has been connected with the Arabi- previously to decrease H3K4me3 (37)] showed a delayed phase dopsis circadian clock (34, 35). Although its mechanism of action and a long-period phenotype that was accompanied by a reduced remains unknown, it is plausible that H3K4me3 could serve as a amplitude of both CCA1::LUC and TOC1::LUC expression substrate for histone demethylases. If that is the case, the oscil- (Fig. 2 A–D and Fig. S7). Similar results were obtained irre- lations in H3K4me3 could be followed by rhythms in H3K4me2. spective of the time of NAM treatment and when gene ex- Indeed, our time-course analyses of H3K4me2 over the diurnal pression was examined by RT–quantitative PCR (RT-Q-PCR) cycle showed that the peak of H3K4me2 was nearly antiphasic (Fig. S7). The inhibitory effect of NAM correlated with a sig- to H3ac, with higher H3K4me2 accumulation at times when H3ac nificant decrease in H3K4me3 both at the peak and the trough is low and vice versa (Fig. S6). This pattern was particularly evi- of gene expression (Fig. 2 I–J).NAMcanalsoinhibithistone dent for the morning-expressed genes. The fact that H3K4me2 deacetylase activity (38). However, our analysis showed that accumulates when H3ac and the mRNA are low suggests that H3K56ac accumulation was reduced in NAM-treated plants H3K4me2 might function as a repressive mark contributing to the (Fig. S8). These results are consistent with the decreased am- low abundance of the morning-expressed genes during the night plitude of oscillator gene expression and with the proposed (Fig. S6). In contrast, H3K4me2 accumulation appears to mark interconnection of methylation and acetylation. Treatment with PLANT BIOLOGY gene repression during the first part of the day for the evening- NAM of plants expressing the promoter of the output gene CCR2 expressed genes. Comparisons of H3K4me2 and H3K4me3 (COLD-CIRCADIAN RHYTHM-RNA BINDING2)fusedto revealed a certain degree of coexistence of both marks around the luciferase (CCR2::LUC) (24) altered the period, as expec- dusk for the morning set of core genes and around dawn for the ted, but did not affect the amplitude of the rhythms (Fig. S7), evening-expressed genes (Fig. S6). Collectively, our studies reveal which suggests that the effects of NAM on the amplitude of the

Fig. 2. Effects of blocking histone K4 trimethylation and acetylation on clock gene expression. CCA1::LUC (A, E, and K) and TOC1::LUC (C, G, and L) luminescence in WT plants entrained under LD cycles and subsequently released to constant light (LL) conditions. Luminescence was examined in the absenceor presence of NAM (A and C), C646 (E and G), or C646 plus NAM (K and L). Period estimates of CCA1::LUC (B and F)andTOC1::LUC (D and H) expression from individual traces examined by fast Fourier transform–nonlinear least-squares (FFT-NLLS) best-ﬁt algorithm analysis. ChIP analysis of H3K4me3 in the presence and in the absence of NAM at ZT3 (I) and ZT15 (J). Enrichment was normalized relative to the control At2g26560 (17). Two-tailed t test analysis with 95% of conﬁdence shows the statistical relevance of the differences: **P < 0.005; ***P < 0.0001. Arrows indicate the circadian time of inhibitor administration. Data are the means ± SEM of the luminescence of 5–10 individual seedlings.

Malapeira et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 oscillator genes are direct and speciﬁc. We favor the scenario in which the oscillator activity is, overall, reduced by NAM, which renders a clock to run slower than in nontreated plants. NAM + + also affects the amplitude of cytosolic Ca2 ([Ca2 ]cyt) oscillation (39). However, our analyses were performed with plants grown + on sucrose, which abolishes the oscillation of [Ca2 ]cyt (40). The effects of NAM are not likely attributable to alteration of poly (ADP ribose) polymerase (PARP) (41) because treatment with the PARP inhibitor thymidine (41) does not phenocopy the effects of NAM (Fig. S7). Consistent with our ChIP experiments, blocking histone acetylation by inhibition with α-methylene- γ-butyrolactone (MB-3) (42) or with C646 (43) reduced histone acetylation and also affected CCA1::LUC and TOC1::LUC expression (Fig. 2 E–H and Fig. S8). The repressing effects were observed very rapidly following treatment (Fig. 2 E and G), suggesting that the decrease in histone acetylation and trimethylation is directly responsible for the repression. Notably, the com- bined use of NAM and C646 completely abolished the rhythmic expression (Fig. 2 K and L), which suggests that acetylation and H3K4me3 are essential marks contributing to the raising phase of CCA1 and TOC1.

Inhibition of H3K4me3 Leads to Increased Clock-Repressor Binding and Gene Repression. Both H3ac and H3K4me3 function as activating marks. However, H3K4me3 extends temporally beyond the peak of H3ac, overlapping with both H3ac and H3K4me2. Ex- tended accumulation of H3K4me3 might be helpful to modulate the progression from gene activation to repression. If that is the case, then the recruitment of clock repressors to the promoters of the oscillator genes might be influenced by the status of H3K4me3. To check this possibility, we examined by ChIP assays the effects of NAM treatments on TOC1 binding to its target genes LHY and CCA1. Our studies revealed a significant increase (P < 0.05) of TOC1 binding to the CCA1 and LHY promoters in plants treated with NAM compared with mock-treated plants (Fig. 3A and Fig. S9), whereas no significant enrichment was observed when toc1-2 mutant plants were used for the ChIP assays (Fig. S9). These results are noteworthy because NAM treatment reduces the expression of TOC1. To avoid possible interferences attributable to the changes in TOC1 expression by NAM, we also checked TOC1 binding in plants overexpressing TOC1 under the 35S promoter. Similar to our results in WT plants, we observed a significant increment (P < 0.01) of TOC1 binding in plants treated with NAM (Fig. 3 B and C). The effects are not restricted Fig. 3. Effects of blocking histone K4 trimethylation on clock-repressor to the evening-expressed repressors, because binding of CCA1 to binding and oscillator gene expression. ChIP assays of TOC1 binding to LHY its target, TOC1, was also significantly increased (P < 0.001) in in WT plants (A) and binding to LHY (B) and CCA1 (C) in TOC1-ox plants. Plants were grown under LD cycles, and samples were analyzed at ZT3, ZT7 CCA1-overexpressing plants treated with NAM (Fig. 3D). and ZT11. (D) ChIP assays of CCA1 binding to TOC1 at ZT3 and ZT15 using To further corroborate our hypotheses, we also compared primers F5 and F10 (Fig. 1). Binding was examined in the absence (-NAM) or binding of the transcriptional repressor PRR5 in the presence or in the presence of NAM (+NAM). ChIP assays of PRR5-ind plants grown under in the absence of NAM. Previous studies have shown that PRR5 LD cycles. Samples were analyzed at ZT3, ZT7, and ZT11. Binding of PRR5 to represses CCA1 and LHY expression by direct binding to their CCA1 (E) and LHY (F) was examined in plants treated with Dex in the ab- promoters (44). We used plants expressing the fusion of PRR5 sence (-NAM) or in the presence of NAM (+NAM). Two-tailed t test analysis to the hormone-binding domain of mouse glucocorticoid re- with 99% of confidence shows the statistical relevance of the differences: < < < ceptor (GR) under the control of the Cauliflower Mosaic Virus *P 0.05; **P 0.005; ***P 0.0001. CCA1::LUC luminescence in PRR5-ind plants entrained under LD cycles and subsequently released to constant (CaMV) 35S promoter [PRR5-inducible (PRR5-ind)] (44). The light (LL) conditions. Luminescence was examined in the absence or in the advantages of using the inducible system are twofold. On one presence of Dex (G). Luminescence in plants treated with Dex without or hand, expression is controlled by the CaMV 35S promoter, which with NAM (H, L, and J). Arrows indicate the circadian time of NAM or Dex avoids the possible effects of NAM on the PRR5 promoter. On administration. the other hand, the fact that the GR fusion protein only becomes biologically functional in the presence of dexamethasone (Dex) (45) avoids the possible clock phenotypes that are inherent in of H3K4me3 might indeed rely on modulating the timing of constant overexpression. Our ChIP assays with the inducible clock-repressor binding. plants revealed a significant increase (P < 0.01) of PRR5 binding As chromatin residency does not always correlate with tran- to the CCA1 and LHY promoters in plants treated with Dex and scriptional regulation, we next examined CCA1::LUC lumines- NAM compared with plants treated only with Dex (Fig. 3 E and cence in PRR5-ind plants in the presence and in the absence of F). The reduction of H3K4me3 accumulation by NAM and the NAM. We reasoned that if H3K4me3 modulates the transition increased repressor binding suggest that the activating function from activation to repression then the increased binding of

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1217022110 Malapeira et al. Downloaded by guest on September 30, 2021 PRR5 by treatment with NAM should be also associated with increased gene repression. Our studies showed that induction of PRR5 led to a signiﬁcant decrease on CCA1::LUC expression (Fig. 3G), consistent with the previously described repressing function of PRR5 (44). Administration of NAM to Dex-treated plants drastically reduced CCA1::LUC luminescence, with lower signals than those observed for plants treated only with NAM (Fig. 3H). The repressing effects were observed very rapidly following treatment of Dex and/or NAM (Fig. 3 I and J), suggesting that the reduced accumulation of H3K4me3 and the function of PRR5 as an effector molecule are directly responsible for the repression. Our results, thus, show that reduction of H3K4me3 leads to increased PRR5 binding and enhanced repression of CCA1, which suggests that H3K4me3 might modulate directly or indirectly the transition from activation to repression. Together, our studies suggest that the changes in H3K4me3 directly or indirectly affect the pattern of clock-repressor binding to its target genes. This notion is supported by our ﬁndings showing that blocking H3K4me3 increases clock-repressor binding and enhances gene repression. Notably, a delay in the rhythmic oscillation of H3K4me3 relative to H3K9ac was reported at the promoter of the clock component mouse albumin D element– binding protein (DBP) (46). It would be interesting to check whether in the animal circadian systems, H3K4me3 also functions as a transition mark facilitating the proper timing from activation to repression. Clock repressors in mammals (47) and in plants (31) seem to antagonize histone acetyltransferase activities at the

core of the clock. It is possible that the observed delayed accu- PLANT BIOLOGY mulation of H3K4me3 might “protect” histone acetylation, buffering against the antagonistic function of clock repressors. Fig. 4. SDG2/ATXR3 might function as a histone methyltransferase at the This notion is consistent with our results showing that H3ac ac- core of the clock. ChIP analysis of H3K4me3 accumulation in WT plants and cumulation was reduced in NAM-treated plants. in heterozygous sdg2 mutants. Plants were grown under LD cycles, and samples were analyzed at ZT3 and ZT15. H3K4me3 enrichment at the CCA1 SDG2/ATXR3 Might Function as a Histone Methyltransferase Respon- and LHY (A) and PRR9 and PRR7 (B) promoters was normalized relative to sible for H3K4me3 at the Core of the Clock. Among more than 30 the control At2g26560 (17). WT(-) and sdg2(-) denote samples processed proteins containing the SDG domain (48), the histone lysine omitting the antibody in the ChIP assay. (C) RT-Q-PCR analysis of oscillator methyltransferase SDG2/ATXR3 was proposed to play an im- gene expression in WT plants and in heterozygous (het) and homozygous portant role in H3K4me3 in Arabidopsis (17). We, therefore, (hom) sdg2 mutants. Plants were grown under LD cycles, and samples were examined the possible function of SDG2/ATXR3 in the regula- analyzed at ZT3. Values were normalized relative to the control At3g01610 tion of histone methylation at the core of the Arabidopsis clock. (17). (D) ChIP analysis of H3K4me3 accumulation in WT plants and in SDG2/ ATXR3 complementation lines (compl) expressing the genomic fragment Loss of SDG2/ATXR3 function results in very severe develop- containing the SDG2/ATXR3 locus (17). Plants were grown under LD cycles, mental phenotypes (17) that complicate the proper interpretation of and samples were analyzed at ZT3. H3K4me3 enrichment at the CCA1, PRR9, the ChIP results. We, therefore, analyzed H3K4me3 accumulation and TOC1 loci was normalized relative to the control At2g26560 (17). WT(-) and in heterozygous sdg2/atxr3 mutants (17). We reasoned that if SDG2compl(-) denote samples processed omitting the antibody in the ChIP SDG2/ATXR3 has an important function regulating the clock, assay. (E) Analysis of TOC1 binding to its target genes in WT and in SDG2/ then a minor reduction of SDG2/ATXR3 activity might lead to ATXR3 complementation lines. Plants were grown under LD cycles, and detectable changes in the pattern of H3K4me3. Indeed, our samples were analyzed at ZT3. TOC1 binding was also examined in SDG2/ fi < ATXR3 complementation lines treated with NAM (+NAM). Two-tailed t test results showed a small but signi cant reduction (P 0.01) fi of H3K4me3 in the heterozygous mutant plants compared analysis with 99% of con dence shows the statistical relevance of the differences: *P < 0.05; **P < 0.01; ***P < 0.001. (F) Scheme depicting the with WT (Fig. 4 A and B and Fig. S9). The changes in H3K4me3 combinatorial sequence of chromatin marks accompanying the waveform of were also accompanied by a reduced expression of the oscillator the oscillator genes. The marks prevalent at every phase are encircled by the genes (Fig. 4C and Fig. S9). Notably, we observed the opposite oval line. Overlapping marks are also denoted. The SDG2 function facilitat- phenotypes (i.e., increased H3K4me3; Fig. 4D) when we used ing the accumulation of H3K4me3 is denoted by the arrows. Modulation of SDG2/ATXR3 complementation lines expressing the genomic the timing of repressor binding by H3K4me3 is depicted by the lines ending fragment containing the SDG2/ATXR3 locus in the sdg2/atxr3 in perpendicular dashes. mutant background (17). Based on our results showing that the pattern of H3K4me3 fi modulates the binding and activity of effector molecules, we next lines) signi cantly affect H3K4me3 accumulation and repressor examined whether the changes of H3K4me3 in the sdg2/atxr3 binding, which suggests that the oscillator genes are very sensi- complementation lines could affect the binding of TOC1 to its tive to slight changes in H3K4me3. target genes. Our ChIP assays showed a slight but significant The current model of the Arabidopsis oscillator comprises a decrease of TOC1 binding in the complementation lines com- myriad of repressors at the core of the clock, but little is known pared with that observed in WT plants (Fig. 4E). The reduced about the mechanisms responsible for activation. Our data de- binding was reverted when the complementation lines were fine histone acetylation and trimethylation as key elements treated with NAM (Fig. 4E), suggesting that, indeed, the changes contributing to oscillator gene activation (Fig. 4F). This con- in H3K4me3 are directly responsible for the phenotypes. Our clusion is consistent with findings in the mammalian clockwork, results, thus, indicate that minor changes in SDG2/ATXR3 ex- showing that CLOCK, the main positive component of the pression (in the heterozygous mutant and in the complementation mammalian oscillator, has histone acetyltransferase activity that

Malapeira et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 is essential for the rhythmic acetylation of histones and non- (52). The list of primers used is shown in Table S1. Details of the analysis and histone circadian proteins and for proper functioning of the the ChIP experiments are described in SI Materials and Methods. clock (49). A recent study has further extended this concept, providing a genome-wide view of chromatin remodeling and Gene Expression Analysis. RNA was isolated (53) using the Purelink Total ﬁ RNA-polymerase II recruitment at the core of the mammalian RNA puri cation system (Invitrogen) as previously described. The list of ﬁ primersusedisshowninTable S1. Details are provided in SI Materials clock (50). Our ndings and those showing that the activation of and Methods. mammalian clock genes is coupled with changes in H3ac and H3K4me3 indicate that despite divergences in oscillator com- ACKNOWLEDGMENTS. We thank Dr. T. Stratmann for comments on the ponents, a chromatin-dependent mechanism of clock gene acti- manuscript; Dr. Xiaoyu Zhang for the SDG2/ATXR3 mutant and complemen- vation is common to both plant and mammal circadian systems. tation lines and for sharing data on SDG2/ATXR3; Dr. N. Nakamichi for the PRR5 seeds; and A. Troncoso for initial help with the ChIP assays and with Materials and Methods the primer design. This work was supported by the Ramón Areces Founda- tion (P.M.), Spanish Ministry of Science and Innovation Grant BIO2010-16483 In Vivo Luminescence Assays and ChIP. Luminescence was monitored as pre- (to P.M.), the European Molecular Biology Organization Young Investigator viously described (51) using a microplate luminometer LB-960 (Berthold Program (P.M.), and a European Young Investigator Award through the Technologies). The ChIP experiments were performed as previously described European Science Foundation (to P.M.).

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