Supporting Information Malapeira et al. 10.1073/pnas.1217022110 SI Materials and Methods Plant Material and Growth Conditions. A. thaliana seedlings were stratified at 4 C in the dark for 3 d on Murashige and Skoog (MS) agar medium supplemented with 3% sucrose and then transferred to LD conditions (12 h light/12 h dark) with 30 μmol m 2 s 1 of cool white fluorescent light at 22 C. For the freerunning experiments, following synchronization, plants were transferred to constant light conditions. Plants expressing the CCA1::LUC (1), TOC1::LUC (2), CCR2::LUC (3), PRR5-ind (4), toc1-2 (3), TOC1-ox (5), CCA1-ox (6), sdg2/atxr3 mutant, and sdg2/atxr3 complementation lines (7) were described elsewhere. In Vivo Luminescence Assays. Luminescence was monitored as described previously (6) using a microplate luminometer LB-960 (Berthold Technologies). Analysis was performed using the software Microwin Version 4.34 (Mikrotek Laborsysteme). Period length and relative amplitude error (RAE) of waveforms were estimated with the FFT-NLLS suite of programs (8) using the Biological Rhythms Analysis Software System (BRASS) (http://www.amillar.org). A time window 96 h was used for the analysis, excluding the first 24 h to avoid transient effects from the synchronizing conditions. Treatment with Inhibitors. For inhibitor treatments, luminescence from reporter lines was recorded in the absence or in the presence of 10 mm NAM (9) (Sigma-Aldrich), 10 mm thymidine (Sigma- Aldrich), 100 μm C646 (10) (Sigma-Aldrich), or 100 μm MB-3 (11) (Sigma-Aldrich) added within the medium at the time of transferring the seedlings to the 96-well plates or at the specified circadian times. For ChIP and RT-Q-PCR experiments, seedlings were transferred for 2 d to new plates containing the inhibitors, and samples were taken at the indicated circadian times. For Dex treatments, seeds were sown on filter paper on top of the agar medium and grown for 8 d under LD conditions (12 h light/12 h dark) with 60 μmol m 2 s 1 of cool white fluorescent light at 22 C. PRR5 was induced following treatment with 10 μm Dex (Sigma-Aldrich). ChIP. The ChIP experiments were essentially performed as described previously (5). Briefly, seedlings were immersed in buffer A [0.4 M sucrose, 10 mm Tris (ph 8), 1 mm EDTA, 1 mm PMSF, 1% formaldehyde, 0.05% Triton X-100] under vacuum for 10 min, followed by an additional 10-min incubation with 0.125 M glycine. Seedlings were ground in liquid nitrogen and resuspended in buffer B [0.4 M sucrose, 10 mm Tris (ph 8), 5mMβ-mercaptoethanol, 1 mm PMSF, 1 μg/ml aprotinin, 1 μg/ ml pepstatin A, 1 μg/ml leupeptin]. Nuclei were then collected by centrifugation, resuspended in lysis buffer [50 mm Tris (ph 8), 10 mm EDTA, 1% SDS, 1 mm PMSF, 1 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 μg/ml leupeptin] and sonicated to generate DNA fragments of 500 800 bp. After centrifugation, the supernatants were incubated in dilution buffer [15 mm Tris (ph 8), 150 mm NaCl, 1% Triton-X-100, 1 mm EDTA, 1 mm PMSF, 1 μg/ml aprotinin, 1 μg/ml pepstatin A, 1 μg/ml leupeptin] overnight at 4 C with Sepharose beads conjugated with the indicated antibodies: H3K56ac (04-1135; Millipore), H3K4me3 (04-745; Millipore), H3K4me2 (ab11946; Abcam), H3K9ac (07-352; Millipore), H3K27me3 (07-449; Millipore), H3k27me2 (07-452; Millipore), and H3k9me3 (07-442; Millipore). The GFP antibody (A-11122; Invitrogen) was used to immunoprecipitate TOC1, PRR5, and CCA1. Before use, the PRR1 antibody (ab93092; Abcam) was incubated previously with protein extracts from toc1 mutant plants. The immune complexes were washed four times with washing buffer (0.1% SDS, 1% Triton X-100, 1mMEDTA,1mMPMSF,1μg/mL aprotinin, 1 μg/ml pepstatin A, 1 μg/ml leupeptin) and eluted from the beads with 1% SDS and 0.1 M NaHCO 3. Immunoprecipitated DNA was isolated using the QIAquick kit (Qiagen) following the manufacturer s instructions. ChIPs were quantified by Q-PCR analysis using a 96-well LightCycler 480 system (Roche) with the LightCycler 480 software (Version 1.5.0.39; Roche). Melting peak analysis using the LightCycler 480 Basic software module (Roche) and gel electrophoresis confirmed that primer dimers or other nonspecific products were not present. The list of primers used for amplification is shown in Table S1. Crossing point (Cp) calculation was used for quantification using the Absolute Quantification analysis by the second derivative maximum method (LightCycler 480 Basic software module; Roche). ChIP values for each set of primers were normalized to input values. In experiments comparing ZT3 and ZT15 and in the time course analysis, enrichment was normalized relative to the control At5g55840. For the SDG2/ ATXR3 ChIP experiments, enrichment was normalized relative to At2g26560 (7). Primers were designed using the PrimerExpress Version 2.0 software (Applied Biosystems). Samples similarly processed but omitting the antibody in the immunoprecipitation incubation were used as negative controls. Analysis of Gene Expression. RNA was isolated using the Purelink Total RNA purification system (Invitrogen) and treated with RNase-free DNase I (Ambion) to reduce genomic DNA contamination (12). Single-strand cdna was synthesized using SuperScript III (Invitrogen) and a mixture of oligo-dt18 and random hexamers (Invitrogen) following the manufacturer s recommendations. The cdna was diluted 10-fold with nuclease-free water and Q-PCR was performed with the SYBR Premix Ex Taq (Takara) in a 96-well LightCycler 480 system (Roche). In the time-course analysis of oscillator gene expression, the IPP2 gene was used as control (13). For the SDG2/ATXR3 experiments, gene expression was normalized relative to At3g01610 (7). Quantification was performed using LightCycler 480 software (Version 1.5.0.39, Roche). Light- Cycler melting curves were analyzed for the reactions. The amplification data were analyzed using the second derivative maximum method. Resulting Cp values were converted into relative expression values using the comparative Ct method. The list of primers used in this study is shown in Table S1. Statistical Analysis. Statistical analyses were performed using Prism software (GraphPad Software). Analyses were performed by twotailed t test with 99% of confidence and by two-way ANOVA test, followed by Bonferroni post test in assays with multiple comparisons. 1. Salomé PA, McClung CR (2005) PSEUDO-RESPONSE REGULATOR 7 and 9 are partially redundant genes essential for the temperature responsiveness of the Arabidopsis circadian clock. Plant Cell 17(3):791 803. 2. Perales M, Más P (2007) A functional link between rhythmic changes in chromatin structure and the Arabidopsis biological clock. Plant Cell 19(7):2111 2123. 3. Strayer CA, et al. (2000) Cloning of the Arabidopsis clock gene TOC1, an autoregulatory response regulator homolog. Science 289(5480):768 771. 4. Nakamichi N, et al. (2010) PSEUDO-RESPONSE REGULATORS 9, 7, and 5 are transcriptional repressors in the Arabidopsis circadian clock. Plant Cell 22(3):594 605. 5. Más P, Kim WY, Somers DE, Kay SA (2003) Targeted degradation of TOC1 by ZTL modulates circadian function in Arabidopsis thaliana. Nature 426(6966):567 570. 1of12
6. Portolés S, Más P (2010) The functional interplay between protein kinase CK2 and CCA1 transcriptional activity is essential for clock temperature compensation in Arabidopsis. PLoS Genet 6(11):e1001201. 7. Guo L, Yu Y, Law JA, Zhang X (2010) SET DOMAIN GROUP2 is the major histone H3 lysine 4 trimethyltransferase in Arabidopsis. Proc Natl Acad Sci USA 107(43):18557 18562. 8. Plautz JD, et al. (1997) Quantitative analysis of Drosophila period gene transcription in living animals. J Biol Rhythms 12(3):204 217. 9. Xydous M, Sekeri-Pataryas KE, Prombona A, Sourlingas TG (2012) Nicotinamide treatment reduces the levels of histone H3K4 trimethylation in the promoter of the mper1 circadian clock gene and blocks the ability of dexamethasone to induce the acute response. Biochim Biophys Acta 1819(8):877 884. 10. Bowers EM, et al. (2010) Virtual ligand screening of the p300/cbp histone acetyltransferase: Identification of a selective small molecule inhibitor. Chem Biol 17(5):471 482. 11. Biel M, Kretsovali A, Karatzali E, Papamatheakis J, Giannis A (2004) Design, synthesis, and biological evaluation of a small-molecule inhibitor of the histone acetyltransferase Gcn5. Angew Chem Int Ed Engl 43(30):3974 3976. 12. Legnaioli T, Cuevas J, Mas P (2009) TOC1 functions as a molecular switch connecting the circadian clock with plant responses to drought. EMBO J 28(23): 3745 3757. 13. Hazen SP, et al. (2005) LUX ARRHYTHMO encodes a Myb domain protein essential for circadian rhythms. Proc Natl Acad Sci USA 102(29):10387 10392. Fig. S1. Spatiotemporal distribution of H3K56Ac and H3K4Me3 along the genomic structure of the oscillator genes. ChIP analysis of H3K56Ac (A F) and H3K4me3 (G L) along the genomic structure of the oscillator genes TOC1 (A and G), LUX (B and H), LHY (C and I), CCA1 (D and J), PRR9 (E and K), and PRR7 (F and L). ChIPs were performed in WT plants grown under LD cycles, and samples were analyzed at ZT3 and ZT15. Values relative to the input are represented as means ± SEM. Enrichment at ZT3 and ZT15 was normalized relative to the control At5g55840. Two-way ANOVA test, followed by Bonferroni post test, shows the statistical relevance of the differences: *P < 0.05; **P < 0.01; ***P < 0.001. The exon intron UTR structure of the gene models and the different fragments amplified by Q-PCR are schematically depicted at the top of the figure. 2of12
Fig. S2. Spatiotemporal distribution of H3K56Ac and H3K4Me3 along the genomic structure of the oscillator genes. ChIP analysis of H3K56Ac (A, C, and E)and H3K4me3 (B, D, and F) along the genomic structure of the oscillator genes TOC1 (A and B), LHY (C and D), and PRR9 (E and F). ChIPs were performed in WT plants grown under LD cycles, and samples were analyzed at ZT3 and ZT15 (+). Control ChIP experiments were performed without antibodies (-). Values relative to the input are represented as means ± SEM. The exon intron UTR structure of the gene models and the different fragments amplified by Q-PCR are schematically depicted at the top of the figure. Experiments were performed as detailed in SI Materials and Methods. 3of12
Fig. S3. Spatiotemporal distribution of H3K27me2, H3K27me3 and H3K9Me3 along the genomic structure of the oscillator genes. ChIP analysis of H3K27me2 (A C), H3K27me3 (D F), and H3K9me3 (G I) accumulation along the genomic structure of the oscillator genes TOC1 (A, D, and G), LHY (B, E, and H), and PRR7 (C, F, and I). ChIPs were performed in WT plants grown under LD cycles, and samples were analyzed at ZT3 and ZT15. Values relative to the input are represented as means ± SEM. The exon intron UTR structure of the gene models and the different fragments amplified by Q-PCR are schematically depicted at the top of the figure. 4of12
Fig. S4. Rhythmic oscillation of H3K9Ac at the core of the oscillator. ChIP assays of H3K9Ac were performed in WT plants sampled every 4 h over a 24-h LD cycle. Primers encompassing the 5 UTR for each gene (Fig. 1) were used to amplify CCA1 (A), LHY (B), PRR9 (C), PRR7 (D), TOC1 (E), and LUX (F). Values are represented as means ± SEM. Enrichment was normalized relative to the control At5g55840. Data were normalized relative to the maximum value. 5of12
Fig. S5. Time-course comparisons of the oscillatory waveforms of H3K56Ac and H3K4Me3 at the core of the clock. ChIP assays of H3K56Ac and H3K4me3 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), CCA1 (B), PRR9 (C), PRR7 (D), TOC1 (E), and LUX (F). Enrichment was normalized relative to the control At5g55840. Values are represented as means ± SEM. Data were normalized relative to the maximum value to facilitate comparisons between the ChIP assays among the different genes at the various time points. 6of12
Fig. S6. Sequential enrichment of H3K56ac-H3K4me3-H3K4me2 correlates temporally with the peak-to-trough oscillator gene expression. Time-course comparisons of the oscillatory waveforms of H3K56Ac, H3K4Me3, and H3K4me2 at the core of the clock. ChIP assays of H3K56Ac, H3K4me3, and H3K4me2 were performed in WT plants sampled every 4 h over a 24-h LD cycle. Primers encompassing the peak of the histone marks for each gene (Fig. S1) were used to amplify LHY (A and B), CCA1 (G and H), PRR9 (C and D), PRR7 (I and J), TOC1 (E and F), and LUX (K and L). Enrichment at ZT3 and ZT15 was normalized relative to the control At5g55840. Values are represented as means ± SEM. Data were normalized relative to the maximum value to facilitate comparisons between the ChIP assays among the different genes at the various time points. H3K4me3 and H3K56ac data from Fig. 1 were replotted to facilitate comparisons with K4me2. 7of12
Fig. S7. Effects of blocking histone methylation on clock gene expression. (A and C) CCA1::LUC (A) and TOC1::LUC (C) luminescence in WT plants under LD cycles analyzed in the absence or presence of NAM. (B) CCA1::LUC luminescence in WT plants under LD cycles analyzed in the absence or presence of thymidine. (D) CCR2::LUC luminescence in WT plants entrained under LD cycles and subsequently released to constant light (LL) conditions. Luminescence was examined in the absence or presence of NAM added at the indicated time (arrow). (E) Period estimates of CCR2::LUC rhythms from individual traces examined by FFT-NLLS best-fit algorithm analysis. Data are the means ± SEM of the luminescence of 5 10 individual seedlings. Data are representative of at least two independent experiments. (F) RT-Q-PCR analysis of oscillator gene expression in WT plants analyzed in the absence or presence of NAM. Plants were grown under LD cycles, and samples were analyzed at ZT3 and ZT15. Values were normalized relative to the control At3g01610 (7), and data were normalized relative to the maximum value. 8of12
Fig. S8. Effects of blocking histone acetylation on clock gene expression. (A and B) CCA1::LUC (A) and TOC1::LUC (B) luminescence in WT plants entrained under LD cycles and subsequently released to constant light (LL) conditions. Luminescence was examined in the absence or presence of MB-3. Arrows indicate the circadian time of inhibitor administration. Data are the means ± SEM of the luminescence of 5 10 individual seedlings. Data are representative of at least two independent experiments. ChIP analysis of H3K56ac in the presence and in the absence of NAM (C and D), C646 (E and F), and MB-3 (G and H) atzt3(c, E, and G) and ZT15 (D, F, and H). 9of12
Fig. S9. Effects of NAM on clock-repressor binding and role of SDG2/ATXR3 as a histone methyltransferase at the core of the clock. (A) ChIP assays of TOC1 binding to CCA1 in WT. Plants were grown under LD cycles, and samples were analyzed at ZT3, ZT7, and ZT11. As control, toc1-2 mutant plants were also assayed at ZT11. Binding was examined in the absence (-NAM) or in the presence of NAM (+NAM). (B) ChIP analysis of H3K4me3 accumulation in WT plants and in heterozygous sdg2 mutants. Plants were grown under LD cycles, and samples were analyzed at ZT3 and ZT15. H3K4me3 enrichment at the TOC1 and LUX loci was normalized relative to the control At2g26560 (1). WT(-) and sdg2(-) denote samples processed omitting the antibody in the ChIP assay. (C F) Time-course analysis by RT-Q-PCR of oscillator gene expression in WT and sdg2 mutant homozygous lines. Plants were grown under LD cycles, and samples were taken every 4 h over the diurnal cycle. Data are represented as the means ± SEM. Values were normalized relative to the control At3g01610 (2). 1. Guo L, Yu Y, Law JA, Zhang X (2010) SET DOMAIN GROUP2 is the major histone H3 lysine 4 trimethyltransferase in Arabidopsis. Proc Natl Acad Sci USA 107(43):18557 18562. 2. Matsushika A, Makino S, Kojima M, Mizuno T (2000) Circadian waves of expression of the APRR1/TOC1 family of pseudo-response regulators in Arabidopsis thaliana: Insight into the plant circadian clock. Plant Cell Physiol 41(9):1002 1012. 10 of 12
Table S1. List of primers used in this study Name Sequence Experiment CCA1-F TCA AGC TTC CAC ATG AGA CTC TA Expression analysis CCA1-R GGA AAC AAA TAC AAA GGC CTC A Expression analysis LHY-F ACA GCA ACA ACA ATG CAA CT Expression analysis LHY-R GAG AGC CTG AAA CGC TAT AC Expression analysis TOC1-F GAA GAT GTT GAT CGA CTG AC Expression analysis TOC1-R GAG CCA ACA TTG CCT TAG AG Expression analysis PRR7-F ACT ATG CAC GGC TCC AAA AG Expression analysis PRR7-R TCG TTG TCT GGT CTG GTT TG Expression analysis PRR9-F TGA GAT ACT GGG GCA ACT TTT Expression analysis PRR9-R GCT TAG CCT GAT CAT TTG CAG Expression analysis IPP2-F CAT GCG ACA CAC CAA CAC CA Expression analysis IPP2-R TGA GGC GAA TCA ATG GGA GA Expression analysis At3g01610-F GCA AGT GCT TCT ACT CAA TGT CA Expression analysis At3g01610-R AAC CGC GTC CTC AGA AGT C Expression analysis CCA1-F1 TCTTCTACCCTTCATGCATGGTT ChIP assays CCA1-R1 GGACCTAAACTTATTTGGGCCAT ChIP assays CCA1-F2 CATTTCCGTAGCTTCTGGTCTCTT ChIP assays CCA1-R2 ATCAGCTTGGATTCGATAAAGATTC ChIP assays CCA1-F4 TCC AGA TAA GAA GTC ACG CTC AGA ChIP assays CCA1-R4 CAT TAA GCC AAT GAA GAT GAG AAC A ChIP assays CCA1-F7 TGG GGG AAT AAC AGG GAA GTC ChIP assays CCA1-R7 CGG ATA AGT CTG AGG TCC TTG C ChIP assays CCA1-F9 GGT GAA AGA AAC GAA TGA AGA CAC T ChIP assays CCA1-R9 GAT CGG TTA TAT TGG AGC TGA TTC T ChIP assays LHY-F1 CGG TTA TTT CAA TTA GAT TCG GGT ChIP assays LHY-R1 TTA GTT CGG TTC GGT TCT AGG TTA A ChIP assays LHY-F2 AGCAAGTTGACCAAAGTTCTCGA ChIP assays LHY-R2 CGGGGAACTTGCGTCTGTATA ChIP assays LHY-F4 TCCTCCATGGCTACTCTCAAGG ChIP assays LHY-R4 TCAGCAGCCAAACAGAGATCTTAG ChIP assays LHY-F5 AAT CTA AAG AGG TTA TCA CAA CGG C ChIP assays LHY-R5 GCT GCT TCA AAT CCT CTC TAA CAA G ChIP assays LHY-F10 GAT GAT TAC CGT TCG TTT CTC CA ChIP assays LHY-R10 TGA GCT GCA GGA TTC TGT AGG A ChIP assays LHY-F11 AAC AAA GTG ACA CGT CAA TGC C ChIP assays LHY-R11 GAGTAACCAAGCTAAATGCCGAC ChIP assays LUX-F1 GTCATGCACAGCGGCAGTAA ChIP assays LUX-R1 CAC AGA TTG GCT GGC ACC TAA CTT T ChIP assays LUX-F3 AGC TTC GAA GAG CTC AAT CTC TAA CTG AA ChIP assays LUX-R3 TCG TAA TCG CTC ATT TGT ACT TCC TCT C ChIP assays LUX-F4 CGG AAG AAG GAG ATT CAG GAA CTG AA ChIP assays LUX-R4 TGT AGC TGC GGT GTC CAC ACT AAA C ChIP assays LUX-F6 AAT CCA GAT CAT GTT TGA TTC ATC TTC G ChIP assays LUX-R6 AAC TCA CAG TGT TAG TTA CCT AGT CCC GAA ChIP assays TOC1-F2 TAA TAT GAG CCA ATC GGT AAT ACG A ChIP assays TOC1-R2 GGT TGG GAA ACA AAT AAT CAA GTT G ChIP assays TOC1-F5 TGT TAA GGG GAT AAA TTA GGC GAC ChIP assays TOC1-R5 GCT ATG ATA CTT CCA TGG CCA AA ChIP assays TOC1-F7 AAT GGC TAA GGG TAT GAA GAT GCT ChIP assays TOC1-R7 GAA GAG CAA CAA AGA ACA CAG CTT A ChIP assays TOC1-F10 AAC AAG CAC ACA GAA GTA GAG GGA C ChIP assays TOC1-R10 AAG GGA ATG TGG ATA GCT GGC ChIP assays PRR7-F1 TGG CCC GAG ACA AAT CTT TCT AAT ATC T ChIP assays PRR7-R1 GAG TGG AAA TCG GAG ACG ACC ATA A ChIP assays PRR7-F2 GCA ATA ATC GAA ATT AGG GTT TAT GGC T ChIP assays PRR7-R2 TTA GCA TTC ATC ACA CCA ACT CTG CTT ChIP assays PRR7-F3 TAT TAA CTC TGT CAT GGC AGT TGT TGA GG ChIP assays PRR7-R3 GGC ATG ATC ACC TCT GTT AGC ACA ATA T ChIP assays PRR7-F5 ACCGATAATCTAAATATACACCCGAGAAGC ChIP assays PRR7-R5 CCACCATTTATAAGCATCTGAGTGCAC ChIP assays PRR7-F8 CGA CTG AGA ACA ACG CTT TCA CAA A ChIP assays PRR7-R8 TGA AAC GAT GAA TGC TTC ACT GAT GA ChIP assays PRR9-F1 TTG GAT TCC AAA GGG GAT CTT ATG AA ChIP assays 11 of 12
Table S1. Cont. Name Sequence Experiment PRR9-R1 CAT TGA CGA AGA AAA AGA TCC GTT TG ChIP assays PRR9-F3 TCC AAT TTG AAT GAT ACA TAG AGC AGC TG ChIP assays PRR9-R3 TGG GTT TCT ATT GTA ATT GTG TGG CTA AGT ChIP assays PRR9-F4 TCT CGG TAG ATT AAG ATC TAA AGC TCG TTG ChIP assays PRR9-R4 CAA CAC TTG GTA AAA CCA ACA AAG CCT A ChIP assays PRR9-F8 GCA GAT TTG AGG AAA GCA AGT CAG C ChIP assays PRR9-R8 TCA TGT GAT TCG GTT GGT GTC TTT G ChIP assays At5g55480-F GAT TCT GCT TCT CAC CAA ChIP assays At5g55480-R ATT CAG CAATAG CCACAA ChIP assays At2g44620-F TAC ATT ATG GGC AGC ATT T ChIP assays At2g44620-R TAG TTC AGG TCA TAC GAG AT ChIP assays At2g26560-F GCT GCT ACT CTT GCG TTC G ChIP assays At2g26560-R GCC TTA GCT GCC ATC AAG G ChIP assays F, forward; R, reverse. 12 of 12