supporting information - pnas · 2012-03-21 · supporting information hu et al....

Supporting InformationHu et al. 10.1073/pnas.1203148109SI Materials and MethodsHistone Extraction and Western Blot Analysis. Rice histone proteinswere extracted from 11-d-old seedlings. After being washed withacetone and dried, the proteins were suspended in SDS/PAGEsample buffer and tested by Western blot with antibodies againstH3K4me1 (Upstate Biotechnology; 07–436), H3K4me2 (Milli-pore; 05–790), H3K4me3 (Millipore; DAM1731494), H3K27me3(Millipore; DAM166204), and H3 (Abcam; ab1791).

Real-Time PCR. Real-time PCR was performed in a total volume of25 μL with 1.0 μL of the reverse transcription (RT) or chromatinimmunoprecipitation (ChIP) products, 0.25 μM primers, and 12.5μL SYBR Green Master mix (Takara) on a 7500 real-time PCRmachine (Applied Biosystems) according to the manufacturer’sinstructions. The rice ACTIN gene was used as the internal con-trol. All primers were annealed at 60 °C and run 42 cycles for RTproducts and 45 cycles for ChIP products. The ChIP enrichmentfor H3K27me3 and H3K4me3 was quantified by comparing thethreshold cycle (Ct) of the ChIP sample with that of the input with2(Ct of input-Ct of sample ChIP). The expression level of target genes wasalso normalized with that of ACTIN 2(Ct of actin-Ct of target).

In Vitro Histone-Binding Assays. Glutathione-agarose beads wereincubated with crude bacterial extract (containingGST fusions) inbinding buffer (50 mM Tris·HCl at pH 8.0, 150 mM NaCl, 0.1%Nonidet P-40, 10 mM ZnCl2, 1 mM DTT, and protease in-hibitors) for 30 min. After three washes, calf histones were addedand incubated overnight at 4 °C. After five washes, bound pro-teins were eluted and analyzed by Western blots with antibodiesagainst H3, H3K4me3, H3K9me3 (Abcam; ab8898), H3K27me3,or H3K36me3 (Abcam; ab9050).Biotinylated histone peptides were purchased from Upstate

Biotechnology. Briefly, 2 μg of peptides were incubated withcrude bacterial GST-plant homeodomain (PHD) and GST pro-tein extracts in binding buffer (50 mM Tris·HCl at pH 7.5, 300mM NaCl, 0.1% Nonidet P-40, 10 mM ZnCl2, 1 mM phenyl-methylsulphonyl fluoride) overnight at 4 °C. After incubationwith the above mixture for 1 h, streptavidin beads (Millipore)were washed three times and subjected to Western analysis withGST Antibody (Abcam; ab19256).

ChIP-seq and Data Analysis. Rice seedlings (11 d old) that weregrown under conditions of 14 h light/10 h dark at 25 °C–28 °C in 1/2 Murashige and Skoog medium were used for ChIP experi-ments. Chromatin was fragmented to 100–500 bp by sonication,and ChIP was performed using antibodies of H3K4me3 andH3K27me3. Briefly, precipitated DNA was end-repaired usinga combination of T4 DNA polymerase and T4 polynucleotidekinase. The blunt, phosphorylated ends were treated with Kle-now enzyme and dATP to yield a protruding 3′ “A” base forligation to Illumina’s adapters, which have a single “T” baseoverhang at the 5′ end, according to the Illumina Paired-EndDNA Sample Prep kit procedure. After adapter ligation, DNA

was PCR-amplified with Illumina primers, and library fragmentsof 100–300 bp (insert plus adaptor and PCR primer sequences)were isolated from an agarose gel. The purified DNA was cap-tured on an Illumina flow cell for cluster generation. Librarieswere sequenced with the equipment Illumina HISEQ2000.Each library had about 12,000,000 raw reads. Sequence reads

from all libraries were mapped to the reference genome of rice(Oryza sativa L. ssp. japonica cv. Nipponbare 6.1) using SOAP2.21software. Reads that could be mapped equally well to multiplelocations without mismatch or with identical mismatches wereassigned to one position at random and were retained for furtheranalysis as described previously (1). Genomic regions associatedwith histone modifications were identified using MACS software(2), in which default parameters (bandwidth: 200 bp; model fold:10, 32; P value: 1.00e-05; largelocal: 5,000) were set to call peaksrepresenting enriched epigenetic marks. MACS software was usedto calculate a dynamic local λ to reflect the local bias due topotential chromatin structure. After the positions of the peaks onthe chromosomes were found, the genes (including the 2-kb up-stream and 2-kb downstream regions) overlapping with the peakswere considered to have the epigenetic marks. The output of theanalysis pipeline was converted to wig files for viewing the data inthe GBrowse 2.0 software. The ChIP-seq data from this publi-cation have been deposited in the Gene Expression Omnibusdatabase (accession no.GSE30490).For annotation of genes and transposable elements and for gene

ontology, classification followed the Rice Genome AnnotationProject 6.1. The Web Gene Ontology Annotation Plotting tool(WEGO) (http://wego.genomics.org.cn/; GO archive: 2009–10-01; input file format: WEGO Native Format) was used to assigngenes to a hierarchical biological process. A particular pathwaythat corresponds to a test statistic was evaluated with a P valuecutoff at 0.05.

Microarray Analysis. For microarray analysis, 11-d-old seedlings ofwild type and mutants were grown in 1/2 Murashige and Skoogmedium under a 14-h light/10-h dark cycle at 25 °C–28 °C. RNAsamples were extracted using TRIzol (Invitrogen) as describedby the manufacturer. Hybridization with Affymetrix GeneChipRice Genome Arrays was performed at CapitalBio. The datasetwas normalized with the option of all probe sets scaled to thetarget signal of 100. The genes with expression calls that wereabsent 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 genesbetween the control and mutant seedlings. The imputation en-gine was set with 10 as the nearest neighbor imputer and thenumber of permutations was 100. The Δ-value in the SAM wasadjusted so that the estimated false discovery rate was < 5% forsignificant genes. The microarray data from this publication havebeen deposited in the Gene Expression Omnibus database (ac-cession no. GSE25073).

1. He G, et al. (2010) Global epigenetic and transcriptional trends among two ricesubspecies and their reciprocal hybrids. Plant Cell 22(1):17–33.

2. Zhang Y, et al. (2008) Model-based analysis of ChIP-Seq (MACS). Genome Biol 9:R137.

3. Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied tothe ionizing radiation response. Proc Natl Acad Sci USA 98:5116–5121.

Hu et al. www.pnas.org/cgi/content/short/1203148109 1 of 10

http://wego.genomics.org.cn/

www.pnas.org/cgi/content/short/1203148109

hCHD8 hCHD9 hCHD7 hCHD6

81

46

97

69Subfamily 1

hCHD1hCHD2dCHD1CHR705 (Os07g46590)

100100

94

100 dCHD3 hCHD3 hCHD4

hCHD5 CHR702 CHR6 (PICKLE) CHR7 (PKR2) ScCHD1 CHR703

98

7999

97

85

1230

8

9

Subfamily 3

CHR705 (Os07g46590)CHR5 (At2g13370)

ScCHD1hCHD6hCHD7hCHD8hCHD9

CHR6 (At2g25170, PICKLE)CHR7 (At4g31900, PKR2)

CHR702 (Os06g08480)

6761

100

100

100100

100

82

56

37

CHR723 CHR744

CHR729 CHR4 (PKR1)

dCHD1 hCHD1

hCHD2 CHR705

CHR5AtMOM

99

93

90

8669

29

3312

Subfamily 2

dCHD3hCHD4hCHD5hCHD3

CHR703 (Os01g65850)CHR729 (Os07g31450)CHR4 (At5g44800, PKR1)

CHR723 (Os06g01320)CHR744 (Os02g02050)CHR15 (At1g08060 MOM1)

100

100

54100

100

98

73

56

AtMOM

0.2

CHR15 (At1g08060, MOM1)

0.2

Fig. S1. Phylogenetic relationship between chromodomain, helicase/ATPase, and DNA-binding domain (CHD) proteins. (Left) Neighbor-joining tree using full-length CHD protein sequences from Saccharomyces cerevisiae (Sc), Homo sapiens (h), Drosophila melanogaster (d), Arabidopsis thaliana (At), and rice (Oryzasativa, Os), using the MEGA3.1 software. The values represent the percentages of sampled trees used in the analysis that contained the consensus partition. Thethree subfamilies are shaded. (Right) Neighbor-joining tree using CHD chromodomain sequences.



A

ZH11 10-16 10-17 10-13 21-2 21-23

CHR729 RNAi21-12

CHR729

Actin

1 2 3 5 9 14 15 16 17WT

CHR729

Actin

B

Fig. S2. Characterization of CHR729 T-DNA mutants and RNAi plants. (A) (Top) Gene structure of CHR729 with exons (black boxes) and introns (lines) and theT-DNA insertion site (triangle). RNAi region is underlined. (Middle) The homozygotes 1, 3, 14, 15, and 17 of chr729 showed no expression of the genes displayedby RT-PCR. (Bottom) Reduction of CHR729 transcripts in RNAi lines. CHR729 mRNA levels in RNAi lines 17–3-3 and 21–3-3 were determined by real-time RT-PCR.(B) Phenotypes produced by CHR729 RNAi plants. (Left) Wild type (left) and CHR729 RNAi (line 21–3-3) (right) plants at mature stage. (Center) A mature leafand 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 secondarybranch as indicated by red arrowheads in the wild type.



BA

6,810917

8,688824

6,128833 6,104616

8

10

80,7 80 82,4 81,760%

80%

100%

ue re

ads

(*10

0000

0)

uniq

ue re

ads

0

2

4

1 2 3 4

19,3 20 17,6 18,30%

20%

40%

HY chr729 HY chr729H3K4me3H3K27me3

HY chr729 HY chr729H3K4me3H3K27me3

Num

ber o

f uni

q

Per

cent

age

of

C

96,9 96,9

11,713,8

3,1 3,1

85%

90%

95%

100%

e of

mod

ified

regi

ons

88,386,2

75%

80%

1 2 3 4HY chr729 HY chr729H3K4me3H3K27me3

Perc

enta

ge

Fig. S3. Analysis of H3K27me3 and H3K4me3 ChIP-seq reads. (A) Number of unique reads from H3K27me3 and H3K4me3 ChIP of wild type (HY) and chr729.(B) Distribution of unique reads in genes (blue) and repetitive (yellow) regions. (C) Distribution of reads in transcribed (red) and nontranscribed (green) regions.



Os01g11000 Os01g16750

1

Os05g28210

H3K27me3 Wild type

chr729

0

0,2

0,4

0,6

0,8

1

0

0,1

0,2

0,3

0,4

0

0,2

0,4

0,6

0,8

1WT H3K4me3

chr729 H3K4me3

WT H3K27me3

chr729 H3K27me3

0 5

1

1,5

1

1,5

2

2,5

Os05g36990Os01g11300

1

1,5

Os07g41014

WT H3K4me3

chr729 H3K4me3

WT H3K27me3

0

0,5

0

0,5

1

0,8 1Os01g06210 Os08g05950

0

0,5

1

Os01g51610

WT H3K27me3

chr729 H3K27me3

0

0,2

0,4

0,6

0

0,2

0,4

0,6

0,8

0

0,2

0,4

0,6

0,8WT H3K4me3

chr729 H3K4me3

WT H3K27me3

chr729 H3K27me3

O 01 10110

Os01g56810

O 01 56810

WT H3K4me3

chr729 H3K4me3

WT H3K27me30,2

0,4

0,6

0,8

Os01g10110

0,1

0,2

0,3

Os01g56810

0,002

0,004

0,006

0,008

Os04g31804

chr729 H3K27me30

21

021

021

Fig. S4. Quantitative PCR validation of H3K27me3 ChIP-seq. Twelve genes were selected for the test (9 with reduced, 2 with unchanged, and 1 with increasedH3K27me3 in ChIP-seq as shown on the left). Levels relative to input are shown.



1

1,5

2

Os01g59090 Os09g25740

1

1,5

2

3

4

Os03g04770

H3K4me3

WT H3K4me3

h 729 H3K4 3

Os03g09170

0,004

0,006

0,008

0,01

Wild type chr729

0

0,5

1

1 2

1,5

Os02g29500

0

0,5

1 2

3

4

Os02g13500

0

1

2

1 2

WT H3K4me3

chr729 H3K4me3

0 1

0,15

Os06g16390Os01g45550

0,003

0,004

0

0,002

0,004

21

0

0,5

1

1 2

0

1

2

3

1 2

chr729 H3K4me3

WT H3K27me3

chr729 H3K27me3

0

0,05

0,1

1 2

Os05g39720Os03g19480

0

0,001

0,002

21

0,0250 4

1,6Os01g02150

WT H3K4me3

chr729

WT H3K27me3

chr7290

0,005

0,01

0,015

0,02

210

0,2

0,4

1 2

0

0,4

0,8

1,2

21

WT H3K4me3

chr729 H3K4me3

WT H3K27me3

Os03g56500Os03g58800

0,5

1

1,5

2

2,5

0

0,5

1

1,5

2

2,5Os11g06390

0,005

0,01

0,015

0,02

chr729 H3K27me30

21

021 0

1 2

Fig. S5. Quantitative PCR validation of H3K4me3 ChIP-seq. Fourteen genes were selected for the test (10 with reduced and 4 with increased or unchangedH3K4me3 levels in ChIP-seq as shown on the left). Levels relative to input are shown.



2WTA

1

1,5chr729 1 Os06g03670

2 Os09g35010

3 Os09g35030

4 Os02g43790

5 Os02g52670

6 Os04g48350

7 Os09g28440ve R

NA

leve

ls

0

0,5

1 2 3 4 5 6 7 8 9 10 11

8 Os03g09170

9 Os01g73770

10 Os02g45450

11 Os08g36920

1 Os06g03670

2 Os09g35010nt

1,5

WT

Rel

ati

3 Os09g35030

4 Os02g43790

5 Os02g52670

6 Os04g48350

7 Os09g28440

8 Os03g09170

9 O 04 52090K4m

e3 e

nric

hme

0,5

1

chr729

s g

H3K

0

1 2 3 4 5 6 7 8 9m

entB 2

HY2,5 HYh 29e v

els

h 729

3K4m

e3 e

nric

hm

0

0,5

1

1,5

0

0,5

1

1,5

2

1 2

chr729

Rel

ativ

e R

NA

l chr729

H3 0

21

Fig. S6. Validation of altered expression levels and H3K4me3 changes of AP2 transcription factor genes in the chr729 mutant. (A) Relative expression levels of11 down-regulated AP2 genes (Upper) and H3K4me3 level for 9 of the 11 genes (Lower) in chr729 compared with wild type. (B) Relative expression (Left) andH3K4me3 (Right) levels of two up-regulated AP2 genes in chr729 compared with wild type.

WTchr729

A le

vels

Rel

ativ

e R

N

OsiEZ1 OsCLF OsEMF2a OsEMF2b OsFIE1 OsFIE2

Fig. S7. Expression of Polycomb group PRC2 genes in chr729 relative to the wild type.



Table S1. Loss or gain of H3K27me3 and H3K4me3 in chr729

H3K27me3 lost H3K27me3 maintained H3K27me3 gained

H3K4me3 loss (%) 12.37 10.31 9.02H3K4me3 gain (%) 1.35 1.18 2.92No H3K4me3 (%) 65.94 66.45 70.95With H3K4me3 (%) 20.34 22.06 17.11

H3K4me3 lost H3K4me3 maintained H3K4me3 gained

H3K27me3 loss (%) 4.89 2.41 3.59H3K27me3 gain (%) 1.39 0.79 3.04No H3K27me3 (%) 90.50 94.73 90.88With H3K27me3 (%) 3.22 2.07 2.49

The percentages of genes that lost, gained, or maintained (with or without) H3K4me3 and H3K27me3 within thecategories of genes that lost, gained, or maintained H3K27me3 and H3K4me3, respectively, in chr729 are shown.



Table S2. Transcription factor genes that are differentially regulated in chr729

Down-regulated Fold change q-value (%)* DNAme K4m3 K9Ac K27m3

LOC_Os09g28440 AP2 domain-containing protein 0.089 0.0590 † † *LOC_Os08g36920 AP2 domain-containing protein 0.143 0.1872 † † †

LOC_Os06g03670 AP2 domain-containing protein 0.146 0.0590 † † †

LOC_Os04g31804 OsMADS64-MADS-box family gene with M-α type-box 0.163 0 †

LOC_Os01g73770 CRT/DRE-binding factor 1 0.176 0.2566 † † †


LOC_Os01g61080 DNA-binding protein 0.226 0.1872 † †

LOC_Os03g53020 Helix–loop–helix DNA-binding domain containing protein 0.237 0.5320 † † †

LOC_Os03g09170 AP2 domain-containing protein 0.252 0 † † †

LOC_Os02g32590 Heat stress transcription factor A3 0.256 0 † † †

LOC_Os01g64310 NAC domain-containing protein 90 0.257 0.1872 † † †

LOC_Os03g60570 Zinc-finger C2H2-type family protein 0.276 0 † †

LOC_Os01g64360 Myb-like DNA-binding domain-containing protein 0.285 0.4217 † †


LOC_Os03g20090 Myb-like DNA-binding domain-containing protein 0.295 0.2566 † † †

LOC_Os05g39720 DNA-binding protein WRKY1 0.299 0.0685 † † †

LOC_Os05g07010 myb-like DNA-binding domain; SHAQKYF class family protein 0.305 0.8319 †


LOC_Os09g28210 Helix–loop–helix DNA-binding domain-containing protein 0.317 0.4217 † † †

LOC_Os02g08440 WRKY transcription factor 0.318 0.2566 † † †

LOC_Os06g44010 WRKY2 protein 0.371 0.8319 † † †

LOC_Os02g26430 WRKY DNA-binding domain-containing protein 0.372 0.2566 † † †

LOC_Os04g23550 Helix–loop–helix DNA-binding domain-containing protein 0.375 0.5320 † † † †

LOC_Os10g39130 Agamous-like MADS box protein AGL19 0.377 0 † † †

LOC_Os03g06630 Heat-shock factor protein 1 0.386 0.0590 † †

LOC_Os09g35030 DREB1A protein 0.395 0.0685 † † †

LOC_Os02g43790 AP2 domain-containing protein 0.396 1.1964 † †

LOC_Os05g03760 Zinc-finger C-x8-C-x5-C-x3-H–type family protein 0.403 0.2566 † † †

LOC_Os01g74040 Zinc-finger C3HC4-type family protein 0.405 1.1964 † † †

LOC_Os04g52090 AP2 domain-containing protein 0.407 0.0590 † †

LOC_Os04g44820 Zinc-finger C3HC4-type family protein 0.41 0 †

LOC_Os01g14440 WRKY DNA-binding domain-containing protein 0.418 0.5320 † †

LOC_Os03g55540 Zinc-finger protein 1 0.423 0.0685 † †

LOC_Os02g41510 Myb-related protein Myb4 0.423 0 † † †

LOC_Os03g03070 MADS-box transcription factor 50 0.424 0 † † †

LOC_Os02g49840 Agamous-like MADS box protein AGL21 0.429 0 † †

LOC_Os08g38460 Zinc-finger C3HC4-type family protein 0.434 0 † †

LOC_Os01g60640 WRKY DNA-binding domain-containing protein 0.436 1.1964 † † †

LOC_Os02g48320 DNA-binding protein 0.438 0.1872 † †


LOC_Os04g43680 Myb-related protein Myb4 0.453 2.5729 † †

LOC_Os05g36930 Histone deacetylase family protein 0.462 0.0685 † †

LOC_Os07g31450 SNF2 family N-terminal domain-containing protein 0.463 0LOC_Os04g48350 CRT/DRE binding factor 1 0.463 0.2566 † †





LOC_Os03g19020 PHD-finger family protein 0.484 0.5320 † †

LOC_Os06g06900 Helix–loop–helix DNA-binding domain-containing protein 0.485 0.0685 † †

LOC_Os08g39450 Multiple stress-responsive zinc-finger protein ISAP1 0.487 0.2566 † † †

LOC_Os01g68160 Zinc finger C2H2-type family protein 0.488 0 †

LOC_Os05g01940 Zinc finger C3HC4-type family protein 0.494 0.0590 † † †

LOC_Os12g10630 ZF-HD protein dimerization region containing protein 0.494 0 † † †

LOC_Os08g36110 Zinc-finger protein 0.496 0 † † †

Up-regulated DNAme K4m3 K9Ac K27m3LOC_Os05g51830 Histone deacetylase 2b, putative 2.01 0 †

LOC_Os04g43560 NAC domain-containing protein 21/22 2.041 1.1964 † † †

LOC_Os03g51690 Homeobox protein OSH1 2.064 0 † † †

LOC_Os07g03770 Homeobox protein rough sheath 1 2.146 0 † †

LOC_Os12g39220 Zinc-finger protein 7 2.156 0.0463 † †

LOC_Os09g11460 AP2 domain containing protein 2.193 0 † † †

LOC_Os03g20550 WRKY DNA-binding domain-containing protein 2.406 0



Table S2. Cont.

Down-regulated Fold change q-value (%)* DNAme K4m3 K9Ac K27m3

LOC_Os07g22730 AP2 domain-containing protein 2.512 0 † † †


LOC_Os10g33760 NAC domain-containing protein 21/22 2.876 0 † †

LOC_Os11g02540 WRKY DNA-binding domain-containing protein 3.079 0.4217 † †

*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 H3K27me3 (K27m3) were detected according to http://www.pyc.pku.edu.cn/.


http://www.pyc.pku.edu.cn/


supporting information - pnas · 2012-03-21 · supporting information hu et al....

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