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Sequence-specific recognition of methylated DNA by human zinc-finger proteins.

Sasai N, Nakao M, Defossez PA - Nucleic Acids Res. (2010)

Bottom Line: The last three proteins can bind either methylated DNA or unmethylated consensus sequences; how this is achieved is largely unclear.Here, we report that the human zinc-finger proteins Kaiso, ZBTB4 and ZBTB38 can bind methylated DNA in a sequence-specific manner, and that they may use a mode of binding common to other zinc-finger proteins.This suggests that many other sequence-specific methyl binding proteins may exist.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR7216, Université Paris-Diderot, Paris, France.

ABSTRACT
DNA methylation is an essential epigenetic mark. Three classes of mammalian proteins recognize methylated DNA: MBD proteins, SRA proteins and the zinc-finger proteins Kaiso, ZBTB4 and ZBTB38. The last three proteins can bind either methylated DNA or unmethylated consensus sequences; how this is achieved is largely unclear. Here, we report that the human zinc-finger proteins Kaiso, ZBTB4 and ZBTB38 can bind methylated DNA in a sequence-specific manner, and that they may use a mode of binding common to other zinc-finger proteins. This suggests that many other sequence-specific methyl binding proteins may exist.

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ZBTB4 has highest affinity for methylated DNA, and its recognition mode is not symmetrical. (A) GST-ZBTB4 was pre-incubated with 10-, 50-, 150- or 400-fold molar excess of unlabeled unmethylated meZ4BS (CCGCCAT), meZ4BS (CMGCCAT) or Z4BS (CTGCCAT) and then incubated with labeled meZ4BS and analyzed by EMSA. (B) GST-ZBTB4 was pre-incubated with 50-, 150- or 400-fold molar excess of the indicated unmethylated competitor DNA before adding the labeled meZ4BS probe.
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Figure 3: ZBTB4 has highest affinity for methylated DNA, and its recognition mode is not symmetrical. (A) GST-ZBTB4 was pre-incubated with 10-, 50-, 150- or 400-fold molar excess of unlabeled unmethylated meZ4BS (CCGCCAT), meZ4BS (CMGCCAT) or Z4BS (CTGCCAT) and then incubated with labeled meZ4BS and analyzed by EMSA. (B) GST-ZBTB4 was pre-incubated with 50-, 150- or 400-fold molar excess of the indicated unmethylated competitor DNA before adding the labeled meZ4BS probe.

Mentions: Finally, we determined the binding preference of ZBTB4 for the different methylated and unmethylated targets we had identified. In a competition experiment, we saw the following order of preference: CMGCCAT > CTGCCAT > CCGCCAT (Figure 3A). Therefore, the methylated sequence meZ4BS is the preferred binding target of ZBTB4 in vitro.Figure 3.


Sequence-specific recognition of methylated DNA by human zinc-finger proteins.

Sasai N, Nakao M, Defossez PA - Nucleic Acids Res. (2010)

ZBTB4 has highest affinity for methylated DNA, and its recognition mode is not symmetrical. (A) GST-ZBTB4 was pre-incubated with 10-, 50-, 150- or 400-fold molar excess of unlabeled unmethylated meZ4BS (CCGCCAT), meZ4BS (CMGCCAT) or Z4BS (CTGCCAT) and then incubated with labeled meZ4BS and analyzed by EMSA. (B) GST-ZBTB4 was pre-incubated with 50-, 150- or 400-fold molar excess of the indicated unmethylated competitor DNA before adding the labeled meZ4BS probe.
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Figure 3: ZBTB4 has highest affinity for methylated DNA, and its recognition mode is not symmetrical. (A) GST-ZBTB4 was pre-incubated with 10-, 50-, 150- or 400-fold molar excess of unlabeled unmethylated meZ4BS (CCGCCAT), meZ4BS (CMGCCAT) or Z4BS (CTGCCAT) and then incubated with labeled meZ4BS and analyzed by EMSA. (B) GST-ZBTB4 was pre-incubated with 50-, 150- or 400-fold molar excess of the indicated unmethylated competitor DNA before adding the labeled meZ4BS probe.
Mentions: Finally, we determined the binding preference of ZBTB4 for the different methylated and unmethylated targets we had identified. In a competition experiment, we saw the following order of preference: CMGCCAT > CTGCCAT > CCGCCAT (Figure 3A). Therefore, the methylated sequence meZ4BS is the preferred binding target of ZBTB4 in vitro.Figure 3.

Bottom Line: The last three proteins can bind either methylated DNA or unmethylated consensus sequences; how this is achieved is largely unclear.Here, we report that the human zinc-finger proteins Kaiso, ZBTB4 and ZBTB38 can bind methylated DNA in a sequence-specific manner, and that they may use a mode of binding common to other zinc-finger proteins.This suggests that many other sequence-specific methyl binding proteins may exist.

View Article: PubMed Central - PubMed

Affiliation: CNRS UMR7216, Université Paris-Diderot, Paris, France.

ABSTRACT
DNA methylation is an essential epigenetic mark. Three classes of mammalian proteins recognize methylated DNA: MBD proteins, SRA proteins and the zinc-finger proteins Kaiso, ZBTB4 and ZBTB38. The last three proteins can bind either methylated DNA or unmethylated consensus sequences; how this is achieved is largely unclear. Here, we report that the human zinc-finger proteins Kaiso, ZBTB4 and ZBTB38 can bind methylated DNA in a sequence-specific manner, and that they may use a mode of binding common to other zinc-finger proteins. This suggests that many other sequence-specific methyl binding proteins may exist.

Show MeSH