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 repres- sing 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