MBD4-Mediated Glycosylase Activity on a Chromatin Template Is Enhanced by Acetylation

Abstract

The ability of the MBD4 glycosylase to excise a mismatched base from DNA has been assessed in vitro using DNA substrates with different extents of cytosine methylation, in the presence or absence of reconstituted nucleosomes. Despite the enhanced ability of MBD4 to bind to methylated cytosines, the efficiency of its glycosylase activity on T/G mismatches was slightly dependent on the extent of methylation of the DNA substrate. The reduction in activity caused by competitor DNA was likewise unaffected by the methylation status of the substrate or the competitor. Our results also show that MBD4 efficiently processed T/G mismatches within the nucleosome. Furthermore, the glycolytic activity of the enzyme was not affected by the positioning of the mismatch within the nucleosome. However, histone hyperacetylation facilitated the efficiency with which the bases were excised from the nucleosome templates, irrespective of the position of the mismatch relative to the pseudodyad axis of symmetry of the nucleosome.Diverse organisms, prokaryotic and eukaryotic, methylate selected cytosines in their genomes (reviewed by Bestor [19]). In bacteria, methylation is usually associated with restrictionmodification systems which protect the cell against attack from bacteriophages. Cytosine methylation is associated with genome defense in some eukaryotes, but it also plays vital roles in development and in the control of gene expression, to the extent that the homozygous knockout of cytosine methyltransferase genes in the mouse is an embryonic lethal mutation (40). Despite its importance, cytosine methylation imposes a genetic and epigenetic cost because 5-methylcytosines deaminate into thymine (reviewed in references 53 and 67). Unrepaired, the resulting T/G mismatches lead to CG-to-TA transition mutations. There is also evidence from work with Escherichia coli that 5-methylcytosines are more prone to mispairing during DNA replication than unmethylated bases and/or are less well repaired and that they are particularly susceptible to other forms of endogenous and exogenous damage which cause them to mispair (24, 54). A similar situation exists for eukaryotes (reviewed in reference 51).Whatever the cause, mutations at methylated cytosines may influence phenotype in two ways: (i) by introducing nonsense or missense mutations which alter the functions of proteins and (ii) by demethylating promoter regions and thereby altering patterns of gene expression. As one line of defense against the genetic and epigenetic effects of mutations associated with methylated cytosines, cells have developed repair systems specific to T/G mismatches. Cytosine methyltransferases methylate both strands of palindromic recognition sequences (CpG or CNG sequences in eukaryotes and CCWGG sequences in E. coli); consistent with their role in preventing mutations at 5-methylcytosines, the DNA repair systems show a strong preference for T/G mismatches which arise in methylase recognition sequences (31,32,46). E. coli initiates repair with a single-stranded endonuc...

Uracil DNA Glycosylase Activity on Nucleosomal DNA Depends on Rotational Orientation of Targets

“…However, the glycosylase NTH1 was shown to efficiently process thymine glycol lesions oriented away from the histone octamer surface at a site about 50 bp from the nucleosome dyad but sites oriented toward the histone octamer or closer to the dyad were removed at a reduced efficiency compared with naked DNA (38). The activity of the glycosylase MBD4 was found to be diminished but not abolished for T/G mismatches within a reconstituted nucleosome in a manner apparently not dependent on position with respect to the dyad, whereas histone hyperacetylation increased the efficiency with which the bases were excised (39). Likewise, two groups (33,34,40) have investigated the activity of human UDGs, SMUG1 and UNG2, in conjunction with AP endonuclease 1, and DNA polymerase ␤ on nucleosomal substrates.…”

Modulation of the activity of methyl binding domain protein 4 (MBD4/MED1) while processing iododeoxyuridine generated DNA mispairs

“…However, our data are supported by a more recently published study which demonstrated that the efficiency of MED1 glycosylase activity was partially dependent on the extent of methylation of the DNA substrate. 37 From a clinical-translational perspective, we have previously shown the MED1 deficient mouse embryonic fibroblasts (mefs) show significantly increased levels of IUdR-DNA incorporation compared to wild-type mefs. 5 Consequently, MED1 deficient cells showed enhanced IUdR-mediated cytotoxicity and radiosensitization, which was not found in wild type mefs.…”

DNA Methylation and Methyl-Binding Proteins Control Differential Gene Expression in Distinct Cortical Areas of Macaque Monkey

“…We aligned the MBD-like regions of the macaque MBDs and MeCP2 proteins, and found that MBD4 and MeCP2 are also more similar to each other than to the other MBD proteins (52.3% identical and 83.1% similarity to MeCP2 vs MBD4; 38.5 Ϯ 2.9% identical and 63.8 Ϯ 4.0% similarity to MBD1, 2, and 3 vs MBD4). It has also been demonstrated previously that both MeCP2 and MBD4 exhibit a significant binding preference for nucleosomes containing methylated DNA (Ishibashi et al, 2008).…”

“…Regarding the question of when the area-selective expression occurs, Bernard et al (2012) reported area-and layer-selective gene expressions using microarray on the macaque neocortex, and Kang et al (2011) on human neocortex. We compared our results with those of others who conducted human (http://humanbraintranscriptome.org) and macaque neocortical expression analyses (http://www.blueprintnhpatlas.org).…”