1022-7954/01/3706- $25.00 © 2001
Russian Journal of Genetics, Vol. 37, No. 6, 2001, pp. 603–609. Translated from Genetika, Vol. 37, No. 6, 2001, pp. 737–744.
Original Russian Text Copyright © 2001 by A.V. Prokhortchouk, Aitkhozhina, Sablina, Ruzov, A.A. Prokhortchouk.
Mammalian DNA is known to contain methylated
CpG dinucleotides. The binding to methylated DNA
sequences has been best demonstrated for MeCP2, a
transcription repressor with the effect mediated by his-
tone deacetylation . The methylated DNA-binding
domain (MBD) is the only well-characterized protein
domain involved in interaction with symmetrically meth-
ylated CpG dinucleotides. In addition, zinc-ﬁnger motifs,
which bind to half-methylated DNA sequences, have been
found in DNA methyltransferase 1 (DNMT 1) .
We have previously described a protein which spe-
ciﬁcally binds in vitro with a symmetrically methylated
DNA sequence of the ﬁrst intron of the
Hence, it was necessary to identify this protein, to study
whether it binds to methylated DNA in vivo, and to
identify the domain involved in its speciﬁc interaction
with methylated DNA. The results of gel ﬁltration,
afﬁnity puriﬁcation, and mass spectrometry, which was
carried out in cooperation with M. Mann (EMBL,
Heidelberg, Germany), showed that the methylated
DNA-speciﬁc protein is a human homolog of murine
Kaiso. A.B. Reynolds and colleagues (Vanderbildt Uni-
versity, Nashville, United States)  have isolated
Kaiso from a yeast two-hybrid system by its ability to
bind with catenin p120, a component of the E-cadherin
complex involved in formation of cell–cell adhesion
and/or signaling. The partner of Kaiso, catenin p120,
has been localized to the contact sites and the cell
nucleus [5, 6].
Kaiso contains zinc-ﬁnger domains . We checked
whether these domains account for methylation-spe-
ciﬁc DNA binding. Experiments showed that the zinc-
ﬁnger domains of Kaiso indeed recognize methylcy-
tosine (mC) in DNA sequences. This is the ﬁrst evi-
dence that the mammalian protein, which belongs to
the family of zinc-ﬁnger proteins, speciﬁcally recog-
nizes symmetrically methylated DNA.
In addition to zinc ﬁngers, Kaiso contains the
BTB/POZ domain, which is potentially able to interact
with histone deacetylases through corepressors N-CoR
and SMRT . Hence, it is possible to assume that
Kaiso is involved in transcription regulation of genes
which are active or inactive depending on whether their
DNA sequences are methylated. To check this assump-
tion, we studied the location of Kaiso in NIH 3T3 cells.
MATERIALS AND METHODS
Gel retardation assay of the binding of nuclear pro-
teins with DNA in the presence or absence of speciﬁc
DNA fragments were 5'-end-labeled at one
strand via enzymic phosphorylation in the presence of
]ATP. The binding of nuclear proteins with the
resulting probes followed a published protocol .
Competitive binding was carried out in the presence of
a 50-fold molecular excess of a nonlabeled methylated
or nonmethylated oligonucleotide.
Gel retardation assays were carried out with a synthetic
double-stranded oligonucleotide used as a
probe and as a speciﬁc competitor. The oligonucleotide
and noncoding strand
and contained a binding site for the methyl-DNA-spe-
ciﬁc protein (boldface).
Kaiso, a New Protein of the BTB/POZ Family,
Specifically Binds to Methylated DNA Sequences
A. V. Prokhortchouk
, D. S. Aitkhozhina
, A. A. Sablina
A. S. Ruzov
, and E. B. Prokhortchouk
Institute of Gene Biology, Russian Academy of Sciences, Moscow, 117334 Russia;
fax: (095) 135-41-05; e-mail: email@example.com
Blokhin Institute of Carcinogenesis, Russian Academy of Medical Sciences, Moscow, 115478 Russia
Received March 28, 2000
—A protein speciﬁcally binding to a symmetrically methylated DNA fragment of the ﬁrst intron of
gene was studied. The protein was puriﬁed by gel ﬁltration and afﬁnity chromatography. Mass spec-
trometry showed that the protein is Kaiso, a new member of the BTB/POZ family. To study the association with
methylated DNA sequences in vivo, the location of Kaiso in NIH 3T3 cells was analyzed. Immunoﬂuorescent
staining with polyclonal antibodies against Kaiso showed that the protein is predominantly associated with the
nucleoli. The causes of its distribution awaits further investigation. The zinc-ﬁnger domains of Kaiso were for
the ﬁrst time demonstrated to speciﬁcally recognize symmetrically methylated DNA sequences in vitro.