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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 binds to methylated DNA in a sequence-specific manner. (A) The Methyl-SELEX procedure. (B) The methylation-dependent ZBTB4-binding site (meZ4BS) identified by Methyl-SELEX, and its alignment with the Z4BS. (C) The MS8 sequence (CCGCTAT) was unmethylated or methylated with SssI and used in EMSA analysis with GST or GST-ZBTB4. (D) GST-ZBTB4 was pre-incubated with 50-, 150- or 400-fold molar excess of the indicated unlabeled competitor oligonucleotides, and then incubated with labeled methylated meZ4BS and used in EMSA. (E) ZBTB38 also binds methylated DNA in a sequence-specific manner. GST-ZBTB38 was used in EMSA as in (D). The oligonucleotides m1, m2 and m3, are the same as in (D).
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Figure 2: ZBTB4 binds to methylated DNA in a sequence-specific manner. (A) The Methyl-SELEX procedure. (B) The methylation-dependent ZBTB4-binding site (meZ4BS) identified by Methyl-SELEX, and its alignment with the Z4BS. (C) The MS8 sequence (CCGCTAT) was unmethylated or methylated with SssI and used in EMSA analysis with GST or GST-ZBTB4. (D) GST-ZBTB4 was pre-incubated with 50-, 150- or 400-fold molar excess of the indicated unlabeled competitor oligonucleotides, and then incubated with labeled methylated meZ4BS and used in EMSA. (E) ZBTB38 also binds methylated DNA in a sequence-specific manner. GST-ZBTB38 was used in EMSA as in (D). The oligonucleotides m1, m2 and m3, are the same as in (D).

Mentions: We next asked whether ZBTB4 has a preferred target on methylated DNA. For this, we modified the methyl-SELEX (Figure 2A; 11).Figure 2.


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

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

ZBTB4 binds to methylated DNA in a sequence-specific manner. (A) The Methyl-SELEX procedure. (B) The methylation-dependent ZBTB4-binding site (meZ4BS) identified by Methyl-SELEX, and its alignment with the Z4BS. (C) The MS8 sequence (CCGCTAT) was unmethylated or methylated with SssI and used in EMSA analysis with GST or GST-ZBTB4. (D) GST-ZBTB4 was pre-incubated with 50-, 150- or 400-fold molar excess of the indicated unlabeled competitor oligonucleotides, and then incubated with labeled methylated meZ4BS and used in EMSA. (E) ZBTB38 also binds methylated DNA in a sequence-specific manner. GST-ZBTB38 was used in EMSA as in (D). The oligonucleotides m1, m2 and m3, are the same as in (D).
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Related In: Results  -  Collection

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Figure 2: ZBTB4 binds to methylated DNA in a sequence-specific manner. (A) The Methyl-SELEX procedure. (B) The methylation-dependent ZBTB4-binding site (meZ4BS) identified by Methyl-SELEX, and its alignment with the Z4BS. (C) The MS8 sequence (CCGCTAT) was unmethylated or methylated with SssI and used in EMSA analysis with GST or GST-ZBTB4. (D) GST-ZBTB4 was pre-incubated with 50-, 150- or 400-fold molar excess of the indicated unlabeled competitor oligonucleotides, and then incubated with labeled methylated meZ4BS and used in EMSA. (E) ZBTB38 also binds methylated DNA in a sequence-specific manner. GST-ZBTB38 was used in EMSA as in (D). The oligonucleotides m1, m2 and m3, are the same as in (D).
Mentions: We next asked whether ZBTB4 has a preferred target on methylated DNA. For this, we modified the methyl-SELEX (Figure 2A; 11).Figure 2.

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