CXXC Domain of Human DNMT1 Is Essential for Enzymatic Activity

Abstract

DNA cytosine methylation is one of the major epigenetic gene silencing marks in the human genome facilitated by DNA methyltransferases. DNA cytosine-5 methyltransferase 1 (DNMT1) performs maintenance methylation in somatic cells. In cancer cells, DNMT1 is responsible for the aberrant hypermethylation of CpG islands and the silencing of tumor suppressor genes. Here we show that the catalytically active recombinant DNMT1, lacking 580 amino acids from the amino terminus, binds to unmethylated DNA with higher affinity than hemimethylated or methylated DNA. To further understand the binding domain of enzyme, we have used gel shift assay. We have demonstrated that the CXXC region (C is cysteine; X is any amino acid) of DNMT1 bound specifically to unmethylated CpG dinucleotides. Furthermore, mutation of the conserved cysteines abolished CXXC mediated DNA binding. In transfected COS-7 cells, CXXC deleted DNMT1 (DNMT1 ∆CXXC ) localized on replication foci. Both point mutant and DNMT1 ∆CXXC enzyme displayed significant reduction in catalytic activity, confirming that this domain is crucial for enzymatic activity. A permanent cell line with DNMT1 ∆CXXC displayed partial loss of genomic methylation on rDNA loci, despite the presence of endogenous wild-type enzyme. Thus, the CXXC domain encompassing the amino terminus region of DNMT1 cooperates with the catalytic domain for DNA methyltransferase activity.

CXXC Finger Protein 1 Contains Redundant Functional Domains That Support Embryonic Stem Cell Cytosine Methylation, Histone Methylation, and Differentiation

“…The MLL1 gene is a frequent target of chromosomal aberrations that are associated with leukemia (55), and the CXXC motif is essential for the transforming capacity of Mll1 fusion proteins (5). Similarly, mutation of the CXXC domain within Dnmt1 abolishes CXXC-mediated DNA binding and results in significant reduction of Dnmt1 catalytic activity and loss of genomic methylation of rDNA loci (42).…”

Diverse Domains of (Cytosine-5)-DNA Methyltransferases: Structural and Functional Characterization

“…The CXXC domain is known to bind specifically unmethylated 5′-CG-3′/3′-GC-5′ sites [98,99]. The structural data confirm this type of specificity: a methyl group presence at the cytosine C5 atom would result in steric clashes between the DNA and the protein atoms [9].…”

“…This domain is shown to bind unmethylated 5′-CG-3′/3′-GC-5′ sites in vitro in the case of MBD1, MLL, CGBP, JHDM1B, and Dnmt1 proteins [3]. The CXXC domain of Dnmt1 is crescent-shaped and contains 8 conservative catalytically important Cys residues [98]. These residues are clustered in two groups and bind two Zn 2+ ions.…”

Total parenteral nutrition induces sustained hypomethylation of DNA in newborn guinea pigs

“…Pradhan et al (22) showed that the activity of DNMT is dependent on the integrity of sensitive thiol functions (R-SH). Different molecules can react with R-SH.…”

“…DNMT3a and 3b are implicated in de novo methylation of DNA (16)(17)(18), whereas DNMT1 is responsible for maintenance of DNA methylation between cell replications (19,20). The activity of DNMT1 is reported to be sensitive to the redox status of its cysteine residues (21,22). We hypothesized that the peroxide content of TPN favors DNA hypomethylation through inhibition of DNMT activity caused by oxidation of its sensitive cysteine residues.…”