Oxidative damage to methyl-CpG sequences inhibits the binding of the methyl-CpG binding domain (MBD) of methyl-CpG binding protein 2 (MeCP2)

Abstract

Cytosine methylation in CpG dinucleotides is believed to be important in gene regulation, and is generally associated with reduced levels of transcription. Methylation-mediated gene silencing involves a series of DNA-protein and protein-protein interactions that begins with the binding of methyl-CpG binding proteins (MBPs) followed by the recruitment of histone-modifying enzymes that together promote chromatin condensation and inactivation. It is widely known that alterations in methylation patterns, and associated gene activities, are often found in human tumors. However, the mechanisms by which methylation patterns are altered are not currently understood. In this paper, we investigate the impact of oxidative damage to a methyl-CpG site on MBP binding by the selective placement of 8-oxoguanine (8-oxoG) and 5-hydroxymethylcytosine (HmC) in a MBP recognition sequence. Duplexes containing these specific modifications were assayed for binding to the methyl-CpG binding domain (MBD) of one member of the MBP family, methyl-CpG binding protein 2 (MeCP2). Our results reveal that oxidation of either a single guanine to 8-oxoG or of a single 5mC to HmC, significantly inhibits binding of the MBD to the oligonucleotide duplex, reducing the binding affinity by at least an order of magnitude. Oxidative damage to DNA could therefore result in heritable, epigenetic changes in chromatin organization.

Figures

Figure 1

(A) Structures of cytosine, 5-methylcytosine, 5-hydroxymethylcytosine, thymine, guanine and 8-oxoG. (B) Sequence of ODN duplex used in EMSA. Replacement of position N with cytosine, 5mC or HmC in both strands yields duplexes C/C, 5mC/5mC and HmC/HmC, respectively. The duplexes are named based upon the modification at the N position, with the slash denoting that the modification is on different strands within the central CpG. Modification in the upper strand is indicated before the slash, whereas modification to the lower strand is indicated after the slash. Four duplexes containing 8-oxoG were used and are named in a manner similar to, e.g. the 5mC/5mC8oxoG duplex contains 5mC at position N in both the upper and lower strands with 8-oxoG replacing the boldface G in the lower strand. The duplex containing 5mC at position N in the upper strand and T at position N in the lower strand was given the name 5mC/T.

Figure 2

Binding of C/C, C/5mC, 5mC/5mC and 5mC/T duplexes to varying concentrations of MBD from 0 to 256 nM assayed via EMSA.

Figure 3

Non-linear regression of the plot of average percentage binding (determined from EMSA with three or more sets of titrations per duplex) and concentration of MBD for duplexes containing (A) normal purines within the CpG site, (B) HmC within the CpG site and (C) 8-oxoG within the CpG site.

Figure 4

Binding of 5mC/5mC, 5mC/HmC and 5mC/5mC8-oxoG duplexes to varying concentrations of MBD from 0 to 256 nM assayed via EMSA.

Figure 5

Molecular model of the sequence 5′-GCmCGGC-3′. The methyl groups of 5mC are depicted in green, and the N7 position of guanines within the methyl-CpG dinucleotide are depicted in dark blue. Multiple sites within the methylated CpG dinucleotide are needed for strong binding by the MBD. All four major sites of contact, two methyl groups and two hydrogen bond accepting nitrogens, are in the major groove of the DNA within close proximity of one another. Disruption of MBD binding results from oxidative damage to any one of these four sites.