VIM1, a methylcytosine-binding protein required for centromeric

VIM1, a methylcytosine-binding

protein required for centromeric

heterochromatinization

Hye Ryun Woo, Olga Pontes, Craig S. Pikaard, and Eric J. Richards

Department of Biology, Washington University, St. Louis, Missouri 63130, USA

Epigenetic regulation in eukaryotes is executed by a complex set of signaling interactions among small RNA

species and chromatin marks, including histone modification and DNA methylation. We identified vim1

(VARIANT IN METHYLATION 1), an Arabidopsis mutation causing cytosine hypomethylation and

decondensation of centromeres in interphase. VIM1 is a member of a small gene family, encoding proteins

containing PHD, RING, and SRA (SET- and RING-associated) domains, which are found together in

mammalian proteins implicated in regulation of chromatin modification, transcription, and the cell cycle.

VIM1 is an unconventional methylcytosine-binding protein that interacts in vitro with 5mCpG- and

5mCpHpG-modified DNA (via its SRA domain), as well as recombinant histones (H2B, H3, H4, and HTR12)

in plant extracts. VIM1 associates with methylated genomic loci in vivo and is enriched in chromocenters.

Our findings suggest that VIM1 acts at the DNA methylation–histone interface to maintain centromeric

heterochromatin.

[Keywords: Cytosine methylation; methylcytosine-binding protein; centromere; epigenetic; SRA domain;

heterochromatin]

Supplemental material is available at http://www.genesdev.org.

Received November 13, 2006; revised version accepted December 14, 2006.

Large regions of higher eukaryotic genomes, particularly

the domains surrounding and encompassing centro-

meres, are composed of repetitive elements, which are

preferential targets for heterochromatin assembly. Het-

erochromatic regions of the genome are characterized by

increased chromatin condensation and decreased or dif-

ferential accessibility to regulatory proteins (Craig 2005;

Huisinga et al. 2006). Most forms of heterochromatin are

stably inherited and contain one or more epigenetic

marks that direct its maintenance during cell division.

Specification and maintenance of heterochromatin and

other functionally distinct chromatin domains rely on

complex interactions among cytosine methylation, his-

tone modification, and RNA interference (RNAi) path-

ways (Tamaru and Selker 2001; Jaenisch and Bird 2003;

Chan et al. 2005; Matzke and Birchler 2005).

Cytosine methylation is an important mechanism for

establishing stable heritable epigenetic marks, thereby

linking primary nucleotide sequence to chromatin orga-

nization (Jaenisch and Bird 2003). The cytosine methyl-

ation reaction is catalyzed by enzymes known as cy-

tosine-DNA-methyltransferases (DNMTs) (Goll and

Bestor 2005). Notably, mammalian cytosine methylation

is mostly restricted to symmetrical CpG sequences, al-

though plant cytosine methylation occurs at CpG,

CpHpG, and CpHpH sequences (where H = A, C, T).

Once established, cytosine methylation can be inherited

through mitosis, and sometimes through meiosis, pro-

viding a stable epigenetic mark (Genereux et al. 2005;

Richards 2006). Cytosine methylation cooperates with

histone modification to generate a self-reinforcing cycle

of epigenetic events that lead to long-term transcrip-

tional repression (Jenuwein and Allis 2001; Nightingale

et al. 2006). The regulatory information encoded by cy-

tosine methylation is deciphered by a family of proteins

that, through a conserved methyl-CpG-binding domain

(MBD), selectively binds methylated CpG dinucleotides

regardless of the sequence context (Klose and Bird 2006).

Some MBD proteins can recruit histone deacetylase

(HDAC) or histone methyltransferase (HMT) complexes

as well as chromatin remodeling factors (Klose and Bird

2006). Chromatin modification can also influence cyto-

sine methylation. For example, methylation at histone

H3K9 creates binding sites for adaptor molecules, such

as HP1 (Heterochromatin Protein 1) and related chromo-

domain proteins, that ultimately recruit DNMTs (Fuks

et al. 2003; Lehnertz et al. 2003). Through these types of

interactions, covalent modifications of DNA and core

Corresponding author.

EMAIL [email protected]; FAX (314) 935-4432.

Article published online ahead of print. Article and publication date are

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histones play important roles in heterochromatin forma-

tion in mammals and plants.

The last several years have witnessed tremendous ad-

vances in the genetic analysis of eukaryotic DNA meth-

ylation, resulting in the identification of proteins and

pathways necessary for specification and maintenance of

genomic cytosine methylation patterns (Jaenisch and

Bird 2003; Rangwala and Richards 2004; Chan et al.

2005). Two major protein categories important for geno-

mic cytosine methylation, identified through both for-

ward and reverse genetic approaches, are DNMTs and

chromatin modification enzymes (e.g., HDACs, HMTs,

nucleosome remodelers). Post-transcriptional silencing/

RNAi pathways mutants also have cytosine hypometh-

ylation phenotypes, highlighting the importance of

RNA-directed DNA methylation mechanisms guided by

small RNA species. However, only a handful of these

mutations affect centromere DNA methylation, despite

the fact that the centromere is a principle target for cy-

tosine methylation and heterochromatin formation in

the cell. In an effort to identify novel alleles or new loci

affecting centromere DNA methylation, we chose an al-

ternative approach of screening natural strains of the

flowering plant Arabidopsis thaliana for changes in ge-

nomic methylation patterns. Through this approach, we

identified a novel genetic locus, VIM1 (VARIANT IN

METHYLATION 1), encoding a non-MBD class methyl-

cytosine-binding protein that is required for full centro-

mere DNA methylation. In addition, loss of VIM1 func-

tion leads to decondensation of the centromere repeat

sequence in interphase and an alteration in the localiza-

tion pattern of the centromere-specific histone H3 vari-

ant, HTR12. Our results suggest that VIM1 acts at the

interface between DNA methylation and chromatin to

maintain centromere heterochromatin.

Results

Identification of a natural variant with

hypomethylated centromere DNA

In a screen for natural variation in cytosine methylation

among 89 different strains of A. thaliana, we identified

one strain that contained hypomethylated centromeric

180-base-pair (bp) repeats (CEN). Centromere repeats

from strain Borky-4 (Bor-4) showed an increased level of

digestion with the restriction endonuclease HpaII (5⬘-

CCGG-3⬘), which is inhibited by either 5mCpG or

5mCpHpG, relative to that seen for repeats in wild-type

Columbia (Col) plants (Fig. 1A). A similar enhanced di-

gestion was seen when the centromere repeats were cut

with MspI, which cleaves the same site as HpaII but is

inhibited only by 5mCpHpG. The effect of two previ-

ously characterized DNA hypomethylation mutations in

strain Col are shown for comparison: ddm1, which re-

duces cytosine methylation in all sequence contexts

(Vongs et al. 1993); and met1, which predominantly re-

duces 5mCpG (Kankel et al. 2003; Saze et al. 2003).

These observations suggest that Bor-4 contains centro-

meric repeats that are hypomethylated at both CpG and

CpHpG sites.

To determine whether reduced cytosine methylation

in Bor-4 occurs at other genomic locations, we examined

the extent of cytosine methylation at three additional

loci that are heavily methylated in wild-type strains: the

FWA gene (Soppe et al. 2000) and the pericentromeric

tandemly repeated 5S rRNA genes (Campell et al. 1992)

and Athila retrotransposable elements (Pelissier et al.

1995). Compared with wild-type Col, we did not find a

significant change in Bor-4 cytosine methylation at the

5S rRNA genes (Fig. 1B), in the Athlia elements or at the

FWA locus (data not shown). In agreement with previous

reports, we observed a strong hypomethylation of all

these loci in Col plants containing either ddm1-2 or

met1-1 alleles. These results indicate that DNA hypo-

methylation in wild strain Bor-4 preferentially affects

the 180-bp centromere repeats.

Altered centromere organization in Bor-4 interphase

nuclei

To investigate whether centromere heterochromatin

was affected in Bor-4, we used fluorescence in situ hy-

bridization (FISH) to detect the 180-bp centromere re-

Figure 1. Reduced cytosine methylation of centromeric re-

peats in Bor-4. (A) Genomic DNA samples from the indicated

genotypes were digested with the isoschizomers HpaII or MspI,

and DNA blot hybridization with a 180-bp centromere repeat

probe (CEN) was performed. (B) The filter shown in A was re-

hybridized with a 5S rRNA probe (5S rRNA). (C) A DNA blot

hybridization pattern with the CEN probe after HpaII digestion

demonstrates that centromeric repeat arrays hypomethylated in

Bor-4 were fully remethylated in F1 hybrids resulting from re-

ciprocal crosses between strains Bor-4 and Ler. The lane labeled

Ler + Bor-4 contains a 1:1 mixture of Ler and Bor-4 HpaII-di-

gested genomic DNA and shows the hybridization pattern ex-

pected if no remethylation occurred in the F1 hybrids.

Woo et al.

2 GENES & DEVELOPMENT

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peats in interphase nuclei. The spatial distribution of

hybridization signal indicated that the centromere re-

peats in Bor-4 were decondensed relative to the repeats

in Col nuclei (Fig. 2; Supplementary Table 1). We also

stained fixed interphase nuclei with anti-HTR12 anti-

body (Talbert et al. 2002) to obtain a more comprehen-

sive understanding of centromere heterochromatin in

Bor-4. HTR12 is the Arabidopsis homolog of human

CENP-A, Drosophila Cid, and yeast Cnp1, which are his-

tone H3 variants that define the specialized chromatin

structure associated with kinetochore assembly (Heni-

koff and Dalal 2005). We found that the area of the

HTR12 immunostained signal was decreased in Bor-4

nuclei compared with the pattern observed in Col nuclei.

We noted that neither the distribution of 180-bp repeats,

nor the pattern of HTR12 staining is affected by the

ddm1 mutation in the Col background (Fig. 2). Thus,

Bor-4 exhibits not only abnormal centromere DNA

methylation, but a unique centromere organization phe-

notype not exhibited by a well-characterized mutation

with severe centromere DNA hypomethylation defects.

A trans-acting mutation causes centromere DNA

hypomethylation in Bor-4

To distinguish whether the Bor-4 centromere pheno-

types result from the action of a trans-acting mutation or

epigenetic inheritance of stochastic DNA hypomethyl-

ation, we outcrossed Bor-4 to standard laboratory strains

Col and Ler and followed the segregation of the DNA

hypomethylation phenotype in subsequent generations.

In the F1 generation, regardless of the direction of the

cross, the hypomethylated 180-bp repeat arrays originat-

ing from Bor-4 were fully remethylated, demonstrating

that centromere DNA hypomethylation is reset effi-

ciently (Fig. 1C; data not shown). This result contrasts

with the persistence of hypomethylated genomes in F1

hybrids resulting from outcrossing ddm1 or met1 mu-

tants (Vongs et al. 1993; Kakutani et al. 1999; Kankel et

al. 2003), and argues against the possibility that Bor-4

centromere repeat hypomethylation is due strictly to

epigenetic inheritance of a hypomethylated state. The

hypomethylated centromere trait segregated as a mono-

genic recessive trait in two independent Ler X Bor-4 F2

families (normal:hypomethylated⬋112:36). Recombina-

tional mapping in Ler X Bor-4 F2 families indicated that

the hypomethylated centromere phenotype of Bor-4 is

caused by variation at a single trans-acting locus, VIM1,

at a map position distinct from known Arabidopsis mu-

tations affecting DNA methylation or chromatin modi-

fication (Supplementary Fig. 1).

Identification of VIM1

Using a Ler X Bor-4 F2 mapping population, we nar-

rowed the region containing the vim1-1 allele from Bor-4

to an interval corresponding to a 113-kb region on the

lower arm of chromosome 1. In Col, this window con-

tains 30 annotated genes (Supplementary Fig. 1). The

Bor-4 allele of one gene in this interval, At1g57820, car-

ries a large deletion. The missing segment spans a 3.2-kb

region from just downstream from the first intron donor

site to the middle of the intergenic region between

At1g57820 and At1g57810 (Fig. 3A; Supplementary Fig.

2). We could not detect At1g57820 transcript in Bor-4

using RT–PCR analysis; however, the predicted tran-

script was observed in Col and Ler (data not shown). To

test whether mutation of At1g57820 causes hypometh-

ylation of the 180-bp centromere repeats, we analyzed

the effect of Agrobacterium T-DNA insertion alleles in

this gene in the Col background. The T-DNA inserts in

At1g57820 disrupt the first intron, the fourth exon, and

the eighth exon, respectively (Fig. 3A). We could not de-

tect full-length transcripts from any Col At1g57820 ho-

mozygous T-DNA mutant (data not shown). All three

T-DNA insertion mutants in Col showed increased

HpaII cleavage of the 180-bp centromere repeats (Fig. 3B),

although the diagnostic ladder-like hybridization pattern

was weaker than that seen in Bor-4 (Fig. 1A). The muted

effect of the T-DNA insertion alleles in At1g57820 may

be due to the action of strain-specific modifiers that par-

tially cover for At1g57820 loss of function in Col. How-

ever, the vim1-2 allele (SALK_050903) in the Col strain

caused distinct centromere defects similar to those seen

in Bor-4 (Fig. 2), arguing that disruption of VIM1 function

is responsible for both the altered centromere heterochro-

matin and centromere DNA hypomethylation phenotypes.

Figure 2. Centromeric heterochromatin is altered in Bor-4.

One-hundred-eighty-base-pair centromeric repeats (CEN) were

detected by FISH, and HTR12 protein was immunolocalized in

interphase nuclei from root tip cells of Bor-4, Col, Col ddm1-2,

and Col vim1-2 (SALK_050903) plants. The DNA was counter-

stained with DAPI; chromocenters are more intensely stained.

Bar, 5 µm.

5mC-binding protein required for DNA methylation

GENES & DEVELOPMENT 3

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We confirmed that At1g57820 corresponds to the

VIM1 locus by two independent genetic tests. Crosses

between Bor-4 and Col plants homozygous for an

At1g57820 T-DNA insertion allele demonstrated a lack

of complementation for the centromere DNA methyl-

ation phenotype in the resulting F1 individuals (Supple-

mentary Fig. 3). Furthermore, a 5.5-kb genomic fragment

containing the Col sequence of At1g57820 was sufficient

to complement the hypomethylation phenotype of Bor-4

(Fig. 3C). The VIM1 locus encodes a 645-amino-acid

protein with a PHD domain, two RING finger domains,

and an SRA (SET- and RING-associated) domain (Fig. 3D;

Supplementary Fig. 4A). Four additional genes encod-

ing related proteins (ⱖ68% amino acid identity to

VIM1) with a similar domain organization exist in the

Arabidopsis genome: At1g57800, At1g66040, At1g66050,

and At5g39550 (Fig. 3D; Supplementary Fig. 4B). Only

three of these genes (At1g57820 [VIM1], At1g66050

[VIM2], and At5g39550 [VIM3]) are expressed at an ap-

preciable level in strain Bor-4, Col and Ler based on our

RT–PCR analysis (data not shown) and cDNA resources

available through public databases (e.g., http://www.

arabidopsis.org, http://mpss.udel.edu/at). Neither the

vim2 nor the vim3 T-DNA insertion allele examined in

Col caused a centromere repeat hypomethylation pheno-

type (Supplementary Fig. 5).

Centromeric small interfering RNA (siRNA)

populations are not significantly changed in Col

vim1 plants

Several characterized Arabidopsis DNA hypomethyl-

ation mutations primarily affect small RNA accumula-

tion, which reduces RNA-directed DNA methylation

Figure 3. Identification of the VIM1 gene. (A) Diagram of the intron–exon structure of the VIM1 gene and positions of T-DNA

insertions in At1g57820 with allele designations. At1g57810 (reverse transcriptase pseudogene) is located between At1g57800 and

At1g57820. The deletion in Bor-4 is shown as an open box (vim1-1 allele). Filled boxes represent exons and thin lines represent introns.

The positions of the start and stop codon are indicated, as are the T-DNA insertion sites in At1g57820.(B) DNA blot hybridization

(HpaII digest, CEN probe) analysis of T-DNA insertion mutants in At1g57820. Genomic DNA was isolated from independent plants

in segregation populations. (+/+) Homozygous wild-type allele; (+/−) heterozygous for mutant allele; (−/−) homozygous for mutant

allele. (C) DNA blot hybridization (HpaII digest, CEN probe) analysis demonstrating complementation of the hypomethylation

phenotype of Bor-4 with a transgene expressing At1g57820 (from Col strain, coding sequence fused to a C-terminal Flag epitope tag).

Analysis of six individuals in the T2 generation segregating the transgene (carrying a Basta herbicide resistance marker: [R] resistant;

[S] sensitive) is shown. (D) Schematic representation of possible homologs of VIM1. The RING (Pfam PF00097), PHD (Pfam PF000628),

and SRA (Pfam PF02182) domains are labeled. The mammalian proteins contain an N-terminal ubiquitin-like domain (UBL), which

is absent in the VIM1-like Arabidopsis proteins, and only one RING domain. (E) Sequence alignment of SRA domains from VIM1 and

three related mammalian proteins. Black shading shows identical residues and gray shading shows similar residues. Arrows denote the

conserved VRV(I/V)RG and YDG motifs in SRA domains.

Woo et al.

4 GENES & DEVELOPMENT

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(Chan et al. 2005). Consequently, we compared the abun-

dance of siRNAs corresponding to the 180-bp centro-

mere repeats in Col and Col vim1-2 plants using RNA

gel blot analysis (Supplementary Fig. 6). We did not find

a significant change in the abundance of centromeric

small RNA populations associated with the VIM1 geno-

type, suggesting that the vim1-2 mutation reduces cen-

tromere DNA methylation without affecting siRNA ac-

cumulation.

VIM1 is a methylcytosine-binding protein

The similarity shared among the SRA domains of VIM1

and three mammalian proteins, human ICBP90 and

NIRF, and murine Np95 (Fig. 3D,E; Fujimori et al. 1998;

Mori et al. 2002) prompted us to investigate whether

VIM1 is a methylcytosine-binding protein because the

SRA domain in these mammalian proteins was shown to

bind methyl-CpG-modified double-stranded DNA

(Unoki et al. 2004). Full-length VIM1 protein and various

VIM1 subdomains (Fig. 4) expressed in vitro were tested

for their ability to bind methylated double-stranded oli-

gonucleotides by electrophoretic mobility shift assay

(EMSA). We demonstrated that full-length VIM1 bound

to a double-stranded oligonucleotide (4x-mCG) contain-

ing four symmetrical 5mCpG sites in a methylation-de-

pendent manner (Fig. 4A–C). VIM1 bound the same oli-

gonucleotide sequence containing only a single 5mCpG

site, but did so more weakly (data not shown). Because

vim1 mutations lead to a reduction in cytosine methyl-

ation in the 180-bp centromere repeats, we tested and

confirmed VIM1’s ability to interact specifically with an

oligonucleotide corresponding to the region of the 180-

bp repeat sequence containing the 5⬘-CCGG-3⬘ site as-

sayed (Fig. 4B,D). Next, we delimited the minimal region

of VIM1 that interacted in a methylation-dependent

manner with the 4x-mCG probe to 279 amino acids com-

prising the entire SRA domain and the interval between

the SRA and second RING domain (Fig. 4A,C, lanes

marked SRA+). While the SRA domain is required for

methylcytosine-binding activity, this domain is not suf-

ficient (Fig. 4A,C, lane marked SRA).

Plant genomes contain cytosine methylation in three

different sequence contexts: two symmetrical sites, CpG

and CpHpG; and the asymmetric site, CpHpH (Chan et

al. 2005). We assayed the specificity of VIM1’s methyl-

cytosine-binding activity using a set of double-stranded

oligonucleotides of identical primary sequence that con-

Figure 4. VIM1 is a methylcytosine-binding protein.

(A) Schematic of the VIM1 protein and derivatives

tested for methylcytosine-binding activity; amino acid

coordinates are shown in the parentheses. Domain des-

ignations include PHD domain, SRA domain, and

RING domains. (B) Oligonucleotide probes used in the

methylcytosine-binding assays shown in C–E. Aster-

isks indicate the positions of 5mC residues. The under-

lined 5⬘-CCGG-3⬘ site corresponds to the HpaII assayed

in Figure 1. (C) VIM1 protein derivatives were expressed

in wheat germ transcription/translation extracts (C,

vector-only control) and incubated with the digoxi-

genin-endlabeled 4x-mCG probe in the presence of 62-

fold excess unlabeled competitor oligonucleotides. (U)

Unmethylated; (M) methylated; (−) no competitor.

EMSA of incubation products is shown; the position of

the unbound free probe is indicated at the bottom of the

image. The same abbreviations and symbols are used in

C–E.(D) EMSA demonstrating the ability of full-length

VIM1 to bind a methylated oligonucleotide probe based

on the 180-bp centromere repeat sequence. (E) EMSA of

interaction of full-length VIM1 with identical probes

methylated in different cytosine contexts. (F) ChIP was

performed on nuclei prepared from Col (N, nontrans-

genic) and transgenic (T) plants expressing Flag-VIM1.

After immunoprecipitation, samples were used as tem-

plates for amplification of different genomic sequences,

which are indicated at the left of the images of the re-

sulting ethidium-stained gels. (Input) 10% of input;

(Mock) precipitation in the absence of antibody; (␣Flag)

precipitation with anti-Flag antibody.

5mC-binding protein required for DNA methylation

GENES & DEVELOPMENT 5

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tained cytosine methylation in only one of the three pos-

sible sequence contexts (Fig. 4B,E). As expected, VIM1

bound the oligonucleotide modified at 5mCpG sites.

VIM1 was unable to bind the 5mCpHpH-containing oli-

gonucleotide, but interacted in a methylation-dependent

manner with the 5mCpHpG-containing oligonucleotide.

Therefore, VIM1 recognizes cytosine methylation in two

different sequence contexts that occur in plant genomes,

including centromere repeat arrays.

After demonstrating that VIM1 has methylcytosine-

binding activity in vitro, we next determined whether

VIM1 associates with methylated DNA in vivo. We em-

ployed a chromatin immunoprecipitation (ChIP) assay

on nuclei prepared from wild-type Col and transgenic

Arabidopsis plants expressing Flag-VIM1. Cross-linked

chromatin complexes were precipitated with or without

anti-Flag antibody, and the precipitated DNA samples

were used as templates to amplify centromeric 180-bp

repeats. As seen in Figure 4F, the 180-bp centromere re-

peat arrays were associated with the VIM1 protein. The

unmethylated locus At4g04040 was not associated with

VIM1 (Gendrel et al. 2002; Lippman et al. 2004). We also

investigated three other methylated genomic loci, in-

cluding the tandemly repeated 5S rRNA genes (5S

rRNA), repetitive Athila retrotransposable elements

(Athila), and a Cinful-like retrotransposon (T5L23.29)

(Gendrel et al. 2002). The anti-Flag antibody precipitated

all methylated genomic sequences tested. Our results

indicate that VIM1 associates with methylated DNA

both in vitro and in vivo. The specificity of vim1 DNA

hypomethylation effects may stem from a heightened

sensitivity of centromeric chromatin to loss of VIM1

function.

VIM1 is concentrated at chromocenters

We next studied the subnuclear localization of VIM1

relative to interphase chromatin organization. The VIM1

genomic coding sequence, expressed under control of its

native promoter, was engineered to express full-length

VIM1 fused to the Flag epitope at the C terminus

(VIM1g-Flag) in transgenic plants. Fixed nuclei from Col,

Bor-4, and transgenic Bor-4 expressing VIM1g-Flag were

immunolabeled with anti-Flag and anti-HTR12 antibod-

ies. Introduction of the VIM1g-Flag construct comple-

mented three centromere-related phenotypes in Bor-4:

decondensation of 180-bp centromere repeats (data not

shown), altered HTR12 localization (Fig. 5, middle col-

umn; Supplementary Table 2), and centromere DNA hy-

pomethylation (Fig. 3C). These results demonstrate that

the epitope-tagged VIM1 is functional. Epitope-tagged

VIM1 protein was localized to the nucleus, where it

showed a broad distribution (excluding the nucleolus)

but enrichment in the heterochromatic chromocenters

in the majority of the cells examined (Fig. 5, top row;

Supplementary Table 3). VIM1 localization is consistent

with our ChIP results (Fig. 4F) and the known distribu-

tion of methylated DNA sequences, which tend to be

concentrated in the chromocenters.

VIM1–histone interaction

The centromere phenotypes of vim1 mutants, including

the apparent changes in HTR12 localization, coupled

with the observation that the SRA domain of murine

Np95 interacts differentially with core histones (Citterio

et al. 2004), led us to test whether VIM1 physically in-

teracts with HTR12. An epitope-tagged HTR12 (N-ter-

minal c-myc tag) was expressed in Escherichia coli and

partially purified using an anti-myc conjugated resin. In

parallel, we expressed and partially purified recombinant

versions of other Arabidopsis histones, including myc-

H2A (HTA2), myc-H2B (HTB2), myc-H3 (HTR3), and

myc-H4 (HFO4). Purified recombinant histones were in-

cubated with extracts from transgenic plants overex-

pressing Flag-VIM1. Proteins associated with the immo-

bilized recombinant histones were subject to Western

blot analysis to monitor the recovery of the Flag-VIM1

protein. As shown in Figure 6A, Flag-VIM1 interacted

with three of the core histones (with the exception of

H2A [HTA2]), as well as the centromere-specific histone

H3, HTR12.

Discussion

We describe the identification and isolation of the Ara-

bidopsis VIM1 gene encoding a methylcytosine-binding

protein required for maintenance of centromere DNA

methylation and proper interphase centromere organiza-

tion. The VIM1 gene was originally identified by a spon-

taneous loss-of-function mutation in a wild accession

that did not exhibit a striking morphological phenotype.

Figure 5. Subnuclear localization of VIM1. Immunolocaliza-

tion of the epitope-tagged VIM1 (␣Flag) and HTR12 (␣HTR12)

was performed on nuclei derived from nontransgenic Bor-4 and

Col plants, as well as transgenic Bor-4 plants expressing VIM1-

Flag under control of the native VIM1 promoter (VIM1g-Flag).

DAPI was used as a DNA counterstain. Bar, 5 µm.

Woo et al.

6 GENES & DEVELOPMENT

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Plants are remarkably tolerant of dramatic alterations in

DNA methylation relative to mammals. However, sev-

eral Arabidopsis mutants with altered cytosine methyl-

ation suffer developmental abnormalities and reduced

fertility under optimal laboratory conditions, suggesting

that these mutants would compete poorly in natural set-

tings. The persistence of the vim1-1 mutation in natural

populations indicates that the alteration in centromere

organization and DNA methylation caused by loss of

VIM1 can be tolerated in the wild, and highlights the

viability of mining natural variation for novel alleles

that affect epigenetic regulation.

Loss of VIM1 function causes CpG and CpHpG hypo-

methylation at the centromere, but has no significant

effect on DNA methylation of the FWA gene or pericen-

tromeric sequences (5S rRNA genes and Athila retro-

transposable elements). The apparent specificity of vim1

effects contrasts with the rather broad spectrum of ge-

nomic targets affected by mutations in other Arabidop-

sis genes involved in cytosine methylation (Rangwala

and Richards 2004; Chan et al. 2005). Another interest-

ing feature of the vim1-induced hypomethylation is that

the centromeric repeats are completely remethylated in

F1 hybrids created by outcrossing with wild-type plants.

This behavior contrasts with the inheritance of the hy-

pomethylated state of the centromere induced by ddm1

or met1 in crosses with wild-type plants. The efficient

re-establishment of DNA methylation on the centro-

mere repeats may reflect the presence in vim1 mutants

of centromeric siRNA populations, which may target

these repeats for de novo methylation (Lippman et al.

2003). The altered interphase organization of the centro-

mere observed in vim1 mutants is also unique. Both

ddm1 and met1 mutations cause a decondensation of

pericentromeric sequences, which become dispersed

from chromocenters while the 180-bp centromere re-

peats remain condensed within the chromocenters

(Soppe et al. 2002). In contrast, vim1 mutants display a

decondensation of the 180-bp centromere repeats, sug-

gesting that VIM1 plays a role in centromere organiza-

tion that is distinct from its function in DNA methyl-

ation.

VIM1 is a methylcytosine-binding protein that

interacts with 5mCpG and 5mCpHpG

Methylcytosine-binding proteins bind 5mCpG dinucleo-

tides regardless of sequence context. These proteins re-

cruit a variety of HDAC complexes and chromatin re-

modeling factors, leading to chromatin compaction, and,

consequently, to transcriptional repression (Klose and

Bird 2006). In mammals, MeCP1 and MeCP2 (methyl-

CpG-binding proteins 1 and 2) were the first protein or

complexes identified with high affinity for methylated

DNA (Meehan et al. 1989). Other mammalian methyl-

CpG-binding proteins have been identified subse-

quently, on the basis of the ability to bind methylated

sequences in vitro and/or the possession of a conserved

MBD motif, including MBD2, MBD4, and Kaiso (which

lacks an MBD) (Hendrich and Bird 1998; Prokhortchouk

et al. 2001). The biological importance of MBD proteins

is demonstrated by the wide range of severe phenotypes

that result when genes encoding these proteins are mu-

tated (Amir et al. 1999; Hendrich et al. 2001). In contrast

to the considerable data on the role of mammalian meth-

ylcytosine-binding proteins, knowledge of plant methyl-

cytosine-binding proteins is very limited. Arabidopsis

contains 13 genes encoding MBD motifs. Three Arabi-

dopsis MBD proteins (MBD5, MBD6, and MBD7) have

been shown to bind symmetrically methylated CpG

sites in vitro and two reports indicate that Arabidopsis

MBD5 can also bind 5mCpHpH (Ito et al. 2003; Scebba et

al. 2003; Zemach and Grafi 2003). The role of these MBD

proteins in plant epigenetic regulation has not yet been

characterized.

VIM1 has several interesting features as a methylcy-

Figure 6. VIM1 at the DNA methylation–chromatin interface.

(A) VIM1 interacts with histones. (Top panel) Immobilized myc-

tagged histones were incubated with protein extracts from

transgenic plants expressing Flag-VIM1, and the associated pro-

teins were collected and detected by Western blot analysis using

an anti-Flag antibody to detect Flag-VIM1. Input lanes corre-

spond to total protein from either nontransgenic (N) or trans-

genic (T) plants (1/240×). The bottom panel shows detection of

myc-tagged histones in the samples using an anti-myc antibody.

(B) A model for the role of VIM1 in the maintenance of centro-

meric heterochromatin. The centromere decondensation phe-

notype is illustrated on the top left. (Blue) 180-bp centromere

repeat; (red) HTR12. The VIM1 biochemical activities illus-

trated at the bottom of the figure are necessary for centromere

condensation (top right). Demonstrated VIM1 activities include

direct binding of methylcytosine (m–C) and histone interaction

(direct or indirect). VIM1 is proposed to act as a ubiquitin (Ub)

ligase to modify centromere chromatin structure through direct

modification of nucleosomal histones or through modification

of one or more chromatin effector proteins (X). An alteration in

centromeric chromatin structure leads to DNA hypomethyl-

ation and decondensation of the centromere repeats.

5mC-binding protein required for DNA methylation

GENES & DEVELOPMENT 7

Cold Spring Harbor Laboratory Presson September 17, 2024 - Published by Downloaded from

tosine-binding protein. First, VIM1 lacks an MBD do-

main and binds methylcytosine through a region encom-

passing the SRA domain. We note that the SRA domain

in murine Np95 and related mammalian proteins is suf-

ficient for 5mCpG binding (Unoki et al. 2004), but the

region in VIM1 required for methylcytosine-binding in-

cludes the SRA domain and an additional segment (up to

61 amino acids) adjacent to this domain. Second, VIM1 is

able to bind oligonucleotides that contain a single

5mCpG site. Third, VIM1 interacts with both 5mCpG

and 5mCpHpG. This is the first report of a methylcyto-

sine-binding protein able to bind 5mCpHpG, suggesting

that VIM1 may play an important role in interpreting

one of the non-CpG methylation systems found in

plants.

Differences between VIM1 and mammalian proteins

sharing structural similarity

VIM1 is a methylcytosine-binding protein that has a

PHD domain, two RING finger domains, and an SRA

domain. Proteins showing structural and amino acid

similarity to VIM1 are found in animals as well as plants.

The mammalian proteins most closely related to VIM1,

human ICBP90 and NIRF, along with murine Np95, ap-

pear to function as transcriptional regulators tied into

both cell cycle control and DNA repair (Hopfner et al.

2000; Bonapace et al. 2002; Muto et al. 2002; Arima et al.

2004). Despite their similarities, VIM1 shows several

distinct differences from these mammalian proteins.

First, the mammalian proteins have only one RING do-

main and an extra ubiquitin-like domain, and amino acid

similarity between VIM1 and the mammalian proteins is

restricted to the SRA domain. Second, VIM1 is associ-

ated with a range of heavily methylated genomic se-

quences, suggesting that VIM1 could play a role in main-

taining heterochromatin throughout the genome. In con-

trast, ICBP90 regulates the transcription of specific

genes, such as topoisomerase-II␣ and Retinoblastoma

protein 1, as well as participates in the maintenance of

silencing of tumor suppressor genes when they become

ectopically methylated (Hopfner et al. 2000; Unoki et al.

2004; Jeanblanc et al. 2005). Third, VIM1 is enriched at

chromocenters, but Np95 and ICBP90 are broadly local-

ized in the nucleus during interphase; Np95 is globally

associated with chromatin (Miura et al. 2001), whereas

ICBP90 is distributed throughout the entire nucleoplasm

(Arima et al. 2004; Jeanblanc et al. 2005). Fourth, loss of

Np95 or ICBP90 function has not yet been reported to

affect DNA methylation of their target sequences.

Model for VIM1 action

Our working model is that VIM1 acts as a bridge be-

tween cytosine methylation and histone modification in

a mutually reinforcing heterochromatin signaling loop

that is required for proper centromere organization and

centromere DNA methylation (Fig. 6B). VIM1 interacts

with core histones and is able to bind 5mC-modified

DNA, placing it physically at the interface between

DNA methylation and chromatin packaging. The possi-

bility that VIM1 is involved in histone modification is

suggested by the demonstration that the SRA domain of

ICBP90, Np95, and NIRF can recruit mammalian

HDAC1 (Unoki et al. 2004). In addition, Np95, via its

RING domain, possesses histone monoubiquitin ligase

activity (Citterio et al. 2004). Although histone ubiqui-

tylation is often associated with transcriptional activa-

tion, there are examples where this modification corre-

lates with transcriptionally silent heterochromatin

(Emre and Berger 2004; Wang et al. 2004). We note the

recent discovery that a cullin 4-dependent E3 ubiquitin

ligase, complexed with Rik1 and the HMT Clr4, plays a

crucial role in formation of centromeric heterochroma-

tin in fission yeast (Horn et al. 2005; Jia et al. 2005; Li et

al. 2005; Thon et al. 2005). The in vivo substrates of the

Rik1-associated E3 ubiquitin ligase complex are not

known, but histone H2B copurified with the Rik1 com-

plex, and this complex exhibited H2B polyubiquitylation

activity in vitro (Horn et al. 2005).

Based on these considerations, we propose that VIM1

binds to methylated centromeric DNA and alters his-

tone modification in the centromeric chromatin by re-

cruiting HDACs and/or by ubiquitylation of core his-

tones. The demonstrated interaction with VIM1 and his-

tones H2B, H3, H4, and HTR12 makes these core

histones good candidates for substrates of RING-domain

mediated ubiquitylation. Alternatively, VIM1 might

ubiquitylate a chromatin effector (or effectors), leading

to an alteration of centromeric heterochromatin. Alter-

ations in centromeric chromatin are postulated to lead to

a reduction in centromeric DNA methylation due to the

inefficient recruitment of DNMTs, such as CMT3 (re-

cruited to heterochromatic histone methylation marks)

(Lindroth et al. 2004) and DRM2 (which contains ubiq-

uitin-binding domains) (Cao et al. 2000). Our results sug-

gest that this new class of methylcytosine-binding pro-

teins may play a different role in epigenetic regulation

compared with MBD-class methylcytosine-binding pro-

teins, which have not been demonstrated to participate

in maintenance of genomic cytosine methylation pat-

terns or centromeric heterochromatin.

Materials and methods

Plant material

Seeds of natural Arabidopsis strains and T-DNA insertion mu-

tants (Alonso et al. 2003) were obtained from the Arabidopsis

Biological Resources Center. The natural strains examined were

part of the 96-member collection assembled by Nordborg et al.

(2005). We also examined the original collection of Bor acces-

sions made by J. Relichová (Masaryk University, Brno, Czech

Republic) to confirm the presence to the vim1-1 allele in natural

isolates. Plants were grown in a controlled environmental

chamber at 22°C under long day conditions (16 h light per day).

DNA blot hybridization

Genomic DNA was digested with HpaII, MspI, or HhaI accord-

ing to the manufacturer’s (New England Biolabs) instructions.

Woo et al.

8 GENES & DEVELOPMENT

Cold Spring Harbor Laboratory Presson September 17, 2024 - Published by Downloaded from

Radiolabeled probes were generated by random priming, and

blots were prepared and hybridized using standard methods.

The following probes were generated from purified cloned in-

serts: 180-bp repeat (CEN) clone, pARR20-1 (Vongs et al. 1993),

and 5S rRNA gene clone pCT4.1 (Campell et al. 1992). The

Athila LTR and FWA probes were derived by PCR from genomic

DNA (Soppe et al. 2000; Lindroth et al. 2001).

Construction of plant expression vectors and generation

of transgenic plants

The 5.5-kb Col genomic DNA fragment containing 2.0 kb of the

promoter region and the whole predicted ORF of VIM1 was

PCR-amplified using two primers: 5⬘-CACCACGACCTTCG

GAAGATGCAAGAA-3⬘ and 5⬘-CCTGATGGTCGCAGAAA

CTGTTGC-3⬘. The fragment was cloned into pENTR-D TOPO

(Invitrogen) and the resulting VIM1 insert was recombined into

pEarlyGate302 (Earley et al. 2006). This construct was trans-

formed into Agrobacterium tumefaciens (LBA4404) and was in-

troduced into Bor-4 plants by in planta transformation (Bechtold

and Pelletier 1998). A full-length VIM1 cDNA clone in

pENTR-D TOPO was recombined into pEarlyGate202 (Earley et

al. 2006) to add an N-terminal Flag tag for the ChIP and histone-

binding assays. The resulting constructs were introduced into

Col plants by standard infiltration protocols.

EMSA

Full-length VIM1 cDNA PCR product (amino acids 1–645) or

deletion mutants—designated PHD/RING1 (amino acids

1–237), SRA/RING2 (amino acids 238–645), SRA (amino acids

238–456), RING2 (amino acids 457–645), and SRA+ (amino ac-

ids 238–517)—were cloned into the pDEST17 vector (Invitro-

gen). We synthesized VIM1 or derivatives in vitro using a T7

RNA polymerase-coupled wheat germ extract system (Promega

). For binding assays, double-stranded probe DNA was

end-labeled using the DIG gel shift kit (Roche) according to the

manufacturer’s instructions. Labeled DNA (62 fmol) were incu-

bated with in vitro translated VIM1 or its derivatives in the

presence or absence of competitors (3.85 pmol). Binding reac-

tions were carried out in 25 mM HEPES (pH 7.6), 50 mM KCl,

0.1 mM EDTA (pH 8.0), 12.5 mM MgCl

, 1 mM DTT, and 5%

(w/v) glycerol; 1.5 µg poly-dIdC was added as a nonspecific com-

petitor. After incubation for 30 min at room temperature, the

reaction mixture was subjected to electrophoresis.

ChIP

ChIP was performed as described in Grendel et al. (2002) on

nuclei prepared from wild-type and Arabidopsis plants express-

ing Flag-VIM1 using anti-Flag (Sigma). Precipitated DNAs were

subjected to PCR using the following primers (see Supplemen-

tary Table 2): CEN-F + CEN-R; 5S rRNA-F + 5S rRNA-R;

Athila-F + Athila-R; T5L23.29-F + T5L23.29-R; At4g04040-

F + At4g04040-R. Amplification of CEN, 5S rRNA, Athila,or

At4g04040 was performed for 26, 22, 30, or 32 cycles, respec-

tively.

Immunolocalization/DNA-FISH

Root meristems from 14-d-old plants were excised and nuclei

were extracted as described previously (Lawrence et al. 2004).

After post-fixation in 4% formaldehyde/PBS (phosphate-buff-

ered saline), washes in PBS, and blocking at 37°C, slides were

exposed overnight to primary antisera mouse anti-Flag (1:100,

Sigma) and rabbit anti-HTR12 (Talbert et al. 2002) in PBS and

0.5% blocking reagent (Roche). After washes in PBS, slides were

incubated at 37°C with anti-mouse-FITC diluted 1:100 (Sigma)

or goat anti-rabbit-TRITC (1:300, Sigma). For combined protein/

DNA-FISH localization experiments, slides were first subjected

to immunolocalization, and then post-fixed in 4% formalde-

hyde/PBS followed by in situ hybridization. FISH using the CEN

repeat probe (pARR20-1) labeled with biotin-dUTP and DIG-

dUTP was performed as described (Pontes et al. 2004). CEN

repeats were detected using either goat anti-biotin conjugated

with avidin (1:200, Vector Laboratories) followed by streptavi-

din-Alexa 543 (Molecular Probes) or anti-digoxeninin (1:200,

Roche) and rabbit anti-mouse Alexa 488 (Molecular Probes).

DNA was counterstained with DAPI (1 µg/mL) in Vectashield

(Vector Laboratories). Nuclei were examined using a Nikon

Eclipse E800i epifluorescence microscope, with images col-

lected using a Photometrics Coolsnap ES Mono digital camera.

The images were pseudocolored, merged, and processed using

Adobe Photoshop (Adobe Systems).

VIM1–histone interaction

Epitope-tagged (myc) recombinant histones were expressed in E.

coli BL21(AI) (Invitrogen) and cells were lysed by sonication in

a buffer containing 500 mM NaCl and 20 mM Tris-HCl (pH 7.5).

For purification, anti-c-Myc agarose conjugate (Sigma) was

added to cleared lysates and incubated for2hat4°C. Beads were

washed in buffer containing 150 mM NaCl, 20 mM Tris-HCl

(pH 7.5), 5 mM MgCl

, and 0.5% NP-40. Total proteins were

extracted from transgenic plants overexpressing Flag-VIM1 by

grinding tissue in an extraction buffer containing 150 mM

NaCl, 20 mM Tris-HCl (pH 7.5), and 5 mM MgCl

. Bead-bound

myc-tagged histones were incubated with extracts from trans-

genic plants overexpressing Flag-VIM1 overnight at 4°C. Beads

were washed as described above. Proteins were eluted by adding

SDS-PAGE sample buffer and were analyzed by SDS-PAGE elec-

trophoresis followed by Western blotting using anti-myc or

anti-Flag antibodies (Sigma).

Acknowledgments

We thank T. Dittmer, H.-f. Kuo, S. Rangwala, T.R. Smith, and

H. Yi for comments on the manuscript. We are grateful to J.

Relichová for providing Bor natural accessions, and J. Haag (his-

tone constructs) and S. Henikoff (anti-HTR12 antibody) for pro-

viding reagents. This work was supported by grants from the

National Science Foundation to E.J.R (MCB-0321990) and the

Monsanto Company. DNA-FISH and immunolocalization was

performed by O.P., who was supported by fellowship SFRH/

BPD/17508/2004 from the FundaçãoparaaCiência e Tecnolo-

gia (Portugal) and by NIH grants R01GM60380 and

R01GM077590 to C.S.P. We thank the Arabidopsis Biological

Resource Center at The Ohio State University for providing

Arabidopsis strains and SALK T-DNA insertion mutants.

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5mC-binding protein required for DNA methylation

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