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

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.

Figure 1.

The optimal binding site for ZBTB4 on unmethylated DNA. (A) The human ZBTB4 protein. The underlined region containing three zinc fingers (ZF, black rectangles) was used in EMSA experiments. BTB: BTB/POZ domain. (B) The consensus ZBTB4-binding sequence (Z4BS) obtained by SELEX. The Z4BS resembles the KBS that has been previously reported (9). (C) Validation of the identified consensus by EMSA. The indicated proteins were used in increasing amounts (2 and 10 ng), with the indicated probes. (D) The relative importance of positions within the Z4BS. Nine unlabeled duplexes (m0–m8), each differing from the Z4BS at a single position, were used in EMSA assay, to outcompete a labeled Z4BS probe.

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).

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.

Figure 4.

ZBTB4 residues involved in DNA recognition. (A) To chelate zinc ions, GST-ZBTB4 was incubated with OPA. Zinc was added back at 0.2 or 0.5 mM of ZnCl₂. The mixtures were then incubated with labeled Z4BS (CTGCCAT) or meZ4BS (CMGCCAT) probes and analyzed by EMSA. (B) Alignment of the four key positions (+1, +2, +3 and +6) in the zinc fingers of human ZBTB4, Kaiso and ZBTB38. The underlined residues were mutated in ZBTB4. The numbers indicate the amino-acid position in human ZBTB4. (C) The ZBTB4 mutants were used in EMSA with Z4BS (CTGCCAT; upper) or meZ4BS (CMGCCAT; lower) probes. The E350A mutant has normal binding to the Z4BS, but is severely affected for binding to methylated DNA. (D) Wild-type or E350 mutant ZBTB4 were used in EMSA analysis with labeled meZ4BS (CMGCCAT), mutated meZ4BS (CMGCCCT), Z4BS (CTGCCAT) and unmethylated meZ4BS (CCGCCAT). (E) NIH3T3 cells were transfected with GFP-ZBTB4 or GFP-ZBTB4 (E350A). DNA was stained with DAPI (top row), and green fluorescence recorded (bottom row).

Figure 5.

Summary of the binding data and interpretation. (A) Two components contribute to the binding of ZBTB4 to an optimal site: the E350 residue is necessary for recognition of the methylated cytosine (black lollipop), while other positions in the zinc fingers recognize the flanking sequences of methylated DNA. (B) Because its affinity for methylated cytosine is high, ZBTB4 can bind methylated DNA even if the flanking sequence is suboptimal. (C) ZBTB4 can also recognize thymine instead of methylated cytosine, but with lower affinity. The E350A mutant of ZBTB4 has lost affinity for methylated cytosine, but maintains the recognition of flanking sequences (D). It cannot bind to methylated DNA lacking the optimal flanking sequence (E), but still binds the consensus unmethylated target (F).