Jung-Nam Cho, Jee-Youn Ryu, Young-Min Jeong, Jihye Park, Ji-Joon Song, Richard M. Amasino, Bosl Noh, and Yoo-Sun Noh

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1 Developmental Cell, Volume 22 Supplemental Information Control of Seed Germination by Light-Induced Histone Arginine Demethylation Activity Jung-Nam Cho, Jee-Youn Ryu, Young-Min Jeong, Jihye Park, Ji-Joon Song, Richard M. Amasino, Bosl Noh, and Yoo-Sun Noh Inventory of Supplemental Information Figure S1, related to Figure 1. Sequence Comparison of the JmjC Domains and the Morphological Phenotypes of jmj20, jmj22 Single, and jmj20 jmj22 Double Mutants Figure S2, related to Figure 2. The Expression of JMJ20, JMJ21, and JMJ22 mrnas upon PHYB Activation Figure S3, related to Figure 5. The H3R2me2 and H3K27me3 Levels at the GA3ox1 and GA3ox2 Loci Figure S4, related to Figure 6. The Binding of SOM to JMJ20/JMJ22 Chromatin and the PHYB-dependent Binding of JMJ20/JMJ22 to GA3ox1/GA3ox2 Chromatin Supplemental Experimental Procedures 1

2 Figure S1, related to Figure 1. Sequence Comparison of the JmjC Domains and the Morphological Phenotypes of jmj20, jmj22 Single, and jmj20 jmj22 Double Mutants (A) A multiple amino acid sequence alignment of the JmjC domains of JMJ20, JMJ21, JMJ22, human JMJD6, and human JMJD4. Asterisks and black dots are conserved catalytic residues required for the binding of the Fe (II) and 2-oxoglutarate cofactors, respectively. (B) Phylogenetic analysis of the JmjC domains of JMJ20, JMJ21, JMJ22, JMJD6, and JMJD4. The amino acid sequence alignment (A) and phylogenetic analysis (B) were performed using CLUSTAL W. (C) Expression of JMJ20 and JMJ22 transcripts in the seedlings of jmj22-1, jmj20-1, and jmj20-1 jmj22-1 as assayed by RT-PCR. The gene structures and T-DNA insertion sites are as explained in Figure 1A. The primers used for RT-PCR assays are marked as arrowheads on the diagram, and their sequences are shown in Supplemental Experimental Procedures. (D) Long-day (LD) grown adult plants of WT, jmj22-1, jmj20-1, and jmj20-1 jmj22-1. (E) Seeds harvested from WT, jmj22-1, jmj20-1, and jmj20-1 jmj

3 Figure S2, related to Figure 2. The Expression of JMJ20, JMJ21, and JMJ22 mrnas upon PHYB Activation Seeds were imbibed for 1 h in the dark and were treated with FR for 5 min with a subsequent 5 min of R treatment. Then, the seeds were incubated in the dark and were harvested at 6 h, 12 h, 24 h, or 36 h for RNA extraction. The transcript levels of GA3ox1, GA3ox2, SOM, JMJ20, JMJ21, and JMJ22 were analyzed by RT-qPCR using gene-specific primers (Supplemental Experimental Procedures). The levels at either 0 h (GA3ox1, GA3ox2, JMJ20, JMJ21, and JMJ22) or 36 h (SOM) were set to 1 after normalization by ACT2. Error bars represent sd. 3

4 Figure S3, related to Figure 5. The H3R2me2 and H3K27me3 Levels at the GA3ox1 and GA3ox2 Loci ChIP-PCR assays using light-treated seeds. 1, FR-treated WT; 2, FR-treated jmj20-1 jmj22-1; 3, R-treated WT; 4, R-treated jmj20-1 jmj22-1. The light treatment regime was as described in Figure 3, and the regions tested for PCR are as described in Figure 4A. INPUT indicates chromatin before immunoprecipitation. MOCK refers to control samples lacking antibody. (A) H3R2me2 levels. (B) H3K27me3 levels. 4

5 Figure S4, related to Figure 6. The Binding of SOM to JMJ20/JMJ22 Chromatin and the PHYB-dependent Binding of JMJ20/JMJ22 to GA3ox1/GA3ox2 Chromatin (A) Germination assay of phyb-9 jmj20-1 jmj22-1 seeds exposed to 5 min of R (left) or pil5-1 jmj20-1 jmj22-1 seeds exposed to 5 min of FR (right). Seeds were imbibed for 1 h before light treatment, and germinated seeds were counted every 24 h. Error bars represent sd (A, B, and D-H). (B) The ChIP-qPCR assay with anti-gfp antibody using WT and 35S::SOM:GFP transgenic seeds. The regions tested are shown in Figure 6F. Experimental conditions and data presentations are as described in Figure 6G. 5

6 (C) Yeast One-Hybrid assay. The promoter regions of JMJ20 and JMJ22 used for reporter constructs (A to D; upper left) and the full-length and truncated SOM proteins used for activation-domain (AD) constructs (lower left) are shown. Black boxes represent zinc-finger (ZnF) domains. Yeast cells co-transformed with each combination of reporter and AD constructs were tested for β-galactosidase assay (right). (D and E) Expression of JMJ20, JMJ22, GA3ox1, GA3ox2 transcripts in 35S::JMJ20:GFP and 35S::JMJ22:GFP transgenic seeds as measured by RT-qPCR using gene-specific primers (Supplemental Experimental Procedures). Seeds were imbibed for 1 h, treated with 5 min of FR, and incubated in the dark for 12 h before RNA extraction. (F) Germination of 35S::JMJ20:GFP and 35S::JMJ22:GFP transgenic seeds. Seeds were exposed to various fluence rate of R for 30 seconds after 5 min of FR pulse, and germinated seeds were scored after 5 d. (G) H4R3me2s levels in 35S::JMJ20:GFP and 35S::JMJ22:GFP transgenic seeds as measured by ChIP-qPCR. Seeds were treated as in (D). Normalization was to AtMu1, and the levels in WT were set to 1. The regions tested are shown in Figure 4A (G and H). (H) ChIP-qPCR analyses for the binding of JMJ20:GFP and JMJ22:GFP to GA3ox1 and GA3ox2. Seeds were imbibed for 1 h, treated with 5 min of FR with (R) or without (F) subsequent 5 min of R, and incubated in the dark for 12 h before harvesting. Normalization was to input, and the levels in F Col were set to 1. 6

7 Supplemental Experimental Procedures RT-qPCR Analysis Possible contamination from genomic DNA during the RNA extraction was removed by treating with RNase-free DNase I (Roche) for 1 h at 37ºC before RT. The first cdna strand was synthesized using M-MuLV reverse transcriptase (Fermentas) according to the manufacturer s recommendation using 3 µg of total RNA. qpcr was performed with the Applied Biosystems 7300 real-time PCR system using the SYBR Green I master mix (Kappa) in a volume of 20 µl. A comparative ΔΔC T method was used to calculate the relative amount of DNA. Normalization was to ACT2. All the RT-qPCR results were presented as means + sd of three biological replicates performed in triplicate. For the RT-qPCR experiments in Figures 3 and 6, seeds were imbibed for 1 h in the dark and were treated with FR for 5 min with (R) or without (F) a subsequent 5 min of R treatment. Then, the seeds were incubated in the dark for 12 h and were used for RNA extraction. Primers used for RT-qPCR or RT-PCR assays are as following. Gene name Sequence (5' 3') JMJ20 CATGTCACGGTTCTCTTTGGGG ATCTTCTGCGAACACGAGCG JMJ21 CCATCTGCGACTTGATACC GTAGGTGTTAAGTAGACTCCAAACA JMJ22 CCGGGGAGGTGATGTTTGTAC GAAAGAAAACTTGAAAGTATCAGTT PIL5 GTGGAATGATGCCAATGATG CTCACCCTGCTCGAACTTGG SOM GCTCTTTCGCCTTCCACTCC TCCTAGATCAGGGTCACCAC DAG1 ACACGGCTACGATGGAAACTAG GTATTGTTGTTCTCTATCTTGGGCAT GA3ox1 CCGAAGGTTTCACCATCACT CCCCAAAGGAATGCTACAGA GA3ox2 TAGATCGCATCCCATTCACA 7

8 TGGATAACTGCTTGGGTTCC GA2ox2 AATAACACGGCGGGTCTTCAAATCT TCCTCGATCTCCTTGTATCGGCTAA GAI AGCGTCATGAAACGTTGAGTCAGTG TGCCAACCCAACATGAGACAGC RGA CATTCCCGGAAACGCGATTTATCAG TCACCGTCGTTCCTATGACTCCA ABA1 GATGCAGCCAAATATGGGTCAAGG GCCATTGCATGGATAATAGCGACTC NCED6 ACCGGGTCGGATATAAATTGGGTTG CCCGGGTTGGTTCTCCTGATTC NCED9 AACCGCCGCTATGGTTTTAGACG CCAGTCACCGGAAGGTTATGCAC CYP707A2 ATGGGGTTGCCTTACATCGGAGA TGGCTTGAACAAGTGAGCTTTGCT ABI3 CTTGAAGCAAAGCGACGTGG TGTCTTACTTTAACCCCTCGTAT ACT2 AGGTCCAGGAATCGTTCACAGAAAAT AGAGGGATCAATTCGATCACTCAGAG JMJ20 a ATGGGAATACAGATTATAGGTCAAA JMJ20 b AATAAAATAGATTTGGGAGGAAAAA JMJ20 b GGTTGTATGCGTTGAGCC JMJ22 c ATGCCAAAGTGCAAGAATCTGTTGC JMJ22 d GAAAGAAAACTTGAAAGTATCAGTT ChIP Assay The antibodies used in the ChIP assays of this study were as follows: α-h3k4me3 (Cell Signaling #9727S), α-h3k9me3 (Abcam ab8898), α-h3k27me3 (Upstate ), α- H3R2me2 (Upstate ), α-h4r3me2s (Abcam ab5823), α-gus (Invitrogen A5790), and α-gfp (Invitrogen A6455). The amount of immunoprecipitated DNA was quantified by qpcr using specific primers. In the ChIP assays measuring histone methylation levels, normalizations were to the reference genes specified in each experiment. For ChIP binding assays, the amount of immunoprecipitated DNA was normalized to input DNA. All the ChIPqPCR results were presented as means + sd of three biological replicates performed in 8

9 triplicate. Primers used for ChIP-qPCR assays are as following. Locus GA3ox1 A GA3ox1 B GA3ox1 C GA3ox1 D GA3ox2 A GA3ox2 B GA3ox2 C GA3ox2 D JMJ20 P1 JMJ20 P2 JMJ20 P3 JMJ20 P4 JMJ22 P1 JMJ22 P2 JMJ22 P3 JMJ22 P4 PIL5 P1 ACT2 AtMu1 Sequence (5' 3') GTTATTGATATGGTCGTACGGTAG CAAAAATACTTTGGAAGGGAACG GACAACCCATCATGTGTATGTATC CATATGTTAAAGCACTTGTTTTGGT GTGTTTAGAGGCCATCCCATTCAC GCATGACCGATTTGGTTAGTCGCG TGAAATCTAGTGTAGTAGTGGACC CATATGTTCCTCGTACTCTTCAAC TTGTAACGGTATAAGGCTTGGC GCCTCTCACTTGCTAGTGTTATA TTGTTTTAAGCTGTCTATTTCCAAG CTTTTGGTGGAGAAGAGGAGTG GCATCCCATTCACATCCCACTCTC TGGTGATCTGGAACGCTCCCC CCTTTGGCTACATGACGATTTCTA GTCATGAGGGTCGAGTCTGT CATCACAACACTCGAGTACTTG CTCAAATCATTCAAAATGTAAGGTTC CACAGACCATGGGCCGTAC GGAGTCACGGGACAAATGC AGAAGGAGTACTTGTAGATGG GCAGCAAGTCACTCATGTAC GGCTTTTCCCTAGAGAGATC GATCAAAGTCGCCGGTAAAG TGATGAAGAGCGGAATGCTG TTTTCTCCTCAGCAAACCGC AGGCGATTATGTCGATGAGG AGACGAATCCGTCAATGAGG ATGCAGATACATGAACGGGG GTAATTCACATTATGCGCTGC CGGTGAGGCTACACTAAAAGC GTTTGACGGAGATCAGCC TCCGGTATTCGATCTTCTATC CGGAGATAAAATAATCAGCCTC AGGTCCAGGAATCGTTCACAGAAAAT AGAGGGATCAATTCGATCACTCAGAG CCGAGAACTGGTTGTGGTTT GCTCTTGCTTTGGTGATGGT 9

10 FLC TTGCATCACTCTCGTTTACCC GCGTCACAGAGAACAGAAAGC In Vitro Histone Demethylase Assay 1 or 2 µg of purified JMJ20:6xHis was incubated with 2 µg of calf thymus histone type II-A (Sigma-Aldrich) in demethylation reaction buffer (50 mm HEPES-KOH ph 8.0, 20 µm (NH 4 ) 2 Fe(SO 4 ) 2, 1 mm 2-oxoglutarate, 500 µm ascorbic acid) for 12 h at 37ºC. Histone methylation levels were measured by western blot using the following antibodies: α- H3R2me2 (Upstate ), α-h3k4me3 (Cell signaling #9727), α-h3k9me2 (Upstate ), α-h3k9me3 (Upstate ), α-h3k27me3 (Upstate ), α-h3k36me2 (Upstate ), α-h3k36me3 (Upstate ), α-h3 (Abcam ab1791), α-h4r3me1 (Abcam 17339), α-h4r3me2s (Abcam ab5823), and α-h4 (Abcam ab7311). Yeast One-hybrid Assay For the reporter constructs, 1-kb and 2-kb promoter regions of JMJ20 and JMJ22 were PCR amplified and cloned into placzi (Clonetech) at the EcoRI and SalI sites. AD constructs were generated by cloning the coding regions of SOM into pb42ad (Clonetech) at the XhoI site. As the induction of the full-length SOM protein resulted in the lethality of yeast cells, we also generated the deleted versions of AD:SOM proteins. Both the reporter and AD plasmids were co-transformed into yeast strain EGY48, and the co-transformed cells were selected on SD media lacking L-Uracil and L-Tryptophan. Selected yeast colonies were spotted on galactose and 5-bromo-4-chloro-indolyl-galactopyranoside (X-Gal)-containing media, and tested for their β-galactosidase activity. Sequences of primers used for constructions are available on request. 10