Báo cáo khoa học: CpG methylation of the CENP-B box reduces human CENP-B binding

CpG methylation of the CENP-B box reduces human

CENP-B binding

Yoshinori Tanaka

1,2,

*, Hitoshi Kurumizaka

1,3

and Shigeyuki Yokoyama

1,2,4

1 Protein Research Group, RIKEN Genomic Sciences Center, Suehiro-cho, Tsurumi, Yokohama, Japan

2 Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan

3 Waseda University School of Science and Engineering, 3-4-1 Okubo, Shinjuku-ku, Tokyo, Japan

4 RIKEN Harima Institute at Spring-8, 1-1-1 Kohto, Mikazuki-cho, Sayo, Hyogo, Japan

The centromere of eukaryotic chromosomes plays an

essential role in the proper segregation of chromo-

somes at mitosis and meiosis, and has a special hetero-

chromatin structure, which is composed of a-satellite

DNA repeats and their associated proteins. The

human centromere proteins A, B and C (CENP-A,

CENP-B and CENP-C, respectively) are such centro-

mere-speciﬁc DNA-binding proteins [1–7]. Neither

CENP-A nor CENP-C shows any sequence speciﬁcity

in DNA binding. In contrast, CENP-B is known to

speciﬁcally bind a 17 base-pair sequence (the CENP-B

box), which appears in every other a-satellite repeat

(171 base-pairs) in human centromeres [8–10].

CENP-B is an 80 kDa protein that contains DNA-

binding and dimerization domains at the N-terminus

and C-terminus, respectively [11–13]. Biochemical ana-

lyses with nucleosomes reconstituted in vitro suggested

that the CENP-B box sequence functions as a cis

element for centromere-speciﬁc nucleosome assembly

[14]. In vivo analyses with cultured human cells

Keywords

CENP-B; centromere; DNA methylation;

chromatin; heterochromatin

Correspondence

H. Kurumizaka, Waseda University School of

Science and Engineering, 3-4-1 Okubo,

Shinjuku-ku, Tokyo 169-8555, Japan

Fax: +81 3 5292 9211

Tel: +81 3 5286 8189

E-mail: kurumizaka@waseda.jp

S. Yokoyama, Protein Research Group,

RIKEN Genomic Sciences Center, 1-7-22

Suehiro-cho, Tsurumi, Yokohama 230-0045,

Japan

Fax: +81 45 503 9195

Tel: +81 45 503 9196

E-mail: yokoyama@biochem.s.u-tokyo.ac.jp

*Present address

Toray Industries, Inc. New Frontiers

Research Laboratories, 1111 Tebiro, Kamak-

ura, Kanagawa 248–0036, Japan

(Received 15 September 2004, accepted

1 October 2004)

doi:10.1111/j.1432-1033.2004.04406.x

In eukaryotes, CpG methylation is an epigenetic DNA modiﬁcation that is

important for heterochromatin formation. Centromere protein B (CENP-

B) speciﬁcally binds to the centromeric 17 base-pair CENP-B box DNA,

which contains two CpG dinucleotides. In this study, we tested complex

formation by the DNA-binding domain of CENP-B with methylated and

unmethylated CENP-B box DNAs, and found that CENP-B preferentially

binds to the unmethylated CENP-B box DNA. Competition analyses

revealed that the afﬁnity of CENP-B for the CENP-B box DNA is reduced

nearly to the level of nonspeciﬁc DNA binding by CpG methylation.

Abbreviations

CENP-B, centromere protein B; RNAi, RNA interference; siRNA, small interfering RNA.

282 FEBS Journal 272 (2005) 282–289 ª2004 FEBS

revealed that the presence of the CENP-B box is essen-

tial for the formation of functional minichromosomes

[15,16]. Therefore, the CENP-B box is required for the

formation of a functional centromere. However,

CENP-B null mice appeared to be normal [17–19].

This discrepancy about the CENP-B dispensibility for

mouse development and the CENP-B box requirement

for minichromosome maintenance in human cells may

be explained by presuming the existence of functional

homologues of CENP-B. In fact, CENP-B-like pro-

teins have been identiﬁed in humans, and the func-

tional redundancy of CENP-B homologues has also

been found in the ﬁssion yeast Schizosaccharomyces

pombe [20–22].

In eukaryotic cells, methylation of the cytosine

within the CpG dinucleotide is an epigenetic DNA

modiﬁcation that is important for heterochromatin

formation. The human a-satellite consensus sequence

Fig. 1. Human a-satellite DNA and the CENP-B(1–129)–DNA complex structure. (A) Schematic diagram showing the organization of human

a-satellite DNA and the CENP-B box. Arrows and circles in the middle row indicate a-satellite DNA repeats and CENP-B boxes, respectively.

The a-satellite consensus sequence [23], containing a 17 bp CENP-B box, is shown in the bottom row. The three boxes, marked as sites 1,

2 and 3, indicate the essential bases for CENP-B binding to the CENP-B box DNA. The three CpG sequences in the a-satellite sequence are

shown in red. (B) The CENP-B(1–129)–DNA complex structure [25]. The essential bases for CENP-B binding are coloured red in the model

structure. The CpG sites that are sharply kinked by the CENP-B binding are encircled in red. The nucleotide sequences of sites 1 and 3 are

indicated in the left column. (C) Model for the interaction between CENP-B and the CpG-methylated CENP-B box. (Left) Interaction between

CENP-B and the methylated CENP-B box DNA at sites 1 and 3. The methyl groups, which are modelled on the C5 atoms of cytosines in the

CpG dinucleotides, are coloured red. The van der Waals radii of the methyl groups (2.0 A

˚) are shown in orange circles. The van der Waals

radii of the Thr44 and Arg125 side chains are shown in grey circles. Distances between the cytosine methyl groups and the nearest Thr44

and Arg125 side chain atoms are indicated by arrows. (Right) Interaction between CENP-B and the unmethylated CENP-B box DNA at sites

1 and 3. The pink dotted line in the right panel indicates the hydrogen bond between the side chain NH

group of Arg125 and the N7 atom

of guanine.

Y. Tanaka et al.CpG methylation reduces human CENP-B binding

FEBS Journal 272 (2005) 282–289 ª2004 FEBS 283

contains only three CpG sequences within its 171 base-

pair sequence [23]. Interestingly, two of the three CpG

sequences in the a-satellite consensus sequence

are located within site 1 (5¢-pTpTpCpG-3¢) and site 3

(5¢-pCpGpGpG-3¢) of the CENP-B box (Fig. 1A; [9]).

Demethylation of satellite DNA sequences, accom-

plished by growing cells in the presence of the DNA

methyltransferase inhibitor, 5-aza-2¢-deoxycytidine,

resulted in the redistribution of CENP-B [24], indica-

ting that the CpG methylation affects the CENP-

B–DNA interaction. Our structural analysis of the

CENP-B DNA-binding domain [CENP-B(1–129)]

complexed with the CENP-B box DNA revealed that

CENP-B induced sharp kinks at the CpG sequences of

sites 1 and 3 upon DNA binding (Fig. 1B; [25]). There-

fore, CpG methylation may regulate CENP-B binding

to the CENP-B box DNA within centromeric a-satel-

lite repeats.

In this study, we tested the complex formation of

CENP-B(1-129) with methylated and unmethylated

CENP-B box DNAs by a complex-reconstitution

assay, and found that the afﬁnity of CENP-B for CpG

methylated CENP-B box DNA is signiﬁcantly reduced.

Results

Model for the interaction between CENP-B and

the CpG-methylated CENP-B box

In eukaryotes, DNA methylation occurs at the C5

atom of cytosine by the action of methyltransferases.

To evaluate the effect of CpG methylation on the

CENP-B–DNA interaction, we modeled additional

methyl groups at the C5 atoms of site 1 (cytosine 6 and

cytosine 7¢) and site 3 (cytosine 15 and cytosine 16¢)in

the CENP-B(1–129)–DNA complex structure (Fig. 1C).

In the crystal structure of the CENP-B(1–129)–DNA

complex [25], the CpG sequences in sites 1 and 3 of the

CENP-B box DNA were sharply kinked by CENP-

B(1–129) binding (16for site 1 and 43for site 3;

Fig. 1B). As shown in Fig. 1C, additional methyl

groups on cytosine 7¢and cytosine 15 caused steric cla-

shes with the side chains of Thr44 and Arg125, respect-

ively. Therefore, the CENP-B(1–129)–methylated DNA

complex model suggested that the methylations of cyto-

sine 7¢and cytosine 15 may sterically interfere with

CENP-B binding to the CENP-B box DNA.

The complex-reconstitution assay

We tested whether the CpG methylations of the

CENP-B box DNA actually affect CENP-B binding

to the DNA, as suggested by the CENP-B(1–129)–

methylated DNA complex model. To do so, we

employed a complex-reconstitution assay with recom-

binant CENP-B(1–129) and the methylated and un-

methylated CENP-B box DNAs (21 bp; Fig. 2A). As

the recombinant CENP-B(1–129) was only detected in

the insoluble fraction when it was expressed in

Escherichia coli cells, CENP-B(1–129) was puriﬁed

under denaturing conditions in the presence of 6 m

urea (Fig. 2B). In the complex-reconstitution assay,

CENP-B(1–129) formed the complex with the CENP-

B box DNA during the refolding process by dialysis

against buffer without urea. The complex-formation

efﬁciency was monitored by a gel-shift assay

(Fig. 2C).

Fig. 2. The complex-reconstitution assay with CENP-B(1–129) and

the CENP-B box DNA. (A) Schematic presentation of the complex-

reconstitution assay. CENP-B(1–129) and the CENP-B box DNA

strands are shown in blue and red, respectively. CENP-B(1–129)

and the CENP-B box DNA were mixed under denaturing conditions

in the presence of 6 Murea. The CENP-B(1–129)–DNA complex

was reconstituted during the refolding process by dialysis against

buffer without urea. (B) The purified recombinant CENP-B(1–129)

protein was analyzed by 15–25% SDS/PAGE. Lane 1 indicates

molecular mass markers (M), and lane 2 indicates CENP-B(1–129)

purified under denaturing conditions in the presence of 6 Murea.

DNA complex, reconstituted by the method shown in panel A, was

analyzed by 20% nondenaturing PAGE. Bands were visualized by

ethidium bromide staining.

CpG methylation reduces human CENP-B binding

1Y. Tanaka et al.

284 FEBS Journal 272 (2005) 282–289 ª2004 FEBS

CENP-B(1–129) preferentially binds to the

unmethylated CENP-B box DNA rather than the

methylated form

Using the complex-reconstitution assay, we tested the

binding of CENP-B(1–129) to the unmethylated and

methylated CENP-B box DNAs (Fig. 3A). As shown

in Fig. 3B, CENP-B(1–129) efﬁciently formed the com-

plex with the unmethylated CENP-B box DNA in a

concentration-dependent manner (lanes 1–5). CENP-

B(1–129) also formed the complex with the methylated

CENP-B box DNA (Fig. 3B, lanes 6–10), but with

slightly reduced efﬁciency, as compared with the

unmethylated CENP-B box DNA (Fig. 3C). Therefore,

CENP-B has the potential to bind to the methylated

CENP-B box DNA.

Fig. 3. CENP-B(1–129) preferentially binds to unmethylated CENP-B box DNA. (A) The 21-mer CENP-B box DNA and the 21-mer nonspecific

DNA (DnaA box DNA [30]), used in this study. The methylated cytosine residues in the methylated CENP-B box DNA are labeled by CH

the middle row. (B) Gel-shift analysis of complex formation between CENP-B(1–129) and the CENP-B box DNA, complexed with increasing

amounts of CENP-B(1–129), by 20% PAGE. The unmethylated CENP-B box DNA (lanes 1–5; 1 lM) and the methylated CENP-B box DNA

(lanes 6–10; 1 lM) were used in this study. The CENP-B(1–129) concentrations were 0 lM(lanes 1 and 6), 0.5 lM(lanes 2 and 7), 1 lM

(lanes 3 and 8), 3 lM(lanes 4 and 9), and 5 lM(lanes 5 and 10). (C) Graphic representation of the complex formation rates shown in panel

B. Unmethylated (d) and methylated (s) CENP-B box DNAs, respectively. (D) Competition analysis for CENP-B binding between the un-

methylated and methylated CENP-B box DNAs. The

P-labeled unmethylated CENP-B box DNA (1 lM)orthe

P-labeled methylated CENP-

B box DNA (1 lM) was mixed with the indicated amounts of the unlabeled competitor methylated or unmethylated CENP-B box DNAs,

respectively, in the presence of CENP-B(1–129) (3 lM). The complexes were analyzed by 20% PAGE. (E) Graphic representation of the com-

plex formation rates shown in panel D. Closed and open circles indicate experiments with the

P-labeled unmethylated (d) and methylated

(s) CENP-B box DNAs. Complex formation rates (%) are plotted against the concentrations of the competitor DNAs.

Y. Tanaka et al.CpG methylation reduces human CENP-B binding

FEBS Journal 272 (2005) 282–289 ª2004 FEBS 285

Next, we tested the binding afﬁnity of CENP-B(1–

129) to the methylated CENP-B box DNA. To do so,

we performed a competition assay. In this assay,

complex formation between CENP-B(1–129) and the

P-labeled unmethylated or methylated CENP-B box

DNAs was tested in the presence of unlabeled compet-

itor DNA. As shown in Fig. 3D, complex formation

between CENP-B(1–129) and the

P-labeled methyla-

ted CENP-B box DNA was signiﬁcantly inhibited in

the presence of only one-third of the amount of unlabe-

led unmethylated CENP-B box DNA (0.33 lm), relat-

ive to the

P-labeled methylated form (1 lm) (lanes

7–12, and E). In contrast, complex formation between

CENP-B(1–129) and the

P-labeled unmethylated

CENP-B box DNA was not affected, even in the pres-

ence of a threefold excess of unlabeled methylated

CENP-B box DNA (3 lm) (Fig. 3D, lanes 1–6, and E).

These results indicate that CENP-B preferentially

forms a complex with the unmethylated CENP-B box

DNA, rather than the methylated CENP-B box DNA.

The CENP-B binding affinity to the methylated

CENP-B box DNA is similar to the level of

nonspecific DNA binding

In order to compare the CENP-B binding to the

CENP-B box DNA and a nonspeciﬁc DNA, we per-

formed a competition assay with the methylated or

unmethylated CENP-B box DNAs and a nonspeciﬁc

DNA. The DnaA box sequence, which is not speciﬁc-

ally bound by CENP-B, was used as nonspeciﬁc DNA

(Fig. 3A, bottom row). The complex formation

between CENP-B(1–129) and the

P-labeled unmethy-

lated CENP-B box DNA was not affected when it was

titrated with unlabeled nonspeciﬁc DNA (Fig. 3A and

4A, lanes 1–6, and B). On the other hand, the complex

between CENP-B(1–129) and the

P-labeled methyla-

ted CENP-B box DNA was signiﬁcantly dissociated in

the presence of an equal amount of unlabeled nonspe-

ciﬁc competitor DNA (1 lm) (Fig. 4A, lanes 7–12, and

B). Therefore, the CpG methylation at sites 1 and 3 of

the CENP-B box DNA reduces the CENP-B binding

afﬁnity almost to the level of nonspeciﬁc DNA binding.

Discussion

In this study, we found that CpG methylation of the

CENP-B box sequence reduces the binding afﬁnity

between CENP-B and the CENP-B box DNA nearly

to the level of nonspeciﬁc binding. In the CENP-B(1–

129)–methylated DNA complex model, the additional

methyl groups on cytosine 7¢and cytosine 15 caused

steric clashes with the side chains of Thr44 and

Arg125, respectively (Fig. 1C). In site 3, the CENP-B

a-helix 8 (120–129 amino acid residues), containing

four Arg residues (Arg125, 127, 128 and 129), penet-

rates perpendicularly into the major groove around the

CpG sequence. Interestingly, the Arg125 side chain

directly formed a hydrogen bond with the N7 atom of

guanine 16, and speciﬁcally recognized the site 3

sequence (Fig. 1C, right). In contrast, the Arg127, 128

and 129 residues bound to the backbone phosphates,

and did not directly interact with the DNA bases.

Steric hindrance of the speciﬁc interaction between

Arg125 and the N7 atom of guanine 16 by the CpG

methylation may be a reason for the reduced speciﬁcity

of CENP-B to the CENP-B box sequence.

What is the functional meaning of the CpG methyla-

tion of the CENP-B box DNA? Recently, a link

between centromeric heterochromatin formation and

the RNA interference (RNAi) machinery was discov-

ered. In ﬁssion yeast, the RNAi machinery is required

for chromosome segregation, gene silencing and nor-

mal centromere function [

326–28]. In chicken–human

Fig. 4. CpG methylation reduces the affinity between CENP-B and

the CENP-B box DNA to nearly the level of nonspecific DNA bind-

ing. (A) The

P-labeled CENP-B box DNA (1 lM) was mixed with

the indicated amounts of the unlabeled nonspecific DNA competitor

in the presence of CENP-B(1–129) (3 lM). The complexes were

analyzed by 20% PAGE. (B) Graphic representation of the complex

formation rates shown in panel A.

P-labeled unmethylated (d)

and methylated (s) CENP-B box DNAs. Formation rates (%) are

plotted against the concentrations of the competitor DNAs.

CpG methylation reduces human CENP-B binding

1Y. Tanaka et al.

286 FEBS Journal 272 (2005) 282–289 ª2004 FEBS

Báo cáo khoa học: CpG methylation of the CENP-B box reduces human CENP-B binding

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