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Molecular Cell

Article

Regulation of Active DNA Demethylationby an a-Crystallin Domain Protein in ArabidopsisWeiqiang Qian,1,2,3 Daisuke Miki,1,3 Mingguang Lei,2,3 Xiaohong Zhu,2 Huiming Zhang,2 Yunhua Liu,2 Yan Li,1

Zhaobo Lang,2 Jing Wang,1 Kai Tang,2 Renyi Liu,1 and Jian-Kang Zhu1,2,*1Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032,

China2Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47906, USA3Co-first author

*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.molcel.2014.06.008

SUMMARY

DNAmethylation patterns are dynamically controlledby DNA methylation and active DNA demethylation,but the mechanisms of regulation of active DNAdemethylation are not well understood. Throughforward genetic screens for Arabidopsis mutantsshowing DNA hypermethylation at specific loci andincreased silencing of reporter genes, we identifiedIDM2 (increased DNA methylation 2) as a regulatorof DNA demethylation and gene silencing. IDM2dysfunction causes DNA hypermethylation andsilencing of reporter genes and some endogenousgenes. These effects of idm2 mutations are similarto those of mutations in IDM1, a regulator of activeDNA demethylation. IDM2 encodes an a-crystallindomain protein in the nucleus. IDM2 and IDM1interact physically and partially colocalize at discretesubnuclear foci. IDM2 is required for the full activityof H3K18 acetylation but not H3K23 acetylationof IDM1 in planta. Our results suggest that IDM2functions in active DNA demethylation and in antisi-lencing by regulating IDM1.

INTRODUCTION

Epigenetic modifications determine the status of chromatin and

consequently control the transcriptional potential (Bender, 2004;

He et al., 2011; Law and Jacobsen, 2010; Matzke and Birchler,

2005; Tariq and Paszkowski, 2004). DNA methylation is an

important epigenetic mark conserved in many eukaryotes. In

plants, DNA methylation occurs in all three cytosine contexts,

namely CG, CHG, and CHH (H represents A, T, or C) (He et al.,

2011; Law and Jacobsen, 2010). Genome-wide mapping of

DNA methylation in Arabidopsis revealed that gene bodies are

mainly associated with CG methylation, whereas transposon-

and DNA-repeat-enriched heterochromatin regions are the ma-

jor targets of CHG and CHH methylation (Zhang et al., 2006).

Although the function of abundant CG methylation within genic

regions remains unclear, DNA methylation, especially CHG and

M

CHH methylation, is generally correlated with transcription-

repressive histonemodifications and confers negative regulation

of transcriptional activities (Law and Jacobsen, 2010; Pikaard,

2013; Zhang and Zhu, 2012).

DNAmethylation at the fifth position of the cytosine pyrimidine

ring is catalyzed by DNA methyltransferases (DNMTs) that use

S-adenosylmethionine as the methyl donor. Plants possess mul-

tiple DNMT proteins that cooperatively establish and maintain

DNA methylation. In Arabidopsis, DRM2 catalyzes methylation

in all cytosine contexts, whereas MET1 and CMT3 catalyze

symmetric CG and CHG methylation, respectively, during DNA

replication to maintain the epigenetic patterns (Law and Jacob-

sen, 2010). Because of its asymmetric nature, CHH methylation

must be established de novo during each cell cycle (Law and

Jacobsen, 2010). In addition to DRM2, CMT2 was recently

demonstrated to be a methyltransferase that catalyzes CHH

methylation (Zemach et al., 2013).

DNA methylation patterns in plants and other eukaryotes

depend not only on DNAmethylation activities but also on active

DNA demethylation (Zhang and Zhu, 2012; Zhu, 2009). In

contrast to passive demethylation, in which DNA methylation is

lost because of a lack of maintenance methylation, active deme-

thylation refers to the enzymatic process in which 5-methylcyto-

sine is replaced with cytosine independently of DNA replication.

In plants, active DNA demethylation is catalyzed by a subfamily

of bifunctional DNA glycosylases/lyases represented by

Repressor of Silencing (ROS)1 and DME (Agius et al., 2006;

Gehring et al., 2006; Gong et al., 2002; Ortega-Galisteo et al.,

2008; Penterman et al., 2007). These DNA demethylases directly

excise the 5-methylcytosine base and then cleave the DNA

backbone at the abasic site. The resultant single-nucleotide

gap is subsequently filled with an unmodified cytosine through

the DNA base excision repair pathway (Zhang and Zhu, 2012;

Zhu, 2009). In Arabidopsis, DME confers global demethylation

during gametogenesis (Gehring et al., 2009; Hsieh et al., 2009;

Huh et al., 2008), whereas ROS1 catalyzes active DNA demethy-

lation at discrete genetic loci across the genome during vegeta-

tive growth (Gong et al., 2002; Lister et al., 2008; Penterman

et al., 2007; Qian et al., 2012; Zhu et al., 2007).

DNA methyltransferases can be targeted to specific loci

through mechanisms such as the RNA-directed DNA methyl-

ation (RdDM) pathway, in which complementary pairing between

24 nt small interfering RNAs and nascent scaffold RNAs guides

olecular Cell 55, 361–371, August 7, 2014 ª2014 Elsevier Inc. 361

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Molecular Cell

A Regulator of Active DNA Demethylation

the methylation complex to its target loci with the aid of protein-

protein interactions within the silencing complex (Gao et al.,

2010; Haag and Pikaard, 2011; He et al., 2009; Law and Jacob-

sen, 2010; Matzke et al., 2009; Pikaard, 2013; Wierzbicki, 2012;

Zhang and Zhu, 2012). In contrast to the establishment of DNA

methylation, little is known about the locus-specific guidance

of active DNA demethylation. Recruitment of ROS1 to its target

loci has been suggested to require assistance by the RNA-

binding protein ROS3, because ros3 mutation disrupts the

subnuclear localization patterns of ROS1 and causes DNA hy-

permethylation of some ROS1 target loci (Zheng et al., 2008).

Increased DNA methylation (IDM)1, a histone acetyltransferase,

was recently identified as another important regulator of active

DNA demethylation (Qian et al., 2012). IDM1 recognizes chro-

matin that contains CG methylation and low histone 3 lysine 4

(H3K4) and arginine 2 (H3R2) methylations and catalyzes

H3K18 and H3K23 acetylation, which then somehow facilitates

ROS1-mediated demethylation (Qian et al., 2012).

The small heat shock protein (HSP) family of proteins is

widely distributed in both prokaryotes and eukaryotes and is

defined by a conserved 100- to 110-amino acid motif known

as the a-crystallin domain (ACD), which is flanked by a variable

N-terminal domain and a short C-terminal extension (Basha

et al., 2012; MacRae, 2000). Small HSPs are known as the para-

medics of cells (Hilton et al., 2013) by functioning as ATP-inde-

pendent molecular chaperones that, under stress conditions,

bind and stabilize denaturing proteins so that these proteins

can later be renatured by ATP-dependent chaperones (Basha

et al., 2012). In mammals, small HSPs not only play critical roles

in modulating vital physiological processes including smooth

muscle relaxation and cardiac contractility but also function

as an innate protector against debilitating pathological condi-

tions such as cardiac hypertrophy and Alzheimer’s disease

(Edwards et al., 2011; Sun and MacRae, 2005). In plants, small

HSPs have been shown in vitro to prevent irreversible protein

aggregation and insolubilization (Basha et al., 2012; Scharf

et al., 2001). Whereas most plant small HSPs are heat shock

inducible, proteins in the ACD family can be involved in other

cellular processes unrelated to heat stress. For instance, the

ACD protein AtRTM2 is not required for heat stress tolerance,

but prevents systemic spreading of tobacco etch virus in

Arabidopsis (Whitham et al., 2000). Although ACD proteins

have been shown to be important for diverse cellular processes,

it is unclear whether any ACD proteins can modulate epigenetic

regulation.

In this study, we found that an atypical small HSP, IDM2, func-

tions in association with IDM1 to regulate active DNA demethy-

lation in Arabidopsis. Identified through forward genetic screens,

the idm2mutants exhibit DNA hypermethylation at thousands of

genetic loci and show increased transcriptional silencing of re-

porter genes and some endogenous genes. Regulation of DNA

methylation by IDM2 requires the conserved ACD. Unlike that

of typical small HSPs, the IDM2 transcript level does not respond

to heat stress. IDM2 interacts and partially colocalizes with IDM1

in vivo. In addition to having DNAmethylation and gene-silencing

phenotypes similar to those of idm1, the idm2mutants are similar

to idm1 in displaying reduced levels of H3K18ac at loci where

DNA methylation is affected. Our results suggest that plants

362 Molecular Cell 55, 361–371, August 7, 2014 ª2014 Elsevier Inc.

have evolved an ACD protein that specifically functions in

epigenetic regulation.

RESULTS

Isolation of idm2 MutantsTo better understand the mechanism of active DNA demethyla-

tion initiated by ROS1, we carried out a genetic screen for

increased DNA methylation mutants using a Chop PCR marker,

which was designed based on the whole-genome DNA methyl-

ation analysis of ros1-1 (Qian et al., 2012). One T-DNA mutant,

referred to here as idm2-1, which shows almost the same

DNA methylation level as the ros1 mutant at the 30 region of

At1g26400 (Figure 1A), was identified by screening a library of

homozygous T-DNA insertion mutants from the Arabidopsis

Biological Resource Center. This mutant has a T-DNA insertion

in the gene At1g54840 (Figures S1A–S1C available online). To

confirm that this mutation results in DNA hypermethylation, we

tested another T-DNA mutant allele (i.e., idm2-2). Chop PCR

results revealed a similar hypermethylation phenotype in the

idm2-2 allele (Figure 1A). Bisulfite sequencing data showed

that the DNA methylation level at the At1g26400 site was

increased in idm2-1, idm2-2, and ros1-4 in all sequence contexts

compared to the wild-type control (Figure 1B). To further confirm

that At1g54840 is indeed the IDM2 gene, we transformed a

2.7 kb wild-type genomic fragment containing the At1g54840

promoter and its coding sequence into idm2 mutant plants. As

shown in Figure S1D, the transgene was able to rescue the

DNA methylation defect of idm2 mutant plants.

IDM2 Prevents the Transcriptional Silencing ofTransgenesPrevious studies showed that ROS1 is required to prevent the

transcriptional silencing of RD29A-LUC and 35S-NPTII trans-

genes (Gong et al., 2002). Mutations in IDM1 cause the silencing

of 35S-NPTII but not of the RD29A-LUC transgene (Li et al.,

2012; Qian et al., 2012). An independent genetic screen for mu-

tants impaired in the prevention of silencing of the 35S-SUC2

transgene led to the isolation of mutant alleles of IDM1 as well

as ROS1 (Wang et al., 2013). This later genetic screen also led

to the isolation of idm2-3 (Figure 1C).Wild-type 35S-SUC2 trans-

genic plants showed an inhibition of root growth on Murashige-

Skoog (MS) mediumwith 2% sucrose due to high levels of SUC2

(sucrose transporter 2) expression (Lei et al., 2011). The root

length in both idm1-9 and idm2-3 plants was similar to that in

the Col-0 control that did not have the 35S-SUC2 transgene.

Both the 35S-SUC2 and 35S-HPTII transgenes were silenced

in idm1-9 and idm2-3 mutant plants (Figure 1D). Bisulfite

sequencing analysis showed that, compared to the wild-type

control, the DNA methylation level of the 35S promoter region

was increased in idm1-9 and idm2-3 plants (Figure S1E). The

At1g26400 region was also hypermethylated in idm1-9 and

idm2-3 according to the Chop PCR results (Figure S1F). Chro-

matin immunoprecipitation (ChIP) assays showed an increased

H3K9me2 level and a decreased H3K4me2 level in the 35S

promoter region in the mutant plants (Figure 1E). In idm2-3, a

single-nucleotide-substitution mutation (from GGG to GAG) in

At1g54840 changed amino acid glycine 276 to glutamic acid.

At1g26400 HhaI

No digestioncontrol

C24 Col

% C

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evitaleR

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Figure 1. Identification of idm2 Mutants, Their Genetic Interactions with ros1, rdr2, and nrpe1 Mutations, and Their Effects on DNA Methyl-

ation, Histone Modification, and Transcriptional Silencing

(A) Analysis of DNA methylation level at the At1g26400 locus by Chop PCR. Because HhaI digestion is sensitive to CG DNA methylation, DNA hypermethylation

results in increased levels of the PCR product. Undigested DNA is shown as a control.

(B) Bisulfite sequencing data showing the effects of idm2mutations on At1g26400 DNAmethylation in different sequence contexts and genetic interactions with

ros1, rdr2, or nrpe1.

(C) Effect of idm1 and idm2 mutations on 35S-SUC2 transgene-mediated root growth suppression. Seedlings were grown on plates with MS medium plus 2%

sucrose for 3 weeks before they were photographed.

(D) Real-time PCR analysis of the expression levels of SUC2 and HPTII transgenes in the different genotypes. TUB8 was used as an internal control. Error bars

represent standard error (n = 3).

(E) ChIP assay of H3K4me2 and K3H9me2 in the wild-type, idm1-9, and idm2-3. Ten-day-old seedlings were used for ChIP assay with antibodies against

H3K9me2 or H3K4me2. The ChIP signal was quantified relative to input DNA. The no-antibody precipitates served as negative control. Two biological replicates

were performed, and very similar results were obtained. Standard errors were calculated from three technical repeats.

(F) The DNA hypermethylation phenotype of idm2-1 plants is rescued by transgenic expression of wild-type IDM2 but not by expression of the D49E, G246D, or

G260D mutant. Chop PCR results for At1g26400 are shown for two representative transgenic lines generated from each construct.

See also Figures S1 and S4 and Tables S4 and S5.

Molecular Cell


Molecular Cell 55, 361–371, August 7, 2014 ª2014 Elsevier Inc. 363

Genotype Hyper-DMR

Hypo-DMR Overlap with rdd

Overlap with ros1-4

Overlap with idm1-1

Overlap with

repeats

rdd 9290 1052 68.8%

ros1-4 4991 106 81.3% 79.5%

idm1-1 1098 75 62.9% 51.5% 60.5%

idm2-1 1218 264 70.3% 49.8% 39.8% 59.3%

0%

20%

40%

60%

80%

100%

ros1-4 rdd idm1-1 idm2-1

TE Intergenic Gene

A

B

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% M

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latio

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CG

CHH

CHG

idm2 hyper-DMRs idm1 hyper-DMRs rdd hyper-DMRs

E856

362

8447idm2-DMRs

Figure 2. Analysis of DMRs Identified in idm2 Mutant

(A) The number of DMRs identified in idm2 and the overlap of hyper-DMRs between different mutant plants.

(B) Composition of the hypermethylated loci in idm2-1, idm1-1, ros1-4, and rdd mutants. TE, transposable element.

(legend continued on next page)

Molecular Cell



Molecular Cell


Consistent with the genetic mapping result (Figure S1G)

suggesting that the idm2-3 mutation was responsible for the

long-root phenotype, all of the F1 plants from genetic crosses

between idm2-3 and idm2-1 (SALK_130656) had a long-root

phenotype like that of idm2-3 (Figure S1H). We also transferred

the wild-type IDM2 genomic fragment into idm2-3 mutant

plants and found that it can complement the root phenotype

(Figure S1H). These results demonstrated that, like IDM1

and ROS1 (Wang et al., 2013), IDM2 is required for prevention

of transcriptional silencing of the 35-SUC2 and 35S-HPTII

transgenes.

IDM2 Prevents DNA Hypermethylation at Thousands ofLociTo test the effect of the idm2mutation on genomic DNA methyl-

ation status, we performed Southern blot analysis on 5S rDNA

and 180 bp centromeric repeat regions. The analysis found

that the idm2 mutation does not affect the DNA methylation at

5S rDNA or at the 180 bp centromeric repeat (Figure S2A). The

results suggest that, like ROS1 and IDM1 mutations, the IDM2

mutation affects the methylation status of specific loci rather

than the overall methylation status of the genome.

To identify the specific loci where DNA methylation is

increased in idm2 mutant plants, we compared the genome-

wide DNA methylation profiles of idm2-1 and wild-type Col-

0 plants by next-generation sequencing after bisulfite conversion

(Lister et al., 2008; Qian et al., 2012). There was no significant dif-

ference between idm2-1 and wild-type plants in their overall

genomemethylation patterns (data not shown). In total, we iden-

tified 1,482 differentially methylated regions (DMRs) in idm2-1.

Among these, 1,218 are hyper-DMRs showing a significantly

increasedDNAmethylation, and only 264 are hypo-DMRs, which

show a significantly reduced methylation (Figure 2A; Tables S1

and S2). About 60% of the hyper-DMRs overlap with repeats

as defined by RepeatMasker (http://www.repeatmasker.org)

(Table S1). Interestingly, genic loci account for approximately

80% of the hyper-DMRs in idm2-1, which is the same as in

idm1-1, whereas genic loci account for only about 30% and

48% of the hyper-DMRs in ros1-4 and rdd (ros1dml2dml3),

respectively (Figure 2B).

We calculated the level of small RNAs at the hyper-DMRs

according to published data sets (Lister et al., 2008) and found

that small RNAs are enriched at the hyper-DMRs compared

to randomly selected regions in the genome (Figure 2C). Of the

1,218 hyper-DMRs in idm2-1, six (DMR-72, DMR-232, DMR-

265, DMR-333, DMR-828, and DMR-946) were selected for

confirmation by individual locus bisulfite sequencing, and all

were confirmed to be hypermethylated in idm2-1 (Figures S2B

and S2C), as in ros1-4 or rdd (Qian et al., 2012). Many of the hy-

permethylated genes in idm2-1 belong to multigene families in

which the member genes are highly homologous (Table S1).

The Chop PCR marker locus At1g26400, which was also identi-

(C) Enrichment of small RNAs (sRNAs) in the 1,218 hypermethylated regions iden

mutant plants by Lister et al. (2008). Black dots indicate the sRNA densities fro

densities in the hypermethylated regions in idm2-1. Horizontal lines indicate the

(D and E) Overlap of hyper-DMRs between idm2 and idm1 (D) and between idm

See also Figure S2 and Tables S1 and S2.

M

fied from the whole-genome bisulfite sequencing data as being

hypermethylated, belongs to a highly homologous and tandemly

arranged flavin adenine dinucleotide-binding berberine gene

family (Figure S2D). According to publicly available microarray

data, the expression of most members from this gene family is

tissue specific (data not shown). As was the case for the hy-

per-DMRs in the idm1 mutant, hyper-DMRs in idm2 correspond

to sequences with low H3K4 mono-, di-, and trimethylation rela-

tive to a comparison group of CG methylated and expressed

genes (Figures S2E–S2K) (Qian et al., 2012; Zhang et al., 2009).

IDM2 Functions in the SameGenetic Pathway with IDM1in Counteracting RNA-Directed DNA Methylation atSome LociDouble mutant analysis indicated that the CHG and CHH hyper-

methylation phenotypes of ros1-4 and idm2-1 or idm2-2 are not

additive at At1g26400 (Figure 1B) or at another two loci (DMR-72

and At5g38550) that we examined (Figure S2C). An examination

of the At5g38550 locus shows that the CG hypermethylation

phenotypes of the twomutants are also not additive (Figure S2C).

Furthermore, analysis of the idm1idm2 double mutant found no

additive effects between the idm1 and idm2mutants (Figure 2D).

These results, together with previous results showing a lack of

additive effects between idm1 and ros1 (Qian et al., 2012), sug-

gest that IDM2 functions with IDM1 and ROS1 in the same

genetic pathway to prevent DNA hypermethylation at some

loci. The hypermethylation in CHG and CHH sequence contexts

in idm2-1 is suppressed by the rdr2 and nrpe1 mutations (Fig-

ure 1B), suggesting that the methylation at the At1g26400 region

is mediated through the RdDM pathway.

According to the whole-genome bisulfite sequencing data,

approximately 40%, 50%, and 70% of the 1,218 hyper-DMRs

in idm2-1 are also hypermethylated in the idm1, ros1-4, and

rdd mutants, respectively (Figure 2A; Table S1). The DNA

methylation level was increased in all sequence contexts for

the overlapped loci in these mutants (Figures 2D and 2E). The

DNA methylation level at the idm2-specific loci also appears

increased in idm1-1 (Figure 2D), although the increases in

idm1-1 were not enough to make the loci count as hyper-

DMRs according to the parameters defined in this study. Simi-

larly, the DNA methylation level at the idm1-specific loci also

appears increased in idm2 (Figure 2D). These results show that

a large percentage of hyper-DMRs in idm2 also have increased

DNA methylation in the idm1, ros1, and rdd mutants, further

supporting that IDM2 functions in active DNA demethylation at

these loci.

IDM2 Prevents the Silencing of Some EndogenousGenesChanges in the DNAmethylation level in or near a gene can affect

the expression level of that gene. To determine whether DNA

hypermethylation affects the expression of nearby genes in

tified in idm2-1. Small RNA reads were generated from the wild-type and rdd

m randomly selected regions in the genome, and red dots indicate the sRNA

99th percentile of sRNA density from 1,000 simulated runs.

2 and rdd (E). Boxplots represent methylation levels of each class of DMRs.


http://www.repeatmasker.org

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idm2-1 vs. idm1-1

B

A

Figure 3. Gene Expression Level in idm1 and idm2 Mutants

(A) Expression of the hypermethylated genes or genes near DMRs in 2-week-

old idm1-1, idm2-1, and ros1-4 seedlings. Error bars represent standard error

(n = 3).

(B) Overlap of differentially expressed genes between the different comparison

groups.

See also Figure S3 and Tables S3 and S4.

Molecular Cell


idm2mutant plants, we examined the expression levels of seven

genes that have transcript levels detectable by quantitative (q)

PCR and show increased DNA methylation near or within the

gene in idm2, idm1, and ros1 (Figures S2F–S2J). We found

that all of the tested genes showed a substantial reduction

in their transcript levels in these mutants (Figure 3A). Our results

suggest that, like ROS1 and IDM1, IDM2 is critical for preventing

the transcriptional silencing of some endogenous plant genes.

The ROS1 transcript level is increased in the idm2 and idm1idm2

double mutants but not in the idm1 mutant, and treatment

with the DNA methylation inhibitor 50-aza-20-deoxycytidine (50-aza-dC) blocks ROS1 expression (Figure S3A). Unlike ROS1,

the IDM1 transcript level does not show a substantial difference

between idm2 mutants and the wild-type (Figure S3B). The

transcript level of IDM2 does not show dramatic differences in

the active DNA demethylation pathway mutants and RdDM

pathway mutants (Figures S3C and S3D).

To test the effect of the idm2 mutation on genome-wide tran-

script levels, we performed a tiling array assay in wild-type,

idm1-1, and idm2-1 plants. Similar numbers of genes are

down- or upregulated in idm1 and idm2 (Figure S3E; Table S3),

and most of the affected genes overlapped between idm1 and


idm2 (Figure 3B). Quantitative PCR assays confirmed that the

genes identified by tiling array are down- or upregulated in

idm1 and idm2 mutant plants (Figures S3F and S3G). The tiling

array assay was not sensitive enough to detect the expression

of the seven genes that have increased methylation in idm2,

idm1, and ros1, although the expression could be detected by

qPCR (Figure 3A). Four (At2g16280, At4g08470, At4g13340,

and At5g24030) of the genes found by the tiling array assay to

have an increased expression in idm1 and idm2 are hypomethy-

lated in the mutants. However, none of the genes found by the

tiling array assay to have decreased expression in idm1 and

idm2 is hypermethylated in the mutants, suggesting that these

genes are not regulated by IDM1 and IDM2 directly through

DNA methylation.

Our previous data showed that IDM1 is important for the

expression of some transposable element genes when these si-

lent genes are released by treatment with the DNA methylation

inhibitor 50-aza-dC or by the ddm1 mutation (Li et al., 2012).

We wondered whether idm2 also affects the expression level

of this type of genes. The qPCR results demonstrated that the

expression level of At3g18250 is lower in idm2 than in the wild-

type control after 50-aza-dC treatment and that there is no addi-

tive effect in the idm1idm2 double mutant (Figure S3H). These

data suggest that IDM2 and IDM1 regulate the expression of

the same groups of genes and, like IDM1, IDM2 is required for

preventing transcriptional silencing of some endogenous genes.

IDM2 Is an Atypical sHsp20 Family MemberIDM2 is predicted to have an ACD in the C-terminal region (Fig-

ures S4A and S4B). IDM2 is highly conserved in plants, because

homologous sequences can be found in monocots as well as di-

cots (Figure S4A). In Arabidopsis, IDM2 belongs to a small gene

family, which has a conserved ACD at the C terminus but a var-

iable N-terminal region (Figure S4B). The sequence of the ACD of

IDM2 has high similarity with that of sHsp20s, which belong to a

class of proteins that occur in all plants and that vary in size from

approximately 16 to 42 kDa (Scharf et al., 2001). Most of the

sHsps are induced by heat shock treatment (Scharf et al.,

2001). Using qPCR, we measured the expression levels of

IDM2 and two closely related genes in Arabidopsis after heat

stress treatment. Interestingly, heat stress did not induce IDM2

or the two closely related genes to express at high levels,

whereas the treatment induced a very high level of expression

of AtHsp17.4 (Figure S4C). Unlike most of the sHsp20s, which

are localized in chloroplasts or mitochondria (Basha et al.,

2012), IDM2 is localized in the nucleus, according to the fluores-

cence signal of GFP-IDM2 in the transgenic Arabidopsis plants

(Figure S4D).

The ACD Is Critical for the Function of IDM2The ACD is highly conserved among different ACD protein family

members (Scharf et al., 2001). Residues G246 and G260 of IDM2

(Figure S4A) correspond to critical positions in the known struc-

tures of ACDs (Scharf et al., 2001). We constructed wild-type

and mutant versions of IDM2 containing the G246D or G260D

mutation and expressed them in idm2-1 mutant plants under

control of the double 35S promoter. The wild-type construct

but not the G246D or G260D mutant complemented the DNA

MYC-IDM2

anti-GFP

anti-MYC

anti-MYC

GFP-IDM1-

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IP: anti-HA

anti-HA

anti-Flag

anti-Flag

D

IDM1-HA X

Figure 4. Protein-Protein Interactions between IDM2 and IDM1

(A) Yeast two-hybrid analysis of IDM1 and IDM2 interaction. AD, activating

domain; BD, binding domain.

(B) LCI assays showing that IDM1 can interact with IDM2 in Arabidopsis pro-

toplasts. 1, IDM1-nLUC+IDM2-cLUC; 2, IDM1-nLUC+cLUC; 3, nLUC+IDM2-

cLUC; 4, nLUC+cLUC. Three biological replicates were performed, and very

similar results were obtained. Standard errors were calculated from three

technical repeats.

(C) Co-IP of IDM1 and IDM2 in tobacco leaves. MYC-tagged IDM2 and GFP-

tagged IDM1 were transiently expressed in N. benthamiana leaves. Anti-GFP

was used for immunoprecipitation (IP); anti-MYC and anti-GFP were used for

immunoblotting. Input, total protein before immunoprecipitation.

(D) The interaction between Flag-IDM2 and IDM1-HA as determined by co-IP.

Transgenic plants expressing Flag-IDM2 or IDM1-HA under their native pro-

moters and their F1 offspring were used for co-IP.

Molecular Cell


hypermethylation phenotype of the idm2-1 mutant (Figure 1F),

even though the mutated proteins were expressed as well as

the wild-type protein (Figure S4E). Additionally, idm2-3 also con-

tains an amino acid change in the ACD and shows a DNA hyper-

methylation phenotype (Figures S1F and S4A). The results show

that the ACD is important for IDM2 function in vivo. In addition to

the conserved ACD at the C-terminal region, the N-terminal re-

gion of IDM2 protein is also conserved across different plant

species (Figure S4A). Expression of the D49E mutant form of

IDM2 driven by the double 35S promoter failed to complement

the DNA hypermethylation phenotype of the At1g26400 region

M

in idm2-1 mutant plants (Figure 1F), even though the mutant

form of IDM2 was expressed at a similar level as the wild-type

protein (Figure S4E). These results suggest that the conserved

N-terminal region is also important for the in vivo function of

IDM2 in prevention of DNA hypermethylation.

IDM2 Interacts with IDM1 In VivoThe effects of the idm2 mutation on DNA methylation and gene

expression resemble those of idm1, and our genetic analysis

indicated that IDM2 and IDM1 function in the same genetic

pathway (Figures 1, 2, and 3; Tables S1, S2, and S3). These re-

sults suggest that IDM2 may function together with IDM1. To

investigate whether IDM1 and IDM2 may interact physically,

we conducted three kinds of assays. First, yeast two-hybrid as-

says showed that IDM2 can interact with IDM1 (Figure 4A). Sec-

ond, a luciferase complementation imaging (LCI) assay (Chen

et al., 2008) showed that nLUC-IDM1 can interact with cLUC-

IDM2 (Figure 4B). Third, in coimmunoprecipitation (co-IP) exper-

iments using GFP-tagged IDM1 and MYC-tagged IDM2 that

were transiently expressed in Nicotiana benthamiana leaves,

IDM2 was detected in the immunoprecipitate from GFP-IDM1-

expressing leaves but not from leaves of control plants (Fig-

ure 4C). The interaction between IDM2 and IDM1 was also

detected by co-IP in plants harboring both the Flag-IDM2 and

IDM1-hemagglutinin (HA) transgenes under their native pro-

moters (Figure 4D). These results suggest that IDM2 associates

with IDM1 in vivo.

IDM2 Partially Colocalizes with IDM1 in Subnuclear FociTo determine the subnuclear localization pattern of IDM2

protein, we generated antibodies specific to IDM2 and used

the antibodies for immunolocalization of IDM2 in Arabidopsis

leaf nuclei. Immunostaining of IDM2 yielded fluorescence signals

dispersed throughout the nucleoplasm without any preferential

accumulation near the 40,6-diamidino-2-phenylindole (DAPI)-

intensive chromocenters (Figure 5A; Figure S5A). No signals

were observed when the antibodies were applied to nuclei iso-

lated from idm2-1 mutant plants, indicating that the observed

immunolocalization signals are specific to IDM2 (Figure 5A; Fig-

ure S5A). To determine whether IDM2 may be colocalized with

other components of the active DNA demethylation pathway,

we performed double immunolocalization in interphase nuclei

from Arabidopsis transgenic lines expressing HA-tagged IDM1

or FLAG-tagged ROS1 under their native promoters. We found

that IDM2 partially colocalized with IDM1 within nucleoplasmic

foci (67%) and nucleolar foci (33%), as shown by the yellow sig-

nals, which resulted from an overlap of the green and red signals

(Figure 5B). A small fraction of nuclei (12%) also showed an over-

lap between some of the IDM2 and ROS1 signals (Figure S5B).

IDM2 Is Important for the In Vivo Function of IDM1The presence of an ACD at the C-terminal region of IDM2 and the

interaction between IDM2 and IDM1 suggest that IDM2 may

function as a molecular chaperone of IDM1 in plants. Previous

data showed that IDM1 is a histone acetyltransferase critical

for H3K18 and H3K23 acetylation in vivo (Qian et al., 2012). We

carried out ChIP assays and found that the H3K18ac marker

was reduced substantially in not only idm1-9 but also idm2-3


WT

Idm

2-1

+DAPI anti-IDM2 anti-HA DAPI Merged

100%n=87

100%n=105

67%

33%n=129

IDM

1-H

A

+DAPI anti-IDM2 anti-HA DAPI Merged

A

B

Figure 5. Subnuclear Localization of IDM2 and Its Colocalization

with IDM1

(A) Detection of IDM2 (red) in the wild-type and idm2-1 mutant nuclei by im-

munostaining using anti-iDM2. Scale bars represent 10 mm.

(B) Dual immunolocalization of IDM2 (red) and IDM1-HA (green). DNA was

stained with DAPI (blue). The frequency of nuclei displaying each interphase

pattern is shown on the right. Scale bars represent 10 mm. See also Figure S5.

Figure 6. The Effect of idm2Mutation onHistoneModificationMarks

H3K18ac and H3K23ac levels at the DMRs and 35S promoter and control

regions were determined by ChIP onwild-type, idm1-9, and idm2-3 plants with

anti-H3K18ac or anti-H3K23ac antibodies. The ChIP signal was quantified

relative to input DNA. The no-antibody precipitates served as negative control.

Two biological replicates were performed, and very similar results were

obtained. Standard errors were calculated from three technical repeats.

See also Figure S6 and Table S4.

Molecular Cell


mutant plants at the tested hyper-DMRs and the 35S promoter

region (Figure 6; Figure S6). Surprisingly, the H3K23ac marker

was reduced in idm1-9 but not in idm2-3mutant plants (Figure 6).

These results show that IDM2 is important for the full function of

IDM1 in planta.

DISCUSSION

Active DNA demethylation regulates many biological processes,

including early development and locus-specific gene expression

in plants and animals (Zhu, 2009). Although the enzymatic

removal of methylated cytosine has been studied extensively,

the mechanisms by which the enzymatic machinery is recruited

to specific target sites and regulated are poorly understood.

IDM1 regulates active DNA demethylation by recognizing the

chromatin marks at ROS1 target sites and then creating histone

acetylation marks such as H3K18ac and H3K23ac that are

necessary for recruiting ROS1 (Qian et al., 2012). By identifying

IDM2, a factor that functions with IDM1 and regulates the

H3K18 acetylation activity of IDM1, this study provides insights

into the regulation of active DNA demethylation.

Our conclusion that IDM2 functions with IDM1 in regulating

active DNAdemethylation is supported by the following observa-

tions. First, IDM2 functions in the same genetic pathway with

IDM1 andROS1 in preventingDNAmethylation at somegenomic

loci, although it is clear that ROS1 also requires other regulatory

factors in addition to IDM1 and IDM2, because themajority of hy-

per-DMRs in ros1 are not hyper-DMRs in idm1 (Qian et al., 2012)

or idm2.Manyof thehyper-DMRs in idm2and idm1mutant plants

overlap. Possible functional redundancywith other proteins in the


IDM2 and IDM1 families may explain the existence of idm1- or

idm2-specific hyper-DMRs. Second, yeast two-hybrid assays,

luciferase complementation assays, and co-IP experiments

showed that IDM2 physically interacts with IDM1. In addition, im-

munostaining revealed a partial colocalization of IDM2 and IDM1

within subnuclear foci. Unlike IDM1, which is a histone acetyl-

transferase, IDM2 does not contain any domain for enzymatic

activity. Yet the levels of H3K18 acetylation are reduced in

both idm2-3 and idm1-9. The result indicates that IDM1 requires

IDM2 to efficiently catalyze H3K18 acetylation in planta, although

we cannot exclude the possibility that IDM2 is required directly or

indirectly for the function of histone acetyltransferases other than

IDM1. Interestingly, H3K23 acetylation levels are decreased in

idm1-9 but not in idm2-3, suggesting that IDM2 is not required

for IDM1 function in H3K23 acetylation in vivo. It is possible that

the H3K18 acetylation activity of IDM1 is more labile in vivo and

needs the protective action of the partner protein IDM2. IDM2

also appears to partially localize with ROS1 in some cell nuclei

(Figure S5B). This indicates that IDM2 may also protect ROS1

for its function in active DNA demethylation and antisilencing at

some genomic loci.

IDM2 belongs to the highly conserved family of ACD proteins.

Our results show that the ACD is critical for IDM2 function in vivo.

ACD proteins have been extensively studied in mammals, where

they play critical roles in diverse cellular processes and have

important protective functions against stress and diseases

such as cataracts and neurodegenerative disorders (Basha

et al., 2012; Hilton et al., 2013; Welsh and Gaestel, 1998). In

plants, most ACD proteins are heat shock-inducible proteins

known as small HSPs (sHSPs) (Scharf et al., 2001). The vast ma-

jority of sHSPs are localized in the cell cytoplasm, chloroplasts,

andmitochondria (Scharf et al., 2001). Many studies have shown

that ACD proteins are molecular chaperones, although few

studies have demonstrated the function of ACD proteins using

knockout mutants (Basha et al., 2012; Horwitz, 1992). Structural

Molecular Cell


studies have suggested that sHSPs form oligomers (Kim et al.,

1998; van Montfort et al., 2001). IDM2 is a nuclear protein and

its transcript levels are not dramatically induced by heat shock.

It is possible that IDM2 may function as a molecular chaperone

for IDM1 and other proteins that are important for active DNA

demethylation and cellular antisilencing.

Our results suggest that plants have evolved a-crystallin

domain proteins that function in epigenetic regulation in the

cell nucleus. It is possible that certain ACD proteins in mammals

also function in epigenetic regulation.

EXPERIMENTAL PROCEDURES

Plant Materials and Mutant Screening by Chop PCR

Arabidopsis homozygous T-DNA insertion lines were obtained from the Arabi-

dopsis Biological Resource Center (http://www.arabidopsis.org). Arabidopsis

seedlings were grown onMS nutrient agar plates at 22�Cwith 16 hr of light and

8 hr of darkness for 2 weeks before DNA or RNA analysis. Genetic screening

was performed as described (Qian et al., 2012).

Map-Based Cloning of IDM2

Based on the root-length phenotype, an ethyl methanesulfonate-mutagenized

wild-type (containing the 35S-SUC2 transgene) population was generated and

screened for mutants with long roots on MS plates with 2% sucrose (Wang

et al., 2013). The idm2-3 mutant was obtained from this screening. The

idm2-3 mutant was crossed to Ler for molecular mapping (Wang et al., 2013).

Plasmid Construction

For complementation of mutants, a 2.7 kb genomic DNA fragment containing

the IDM2 gene was amplified from wild-type Col-0 genomic DNA by PCR and

cloned into the pCAMBIA1305 vector for plant transformation. All mutation

sites were introduced into 35S::6MYC-IDM2 (pCAMBIA1307 vector) through

site-directed mutagenesis with the QuikChange Kit according to the manufac-

turer’s instructions (Stratagene). Agrobacterium tumefaciens strain GV3101

carrying various IDM2 constructs was used to transform mutant plants via

the standard floral dipping method. Primary transformants were selected on

MS plates containing hygromycin.

Individual Locus Bisulfite Sequencing

Genomic DNAwas extracted using the DNeasy Plant Mini Kit (QIAGEN). About

200 ng of genomic DNA was treated with sodium bisulfite using the BisulFlash

DNA Modification Kit (Epigentek) following the manufacturer’s protocol. For

each PCR reaction, a 2 ml aliquot of bisulfite-treated DNA was used in a

20 ml reaction. PCR products were purified using the Wizard SV Gel and

PCR Clean-Up System Kit (Promega) and then subcloned into the T-easy vec-

tor (Promega) following the supplier’s instructions. For each DMR locus from

each genetic background, at least 20 independent clones were sequenced

(Table S5).

Real-Time RT-PCR

Total RNA was extracted from 2-week-old seedlings using the RNeasy Plant

Mini Kit (QIAGEN), and contaminating DNA was removed with the DNA-free

Kit (Ambion). mRNA (2 mg) was used for first-strand cDNA synthesis with

the SuperScript III First-Strand Synthesis System (Invitrogen) for RT-PCR

following the manufacturer’s instructions. The cDNA synthesis reaction was

then diluted five times, and 1 ml was used as template in a 20 ml PCR reaction

with iQ SYBR Green Supermix (Bio-Rad). All reactions were carried out on the

iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad). The comparative

threshold cycle method was used for determining relative transcript levels

(Bulletin 5279, Real-Time PCR Applications Guide; Bio-Rad), with TUB8 as

an internal control.

DNA Methylation Assay by Southern Hybridization

The Southern hybridization assay was performed as described (Gong et al.,

2002). Briefly, 5 mg of genomic DNA was digested with MspI, HpaII, or HaeIII.

M

The digested DNA was loaded onto a 1.2% agarose gel and transferred to

Hybond-N+ membranes. The 180 bp centromeric repeat and 5S rDNA repeat

were labeled with digoxin by PCR (Roche). Southern hybridization was done

following the manufacturer’s instructions.

Whole-Genome Bisulfite Sequencing and DMR Analysis

Bisulfite conversion, Illumina library construction, sequencing, and bio-

informatic analysis were performed as described in Qian et al. (2012).

Tiling Array Analysis

To identify genes that are differentially expressed in idm1-1, idm2-1, and the

wild-type, we performed pairwise comparisons of the tiling array data from

the three genotypes (two replicates from each genotype). We first remapped

the tiling array probes to the Arabidopsis genome (TAIR9) using SOAP2 (Li

et al., 2009) and retained only probes that were perfectly mapped to a unique

position in the genome. We created a custom chip definition file based on

the probe mapping result and the TAIR9 gene annotation, and used the

aroma.affymetrix framework (Bengtsson et al., 2008) for quantile normalization

of the tiling array data. Only genes with three or more probes were considered

for subsequent analysis. We used the genefilter package in Bioconductor

(http://www.bioconductor.org) to remove genes expressed at low levels

(normalized signal intensity <100 in all samples) and genes whose expression

showed little change across samples (interquartile range of log2 inten-

sities <0.5). We applied the linear model method implemented in the limma

package in Bioconductor to identify genes that showed differential expression.

The Benjamini and Hochberg method (Hochberg and Benjamini, 1990) was

used for adjustment for multiple comparisons.

Coimmunoprecipitation

A 2 g quantity of leaves from N. benthamiana or transgenic Arabidopsis plants

was harvested and ground to a fine power in liquid N2. Total protein was ex-

tracted with protein extraction buffer (20 mM Tris,HCl [pH 8.0], 150 mM

NaCl, 1 mM EDTA, 10% glycerol, 5 mM dithiothreitol, 1 mM phenylmethane-

sulfonylfluoride, and 13 protease inhibitor cocktail [Roche]). The samples

were centrifuged at 12,000 3 g at 4�C for 30 min. Immunoprecipitation

was then performed by incubating approximately 10 mg of total protein with

an excess amount of anti-GFP antibody (A-11122; Invitrogen) or anti-HA

(H3663; Sigma) at 4�C for 3 hr before 50 ml protein A agarose beads (Millipore)

was added for an additional 2 hr. The beads were washed three times with

extraction buffer. The immunoprecipitates were analyzed by western blotting

with an anti-MYC (M4439; Sigma) or anti-Flag (F3165; Sigma) antibody.

Immunolocalization

Immunofluorescence localization was performed in 2- to 3-week-old leaves as

described by Pontes et al., 2006). Nuclei preparations were incubated over-

night at room temperature with rabbit anti-iDM2 (anti-IDM2 peptide antibody

was custom made by YenZym Antibodies) and mouse anti-HA (H3663) or

anti-Flag (F3165; Sigma). Primary antibodies were visualized using mouse

Alexa 488-conjugated and rabbit Alexa 594 secondary antibody at 1:200 dilu-

tion (Molecular Probes) for 2 hr at 37�C. DNAwas counterstained using DAPI in

ProLong Gold (Invitrogen). Nuclei were examined with a Nikon Eclipse E800i

epifluorescence microscope equipped with a Photometrics CoolSNAP ES.

Luciferase Complementation Imaging Assay

LCI assays were performed as described (Chen et al., 2008).

Yeast Two-Hybrid Assay

Yeast two-hybrid assays were conducted as described previously (Xia et al.,

2006).

ChIP Assays

Chromatin immunoprecipitation assays were performed according to a pub-

lished protocol (Saleh et al., 2008). The following antibodies were used for

ChIP assays: anti-H3K4me2 (07-030; Millipore), anti-H3K9me2 (308-32361;

Wako), anti-H3K18ac (ab1191; Abcam), anti-H3K23ac (07-355; Millipore),

and anti-HA (H9658; Sigma). ChIP products were eluted into 50 ml of Tris-

EDTA buffer, and a 2 ml aliquot was used for each qPCR reaction.


http://www.arabidopsis.org

http://www.bioconductor.org

Molecular Cell


ACCESSION NUMBERS

We used whole-genome bisulfite sequencing data to analyze the genome-

wide methylation status in idm2-1 and idm1-1idm2-1 mutant plants. The

data set has been deposited in the Gene Expression Omnibus at the National

Center for Biotechnology Information (accession number GSE49421).

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

six figures, and five tables and can be found with this article online at http://

dx.doi.org/10.1016/j.molcel.2014.06.008.

AUTHOR CONTRIBUTIONS

W.Q. and J.-K.Z. designed the study and wrote the manuscript together with

H.Z.; W.Q. isolated the idm2-1 and idm2-2 alleles and characterized the mu-

tants and performed the co-IP and transgenic experiments; D.M. performed

the bisulfite sequencing and ChIP assays; M.L. performed the antisilencing

mutant screen and isolated the idm2-3 allele; X.Z. performed the immunoloc-

alization experiments; H.Z. did the split luciferase assays; Y. Liu, Y. Li, Z.L., and

J.W. assisted with the experiments on mutant characterization and protein in-

teractions; and K.T. and R.L. analyzed the whole-genome bisulfite sequencing

and tiling array data.

ACKNOWLEDGMENTS

We thank Rebecca A. Stevenson for technical assistance. This work was sup-

ported by NIH grants R01GM070795 and R01GM059138 (to J.-K.Z.) and by

the Chinese Academy of Sciences.

Received: December 31, 2013

Revised: April 3, 2014

Accepted: May 20, 2014

Published: July 3, 2014

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Molecular Cell, Volume 55

Supplemental Information

Regulation of Active DNA Demethylation by an -Crystallin Domain Protein in Arabidopsis

Weiqiang Qian, Daisuke Miki, Mingguang Lei, Xiaohong Zhu, Huiming Zhang, Yunhua Liu,

Yan Li, Zhaobo Lang, Jing Wang, Kai Tang, Renyi Liu, and Jian-Kang Zhu

F+R

TUB8

LBa1+R

F+R

LBa1+F

F+R

At1g26400 HhaI

No digestion control

idm2-1 (salk_130656) idm2-2 (salk_138229)

ATG (1) TAA (1132)

F R

idm2-3 (G276E)

A

B C

D

E

% C

yto

sin

e M

eth

ylation

0

20

40

60

80

100

CG CHG CHH

WTidm1-9idm2-3

F

At1g26400

HhaI

No digestion

control

Figure S1

G

WT AGC TGT GAA GTA GAA GAC AAT GGG AAG GTA CTA GTT AGAidm2-3 AGC TGT GAA GTA GAA GAC AAT GAG AAG GTA CTA GTT AGA

Chr.1

Physical distance (Mb)

Recom. Rate (%) n=96

16.37 20.121

20.412

20.625

20.860

22.94

12 3.1 0.0 0.5 1.0 4.7

Candidate region (0.504 Mb)

ATG(1) TAA(1132)

At1g54840

G908A

H

Col-0 idm2-3WT idm2-1X idm2-3 F1 IDM2/ idm2-3

35S-SUC2

A

0

20

40

60

80

CG CHG CHH

Colrddidm2-1idm2-2

At3g7232211-2628

0

20

40

60

80

CG CHG CHH

Colrddidm2-1idm2-2

DMR-265

0

20

40

60

80

CG CHG CHH

Colrddidm2-1idm2-2

DMR-232

0

20

40

60

80

100

CG CHG CHH

Colrddidm2-1idm2-2

DMR-828

0

20

40

60

80

100

CG CHG CHH

Colrddidm2-1idm2-2

DMR-333

0

20

40

60

CG CHG CHH

Col

rddidm2-1

idm2-2

DMR-946

B

% C

yto

sin

e M

eth

yla

tion

% C

yto

sin

e M

eth

yla

tion

Figure S2

CAt5g38550

0

20

40

60

80

100

CG CHG CHH

WTros1-4idm2-1idm2-2idm2-1ros1-4idm2-2ros1-4

% C

yto

sin

e M

eth

yla

tion

DMR-72

Gene

WT_rep1

idm2-1_rep1

ros1-4_rep1

idm1_rep1

Repeats

WT_rep2

idm2-1_rep2

ros1-4_rep2

idm1_rep2

H3K4me1

H3K4me2

H3K4me3

DMR-77 DMR-78 DMR-79 DMR-80 DMR-81 DMR-82

D

WT_CG

idm2-1_CG

ros1-4_CG

idm1-1_CG

WT_CHG

idm2-1_CHG

ros1-4_CHG

idm1-1_CHG

WT_CHH

idm2-1_CHH

ros1-4_CHH

idm1-1_CHH

G

F

E

Gene

WT_rep1

idm2-1_rep1

ros1-4_rep1

idm1-1_rep1

Repeats

WT_rep2

idm2-1_rep2

ros1-4_rep2

idm1-1_rep2Coordinates

H3K4me1

H3K4me2

H3K4me3

DMR-319

J

Gene

WT_rep1

idm2-1_rep1

ros1-4_rep1

idm1-1_rep1

Repeats

WT_rep2

idm2-1_rep2

ros1-4_rep2

Coordinates

H3K4me1

H3K4me2

H3K4me3

DMR-259

idm1-1_rep2

I

Gene

WT_rep1

idm2-1_rep1

ros1-4_rep1

idm1-1_rep1

Repeats

WT_rep2

idm2-1_rep2

ros1-4_rep2

Coordinates

H3K4me1

H3K4me2

H3K4me3

DMR-256

idm1-1_rep2

H

ROS1

0.0

0.5

1.0

1.5

2.0

Col

nrpd

2a

nrpd

1a-3

nrpd

1b-1

1

rdm1-3

rdm3-3

met1-1R

ela

tive

exp

ressio

n leve

l

IDM2

0.0

0.5

1.0

1.5

Col zdp-1 zdp-2

0.0

0.5

1.0

1.5

C24 ros1 ros3

Rela

tive

exp

ressio

n leve

l

0.0

0.5

1.0

1.5

Col_0 phd-1 phd-2

IDM2 IDM2 IDM2

A

C

D

Up-regulated

genes

Down-regulated

genes

idm2-1 133 53

idm1-1 83 63

E

0.0

0.5

1.0

1.5 IDM1

B

Figure S3

Rela

tive e

xpre

ssio

n level

Up-regulated genes

Rela

tive e

xpre

ssio

n level Down-regulated genes

F

GAt3g18250

0

5

10

15

20 Control

5'Aza

Rela

tive e

xpre

ssio

n level

H

Medicago -----------------------MSKSQNRMEQPNQYQTTLALLEPKPKNLNSNNLNDDQ 37Soybean ------------------------------------------------------------Papaya MDLPCGTKPYIQTIINILQLISCIAAMNKLSRTTNSFAAVGSGSINSLRPGSEDTINDDQ 60Arabidopsis --------------------------MSDTMPEIVSPLVAENSQEQEESVLISLDIEEDK 34

Medicago CFLLYFIMGTYFGPDINGD--KKSVLQIVAEGLPSYTREQLTNSYMKVSELERIYYYILR 95Soybean -------MGTYFGPDIKGETTKKSILQRVAEGLPPYTLDQLTHSCIKIVELERVYYYILR 53Papaya FFLLYFIIGTYFGPDLKGERQPKSVLQRKDEGLSPYTSDQLANSHMKTEELKQVYYYVLR 120Arabidopsis LFLLHFIIGTYFGPDLRKQHHRPKQSAFQIQALKNVVVDELSGSLMKRAELERVYYHIIR 94

:*******:. : . :.* . ::*: * :* **:::**:::*

Medicago NVDKSLTVKLTFLRRFIQG--LEGS-----SNCNYPQFTDLFPLELHPQSMFKGNRFKII 148Soybean NTEKSLILKLTSLRRFFQGQALGGDGNGNGNNNNYPQFPELFHPGFHPQYRFK-NKHKVV 112Papaya KADQHISSKLLYL--FVRG-KLSALGCG--HTASYPEFPDLFPLELHPQTWHD-DHNAVI 174Arabidopsis NVDPSLVMKPKKLREYFNA--KRND-----SNRDYPLFVDLFPRKLHPETHVR-HKFKFI 146

:.: : * * :... . .** * :** :**: .: .:

Medicago ENIVFIDNPEVFFFSQEDIERFKRLSGLEDFVVDKDVARLYN--CMDGSGLRNKSVVKVE 206Soybean DNVVFINNPDSFYIRIEDVERFKRLSGVEELHVDRDAARLQLGICFDDNRVPSNTDISVG 172Papaya DSIVFINNPDFSYMKLKDFQRFRRLTGLDDFLLDKDSGQLNVS---EDSDRYNTRIQVVE 231Arabidopsis RSIVFINDPDTSCMREECVARFKRLTGLDSFALS-----------------LSVDVTK-- 187

.:***::*: : : . **:**:*::.: :. .

Medicago HKKIIPLPLPELQSSSRKTSSRKVTESDDFSDLKYQLPHAHAVTPISCVPFNGGMGLDGE 266Soybean NVE------PDGNVESCGGGGGGSSEPEDRVNVDASRSGGATCRGGTNVMYDYMDTEDDE 226Papaya YNK----GSSPLGSSPHSRRKRNVSESED-QNIHAPACSSMYSHDAPSVPPK------DE 280Arabidopsis -------------------SNGVVAANEVKVEIDESVEPVKEDNAGTCTSGE-------E 221

: : ::. . . . *

Medicago GDSVKVGAPAALFLPSRPTKKEWSNIVAATNSGFALTGSAAMGQIGPIMGLVDIGECEDS 326Soybean SDHDKVG-PAMLFLPSRPSKKEWSDIVAATKNGFGLTGTVATGGIGPTMGLVDIGECEDA 285Papaya NNSAERCSSAMIFLPSHPTKQELADVIAATKNGFALTGSAAMGQVGPVVGKVDIGECEDS 340Arabidopsis SD-----------VAAKPEVK-----------------SEAHG--GLMVGLMDIGECDDA 251

.: :.::* : : * * * :* :*****:*:

Medicago YLFRMSLPGVKRDDKEFSCEVDTDGKVFIQGITT-TGEKTVSMRTQVFEMQTQNLCPAGQ 385Soybean YLFRLSLPGVKRNEREFSCEVGTDGKVLISGVTT-TGENTVSRYSQVFEMQTRNLCPPGQ 344Papaya YLFRVSLPGVKRDERDFSCEIQSDGKVLIRGVTVATGEKRVCMYSQVFEMQSQNLCPPGH 400Arabidopsis YLFRVSLPGVKRDERYFSCEVEDNGKVLVRGVTT-TGGKRVKRYSHVFEMQTRSLCPPGN 310

****:*******::: ****: :***:: *:*. ** : * ::*****::.***.*:

Medicago FSITFQLPGPVDPHQFSGNFGTDGILEGIVVK--RKPR- 421Soybean FSVSFQLPGPVDPHQFSGNFGIDGILEGVVMK--GKCT- 380Papaya FSISFQLPGPVDPQLFTGDFGIDGIFEGIVMK--KKK-- 435Arabidopsis FSVSFRLPGPVHPHEFSGNFGTDGILEGVVMKNLQKQTV 349

**::*:*****.*: *:*:** ***:**:*:* *

G246D

G260D

D49E

idm2-3 (G276E)

A

Figure S4

ACD domain

B

IDM

2

At1g54850

At1g20870

AtHsp

17.4

Rela

tive t

ranscript

level

0

10

20

15000

16000

17000

18000 RT

HS

C

D

250

130

100

70

55

35

25

Col

idm

2-1

D49E

G2

46

D

G260D

WT

idm2-1

Col

idm

2-1

D4

9E

G2

46

D

G2

60

D

WT

idm2-1

Mark

er

(kD

)

Mark

er

WB: anti-MYC Ponceau S stain

E

F

anti-HA

anti-H3

WT

idm

2-1

IDM2 ROS1

IDM2

ROS1

100%

n=100

100%

n=120

IDM2

ROS1

DAPI

88%

12%

n=125

A

B

Figure S5

Rela

tive

to

In

pu

t

H3K18Ac

No antibody control

Rela

tive

to

In

pu

t

Figure S6

Supplemental Figure legends

Figure S1. Characterization of idm2 mutants, related to Figure 1.

(A) Schematic diagram showing the positions of the T-DNA insertions at the IDM2 locus. Black

rectangles represent exons. Arrows indicate PCR primers used in genotyping and RT-PCR

analysis. (B) Genotyping of idm2 mutants. (C) RT-PCR analysis of IDM2 transcript levels in

idm2 mutants. TUB8 served as a control. (D) Complementation assay. CHOP PCR results for the

At1g26400 locus are shown for three representative transgenic lines. (E) Bisulfite sequencing

results for the 35S promoter. (F) Analysis of DNA methylation level at the At1g26400 locus by

CHOP PCR. (G) Map-based cloning of IDM2. (H) Complementation and allelism analyses of

the idm2-3 mutant. Seedlings were grown on plates with MS medium plus 2% sucrose for 7 days

before they were photographed. See also Table S5.

Figure S2. Methylation status of 5S rDNA and centromeric DNA and DNA methylation

levels assayed by individual locus bisulfite sequencing in wild-type, idm2, and rdd mutant

plants and Examples of whole-genome bisulfite sequencing data and other data showing

DNA hypermethylation and other features in idm2-1 and other mutant plants , related to

Figure 2.

(A) Genomic DNA from wild-type, ros1, idm1, idm2, idm1idm2 double and ddm1 mutant plants

were digested with the methylation sensitive enzyme MspI (CHG methylation), HaeIII (CHH

methylation) or HpaII (CG & CHG methylation) and hybridized with 5S rDNA, or180-bp repeat

probes. (B) Bisulfite sequencing results in wild-type, idm2, and rdd mutant plants. (C) Bisulfite

sequencing data showing the effects of idm2 mutation on the DNA methylation of two tested loci

and genetic interactions with ros1. (D) Upper: Five members of the FAD-binding berberine gene

family are hypermethylated in idm2, idm1-1 and ros1-4 mutant plants. Hypermethylated regions

are highlighted with red boxes. Repeats were annotated according to RepeatMasker. H3K4

methylation data was downloaded from Zhang et al (2009). Lower: The marker locus,

At1g26400, was also identified as one of the hypermethylated loci in the mutants by the whole-

genome bisulfite sequencing (DMR-79). (E) Four members of the cysteine/histidine-rich C1

domain gene family are hypermethylated in idm2-1, idm1-1 and ros1-4 mutant plants.

Hypermethylated regions are highlighted with red boxes, where all idm2-dependent

hypermethylation sites show a lack of H3K4 methylation; in contrast, a ros1-dependent but

idm2-independent hypermethylated region (between DMR-1169 and DMR-1170) does have high

levels of H3K4me2 and H3K4me3, which is highlighted with a blue box. (F-J) Examples of

hyper-DMRs. The data for idm1-1 and ros1-4 were from Qian et al (2012). (K) Analysis of

H3K4 methylation levels at hyper-DMRs and at a comparison group of expressed genes with

high CG methylation, see Qian et al (2012) for details. The X axis represents the arbitrary

segment along the region of hyper DMRs or gene controls.

Figure S3. Real-time PCR analysis of transcript levels and Transcript profiles for idm1 and

idm2 plants by tiling array analysis, related to Figure 3.

(A) ROS1 mRNA levels in wild-type, idm1, idm2 and idm1idm2 double mutant plants. Three

biological replicates were performed, and very similar results were obtained. The transcript

levels were normalized using TUB8 as an internal standard. (B) IDM1 transcript level in idm2

mutant plants. (C) IDM2 transcript level in different mutant plants. (D) IDM2 transcript levels in

various RdDM pathway mutants and the met1-1 mutant. (E) Differentially expressed genes

identified from idm1 and idm2 mutant plants. (F-G) Validation of the tiling array data by q-PCR.

(H) q-PCR analysis of At3g18250 transcripts in different genotypes after 5’-aza-dC treatment.

Standard errors were calculated from three technical repeats.

Figure S4. IDM2 is an atypical sHSP family member, related to Figure 1.

(A) Alignments of the deduced amino acid sequences of IDM2 and its paralogs from Arabidopsis.

(B) Alignment of the deduced amino acid sequences of IDM2 orthologs from different plant

species. Asterisks indicate invariant residues. Conserved amino acids for site-direct mutagenesis

are marked by blue boxes. ACD is marked by red underline. (C) Real-time PCR analysis of gene

transcript levels after heat stress. RNA was extracted from 2-week-old seedlings grown on MS

medium. Heat treatment at 42ºC was for 1 h. RT, growth room temperature; HS, heat stress

treatment. TUB8 was used as an internal control. Error bars represent standard error (n=3). (D)

Fluorescence images of GFP-IDM2 in hypocotyl cells of transgenic Arabidopsis plants. (E)

Western blot detection of IDM2 protein with anti-MYC antibody. Ponceau S stain was shown as

loading control. (F) Western blot detection of IDM1-HA protein with anti-HA antibody. Histone

H3 was used as loading control.

Figure S5. Sub-nuclear localization of IDM2 and co-localization with ROS1, related to

Figure 5.

(A) Detection of IDM2 (red) in wild-type and idm2-1 mutant nuclei by immunostaining using

anti-IDM2 antibody. (B) Dual immunolocalization of IDM2 (red) and Flag-ROS1(green). DNA

was stained with DAPI (blue). The frequency of nuclei displaying each interphase pattern is

shown on the right.

Figure S6. The effect of idm2 mutation on the H3K18ac mark, related to Figure 6.

H3K18ac levels at the DMRs and control regions. ChIP was performed in wild-type, idm1-1 and

idm2-1 plants with anti-H3K18ac antibodies. The ChIP signal was quantified as relative to input

DNA. The no-antibody precipitates served as negative control. Two biological replicates were

performed, and very similar results were obtained. Standard errors were calculated from three

technical repeats.

Table S1. Hyper-DMRs in idm2-1, related to Figure 2.

Table S2. Hypo-DMRs in idm2-1, related to Figure 2.

Table S3. Differentially expressed genes in idm1 and idm2 mutant plants, related to Figure

3.

Table S4. List of primers used in this study, related to Figure1, Figure 3, and Figure 6.

Table S5. Detailed information for individual locus bisulfite sequencing, related to Figure1

and Figure S2.

Table S5. Detailed information for individual locus bisulfite sequencing,

related to Figure1 and Figure S1.

At1g26400 clone number CG CHG CHH

WT 20 11.29 2.35 1.21

ros1-4 21 92.86 66.00 34.52

idm2-1 23 84.62 48.46 17.58

idm2-2 20 67.35 33.57 13.69

rdr2 25 14.73 0.57 1.56

nrpe1-11 23 43.41 0.77 0.09

idm2-1ros1-4 21 93.57 59.00 28.10

idm2-2ros1-4 20 90.48 68.89 36.9

idm2-1rdr2 27 30.95 5.00 2.29

idm2-1nrpe1 22 81.99 2.31 0.18

35S promoter clone number CG CHG CHH

WT 23 72.14 46.89 23.12

idm1-4 25 82.33 70.23 21.99

idm2-3 20 80.19 66.54 24.69

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