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1 Supporting Information Hu et al..73/pnas.3489 SI Materials and Methods Histone Extraction and Western Blot Analysis. Rice histone proteins were extracted from -d-old seedlings. After being washed with acetone and dried, the proteins were suspended in SDS/PAGE sample buffer and tested by Western blot with antibodies against H3K4me (Upstate Biotechnology; 7 436), H3K4me (Millipore; 5 79), H3K4me3 (Millipore; DAM73494), H3K7me3 (Millipore; DAM664), and H3 (Abcam; ab79). Real-Time PCR. Real-time PCR was performed in a total volume of 5 μl with. μl of the reverse transcription (RT) or chromatin immunoprecipitation (ChIP) products,.5 μm primers, and.5 μl SYBR Green Master mix (Takara) on a 75 real-time PCR machine (Applied Biosystems) according to the manufacturer s instructions. The rice ACTIN gene was used as the internal control. All primers were annealed at 6 C and run 4 cycles for RT products and 45 cycles for ChIP products. The ChIP enrichment for H3K7me3 and H3K4me3 was quantified by comparing the threshold cycle (C t ) of the ChIP sample with that of the input with (Ct of input-ct of sample ChIP). The expression level of target genes was also normalized with that of ACTIN (Ct of actin-ct of target). In Vitro Histone-Binding Assays. Glutathione-agarose beads were incubated with crude bacterial extract (containing GST fusions) in binding buffer (5 mm Tris HCl at ph 8., 5 mm NaCl,.% Nonidet P-4, mm ZnCl, mm DTT, and protease inhibitors) for 3 min. After three washes, calf histones were added and incubated overnight at 4 C. After five washes, bound proteins were eluted and analyzed by Western blots with antibodies against H3, H3K4me3, H3K9me3 (Abcam; ab8898), H3K7me3, or H3K36me3 (Abcam; ab95). Biotinylated histone peptides were purchased from Upstate Biotechnology. Briefly, μg of peptides were incubated with crude bacterial GST-plant homeodomain (PHD) and GST protein extracts in binding buffer (5 mm Tris HCl at ph 7.5, 3 mm NaCl,.% Nonidet P-4, mm ZnCl, mm phenylmethylsulphonyl fluoride) overnight at 4 C. After incubation with the above mixture for h, streptavidin beads (Millipore) were washed three times and subjected to Western analysis with GST Antibody (Abcam; ab956). ChIP-seq and Data Analysis. Rice seedlings ( d old) that were grown under conditions of 4 h light/ h dark at 5 C 8 C in / Murashige and Skoog medium were used for ChIP experiments. Chromatin was fragmented to 5 bp by sonication, and ChIP was performed using antibodies of H3K4me3 and H3K7me3. Briefly, precipitated DNA was end-repaired using a combination of T4 DNA polymerase and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow enzyme and datp to yield a protruding 3 A base for ligation to Illumina s adapters, which have a single T base overhang at the 5 end, according to the Illumina Paired-End DNA Sample Prep kit procedure. After adapter ligation, DNA was PCR-amplified with Illumina primers, and library fragments of 3 bp (insert plus adaptor and PCR primer sequences) were isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced with the equipment Illumina HISEQ. Each library had about,, raw reads. Sequence reads from all libraries were mapped to the reference genome of rice (Oryza sativa L. ssp. japonica cv. Nipponbare 6.) using SOAP. software. Reads that could be mapped equally well to multiple locations without mismatch or with identical mismatches were assigned to one position at random and were retained for further analysis as described previously (). Genomic regions associated with histone modifications were identified using MACS software (), in which default parameters (bandwidth: bp; model fold:, 3; P value:.e-5; largelocal: 5,) were set to call peaks representing enriched epigenetic marks. MACS software was used to calculate a dynamic local λ to reflect the local bias due to potential chromatin structure. After the positions of the peaks on the chromosomes were found, the genes (including the -kb upstream and -kb downstream regions) overlapping with the peaks were considered to have the epigenetic marks. The output of the analysis pipeline was converted to wig files for viewing the data in the GBrowse. software. The ChIP-seq data from this publication have been deposited in the Gene Expression Omnibus database (accession no.gse349). For annotation of genes and transposable elements and for gene ontology, classification followed the Rice Genome Annotation Project 6.. The Web Gene Ontology Annotation Plotting tool (WEGO) ( GO archive: 9 - ; input file format: WEGO Native Format) was used to assign genes to a hierarchical biological process. A particular pathway that corresponds to a test statistic was evaluated with a P value cutoff at.5. Microarray Analysis. For microarray analysis, -d-old seedlings of wild type and mutants were grown in / Murashige and Skoog medium under a 4-h light/-h dark cycle at 5 C 8 C. RNA samples were extracted using TRIzol (Invitrogen) as described by the manufacturer. Hybridization with Affymetrix GeneChip Rice Genome Arrays was performed at CapitalBio. The dataset was normalized with the option of all probe sets scaled to the target signal of. The genes with expression calls that were absent from at least two arrays were filtered for further analyses. The significance analysis of microarrays (SAM) Excel add-in (3) was used to identify significantly differentially expressed genes between the control and mutant seedlings. The imputation engine was set with as the nearest neighbor imputer and the number of permutations was. The Δ-value in the SAM was adjusted so that the estimated false discovery rate was < 5% for significant genes. The microarray data from this publication have been deposited in the Gene Expression Omnibus database (accession no. GSE573).. He G, et al. () Global epigenetic and transcriptional trends among two rice subspecies and their reciprocal hybrids. Plant Cell (): Zhang Y, et al. (8) Model-based analysis of ChIP-Seq (MACS). Genome Biol 9:R Tusher VG, Tibshirani R, Chu G () Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98:56 5. Hu et al. of

2 hchd hchd dchd Subfamily CHR75 (Os7g4659) CHR5 (Atg337) ScCHD hchd6 hchd7 Subfamily 3 hchd8 hchd9 CHR6 (Atg57, PICKLE) CHR7 (At4g39, PKR) CHR7 (Os6g848) dchd3 hchd4 hchd5 Subfamily hchd3 CHR73 (Osg6585) CHR79 (Os7g345) CHR4 (At5g448, PKR) CHR73 (Os6g3) CHR744 (Osg5) CHR5 (Atg86, MOM) hchd8 hchd9 hchd7 hchd6 dchd3 hchd3 hchd4 hchd5 CHR7 CHR6 (PICKLE) CHR7 (PKR) ScCHD CHR73 CHR73 CHR744 CHR79 CHR4 (PKR) dchd hchd hchd CHR75 CHR5 AtMOM.. Fig. S. Phylogenetic relationship between chromodomain, helicase/atpase, and DNA-binding domain (CHD) proteins. (Left) Neighbor-joining tree using fulllength CHD protein sequences from Saccharomyces cerevisiae (Sc), Homo sapiens (h), Drosophila melanogaster (d), Arabidopsis thaliana (At), and rice (Oryza sativa, Os), using the MEGA3. software. The values represent the percentages of sampled trees used in the analysis that contained the consensus partition. The three subfamilies are shaded. (Right) Neighbor-joining tree using CHD chromodomain sequences. Hu et al. of

3 A CHR79 Actin WT CHR79 RNAi ZH CHR79 Actin B Fig. S. Characterization of CHR79 T-DNA mutants and RNAi plants. (A) (Top) Gene structure of CHR79 with exons (black boxes) and introns (lines) and the T-DNA insertion site (triangle). RNAi region is underlined. (Middle) The homozygotes, 3, 4, 5, and 7 of chr79 showed no expression of the genes displayed by RT-PCR. (Bottom) Reduction of CHR79 transcripts in RNAi lines. CHR79 mrna levels in RNAi lines and 3-3 were determined by real-time RT-PCR. (B) Phenotypes produced by CHR79 RNAi plants. (Left) Wild type (left) and CHR79 RNAi (line 3-3) (right) plants at mature stage. (Center) A mature leaf and the flag leaf of wild type (left) and RNAi plant (right). (Right) Main panicles from wild type and RNAi plants. Note that the RNAi panicle has no secondary branch as indicated by red arrowheads in the wild type. Hu et al. 3of

4 A C Percentage e of modified regions Number of uniq ue reads (*) % 95% 9% 85% 8% 75% 6,897 8, ,8833 6,46 HY chr79 HY 3 chr79 4 H3K7me3 H3K4me3,7 88,3 3,8 86, 3, 3, 96,9 96,9 HY chr79 HY 3 chr79 4 H3K7me3 H3K4me3 B unique reads Percentage of % 8% 6% 4% % % 8,7 8 8,4 8,7 9,3 7,6 8,3 HY chr79 HY chr79 H3K7me3 H3K4me3 Fig. S3. Analysis of H3K7me3 and H3K4me3 ChIP-seq reads. (A) Number of unique reads from H3K7me3 and H3K4me3 ChIP of wild type (HY) and chr79. (B) Distribution of unique reads in genes (blue) and repetitive (yellow) regions. (C) Distribution of reads in transcribed (red) and nontranscribed (green) regions. Hu et al. 4of

5 H3K7me3 Wild type chr79 Osg Osg675 Os5g8 chr79 H3K4me3 WT H3K7me3 chr79 H3K7me3,8,6,4,,4,3,,,8,6,4, chr79 H3K4me3 WT H3K7me3 chr79 H3K7me3 Os7g44,5,5 Osg3,5,5,5 Os5g3699,5,5 5 chr79 H3K4me3 WT H3K7me3 chr79 H3K7me3 Osg56,8,6,4, Osg6 Os8g595,8,6,4, Osg568,8,6,4, Osg O Osg568 Os4g384 chr79 H3K4me3 WT H3K7me3 chr79 H3K7me3,8,6,4,,3,,,8,6,4, Fig. S4. Quantitative PCR validation of H3K7me3 ChIP-seq. Twelve genes were selected for the test (9 with reduced, with unchanged, and with increased H3K7me3 in ChIP-seq as shown on the left). Levels relative to input are shown. Hu et al. 5of

6 H3K4me3 Wild type chr79 chr79 H3K4me33 chr79 H3K4me3 WT H3K7me3 chr79 H3K7me3 Osg599,5,5 Osg95,5,5 Os9g574,5,5 Osg Os3g Os6g639,5,,5 Os3g97 Osg4555,,8,6,4,,4,3,, chr79 WT H3K7me3 chr79 Os3g948,4 4, Os5g397,5,,5,,5 Osg5,6,,8,4 chr79 H3K4me3 WT H3K7me3 chr79 H3K7me3 Os3g588,5,5,5 Osg639,,5,,5 Os3g565,5,5,5 Fig. S5. Quantitative PCR validation of H3K4me3 ChIP-seq. Fourteen genes were selected for the test ( with reduced and 4 with increased or unchanged H3K4me3 levels in ChIP-seq as shown on the left). Levels relative to input are shown. Hu et al. 6of

7 A Relative RNA levels nt H3K K4me3 enrichme,5,5,5,5 WT chr WT chr Os6g367 Os9g35 3 Os9g353 4 Osg Osg567 6 Os4g Os9g844 8 Os3g97 9 Osg7377 Osg4545 Os8g369 Os6g367 Os9g35 3 Os9g353 4 Osg Osg567 6 Os4g Os9g844 8 Os3g97 9 Os4g59 B,5 HY chr79,5 evels Relative RNA l,5,5 H3 3K4me3 enrichm ment,5 HY chr79 Fig. S6. Validation of altered expression levels and H3K4me3 changes of AP transcription factor genes in the chr79 mutant. (A) Relative expression levels of down-regulated AP genes (Upper) and H3K4me3 level for 9 of the genes (Lower) inchr79 compared with wild type. (B) Relative expression (Left) and H3K4me3 (Right) levels of two up-regulated AP genes in chr79 compared with wild type. Relative RN A levels WT chr79 OsiEZ OsCLF OsEMFa OsEMFb OsFIE OsFIE Fig. S7. Expression of Polycomb group PRC genes in chr79 relative to the wild type. Hu et al. 7of

8 Table S. Loss or gain of H3K7me3 and H3K4me3 in chr79 H3K7me3 lost H3K7me3 maintained H3K7me3 gained H3K4me3 loss (%) H3K4me3 gain (%) No H3K4me3 (%) With H3K4me3 (%) H3K4me3 lost H3K4me3 maintained H3K4me3 gained H3K7me3 loss (%) H3K7me3 gain (%) No H3K7me3 (%) With H3K7me3 (%) The percentages of genes that lost, gained, or maintained (with or without) H3K4me3 and H3K7me3 within the categories of genes that lost, gained, or maintained H3K7me3 and H3K4me3, respectively, in chr79 are shown. Hu et al. 8of

9 Table S. Transcription factor genes that are differentially regulated in chr79 Down-regulated Fold change q-value (%)* DNAme K4m3 K9Ac K7m3 LOC_Os9g844 AP domain-containing protein * LOC_Os8g369 AP domain-containing protein LOC_Os6g367 AP domain-containing protein LOC_Os4g384 OsMADS64-MADS-box family gene with M-α type-box.63 LOC_Osg7377 CRT/DRE-binding factor LOC_Os9g35 AP domain-containing protein LOC_Osg68 DNA-binding protein.6.87 LOC_Os3g53 Helix loop helix DNA-binding domain containing protein LOC_Os3g97 AP domain-containing protein.5 LOC_Osg359 Heat stress transcription factor A3.56 LOC_Osg643 NAC domain-containing protein LOC_Os3g657 Zinc-finger CH-type family protein.76 LOC_Osg6436 Myb-like DNA-binding domain-containing protein LOC_Osg4545 AP domain-containing protein LOC_Os3g9 Myb-like DNA-binding domain-containing protein LOC_Os5g397 DNA-binding protein WRKY LOC_Os5g7 myb-like DNA-binding domain; SHAQKYF class family protein LOC_Osg567 AP domain-containing protein LOC_Os9g8 Helix loop helix DNA-binding domain-containing protein LOC_Osg844 WRKY transcription factor LOC_Os6g44 WRKY protein LOC_Osg643 WRKY DNA-binding domain-containing protein LOC_Os4g355 Helix loop helix DNA-binding domain-containing protein LOC_Osg393 Agamous-like MADS box protein AGL9.377 LOC_Os3g663 Heat-shock factor protein LOC_Os9g353 DREBA protein LOC_Osg4379 AP domain-containing protein LOC_Os5g376 Zinc-finger C-x8-C-x5-C-x3-H type family protein LOC_Osg744 Zinc-finger C3HC4-type family protein LOC_Os4g59 AP domain-containing protein LOC_Os4g448 Zinc-finger C3HC4-type family protein.4 LOC_Osg444 WRKY DNA-binding domain-containing protein LOC_Os3g5554 Zinc-finger protein LOC_Osg45 Myb-related protein Myb4.43 LOC_Os3g37 MADS-box transcription factor 5.44 LOC_Osg4984 Agamous-like MADS box protein AGL.49 LOC_Os8g3846 Zinc-finger C3HC4-type family protein.434 LOC_Osg664 WRKY DNA-binding domain-containing protein LOC_Osg483 DNA-binding protein LOC_Os5g378 NAC domain-containing protein LOC_Os4g4368 Myb-related protein Myb LOC_Os5g3693 Histone deacetylase family protein LOC_Os7g345 SNF family N-terminal domain-containing protein.463 LOC_Os4g4835 CRT/DRE binding factor LOC_Osg66 NAC domain-containing protein LOC_Osg4578 Zinc-finger C3HC4-type family protein LOC_Osg457 Zinc-finger C3HC4-type family protein LOC_Os8g3776 Zinc-finger C3HC4-type family protein LOC_Os3g9 PHD-finger family protein LOC_Os6g69 Helix loop helix DNA-binding domain-containing protein LOC_Os8g3945 Multiple stress-responsive zinc-finger protein ISAP LOC_Osg686 Zinc finger CH-type family protein.488 LOC_Os5g94 Zinc finger C3HC4-type family protein LOC_Osg63 ZF-HD protein dimerization region containing protein.494 LOC_Os8g36 Zinc-finger protein.496 Up-regulated DNAme K4m3 K9Ac K7m3 LOC_Os5g583 Histone deacetylase b, putative. LOC_Os4g4356 NAC domain-containing protein / LOC_Os3g569 Homeobox protein OSH.64 LOC_Os7g377 Homeobox protein rough sheath.46 LOC_Osg39 Zinc-finger protein LOC_Os9g46 AP domain containing protein.93 LOC_Os3g55 WRKY DNA-binding domain-containing protein.46 Hu et al. 9of

10 Table S. Cont. Down-regulated Fold change q-value (%)* DNAme K4m3 K9Ac K7m3 LOC_Os7g73 AP domain-containing protein.5 LOC_Os7g4868 Zinc-finger C3HC4-type family protein LOC_Osg3376 NAC domain-containing protein /.876 LOC_Osg54 WRKY DNA-binding domain-containing protein *False discovery rate was calculated by using the SAM software as indicated in SI Materials and Methods. Genes with DNA methylation, H3K4me (K4m 3), H3K9 acetylation (K9ac), and H3K7me3 (K7m3) were detected according to Hu et al. of