Disease-Associated Mutations G589A and V590F Relieve Replication Focus Targeting Sequence-Mediated Autoinhibition of DNA Methyltransferase 1.

In eukaryotes, the most common epigenetic DNA modification is methylation of carbon 5 of cytosines, predominantly in CpG dinucleotides. Methylation patterns are established and maintained by a family of proteins known as DNA methyltransferases (DNMTs). DNA methylation is an important epigenetic mark associated with gene repression, and disruption of the normal DNA methylation pattern is known to play a role in several disease states. Methylation patterns are primarily maintained by DNMT1, which possesses specificity for methylation of hemimethylated DNA. DNMT1 is a multidomain protein with a C-terminal catalytic methyltransferase domain and a large N-terminal regulatory region. The replication focus targeting sequence (RFTS) domain, found in the regulatory region, is an endogenous inhibitor of DNMT1 activity. Recently, several mutations in the RFTS domain were shown to be causal for two adult onset neurodegenerative diseases; however, little is known about the impact of these mutations on the structure and function of DNMT1. Two of these mutations, G589A and V590F, are associated with development of autosomal dominant cerebellar ataxia, deafness, and narcolepsy (ADCA-DN). We have successfully expressed and purified G589A and V590F DNMT1 for in vitro studies. The mutations significantly decrease the thermal stability of DNMT1, yet the mutant proteins exhibit 2.5-3.5-fold increases in DNA binding affinity. In addition, the mutations weaken RFTS-mediated inhibition of DNA methylation activity. Taken together, these data suggest these disease-associated mutations decrease protein stability and, at least partially, relieve normal RFTS-mediated autoinhibition of DNMT1.

Disease-Associated Mutation A554V Disrupts Normal Autoinhibition of DNMT1.

DNA methyltransferase 1 (DNMT1) is the enzyme primarily responsible for propagation of the methylation pattern in cells. Mutations in DNMT1 have been linked to the development of adult-onset neurodegenerative disorders; these disease-associated mutations occur in the regulatory replication foci-targeting sequence (RFTS) domain of the protein. The RFTS domain is an endogenous inhibitor of DNMT1 activity that binds to the active site and prevents DNA binding. Here, we examine the impact of the disease-associated mutation A554V on normal RFTS-mediated inhibition of DNMT1. Wild-type and mutant proteins were expressed and purified to homogeneity for biochemical characterization. The mutation increased DNA binding affinity ~8-fold. In addition, the mutant enzyme exhibited increased DNA methylation activity. Circular dichroism (CD) spectroscopy revealed that the mutation does not significantly impact the secondary structure or relative thermal stability of the isolated RFTS domain. However, the mutation resulted in changes in the CD spectrum in the context of the larger protein; a decrease in relative thermal stability was also observed. Collectively, this evidence suggests that A554V disrupts normal RFTS-mediated autoinhibition of DNMT1, resulting in a hyperactive mutant enzyme. While the disease-associated mutation does not significantly impact the isolated RFTS domain, the mutation results in a weakening of the interdomain stabilizing interactions generating a more open, active conformation of DNMT1. Hyperactive mutant DNMT1 could be responsible for the increased DNA methylation observed in affected individuals.

Substituted anthraquinones represent a potential scaffold for DNA methyltransferase 1-specific inhibitors.

In humans, the most common epigenetic DNA modification is methylation of the 5-carbon of cytosines, predominantly in CpG dinucleotides. DNA methylation is an important epigenetic mark associated with gene repression. Disruption of the normal DNA methylation pattern is known to play a role in the initiation and progression of many cancers. DNA methyltransferase 1 (DNMT1), the most abundant DNA methyltransferase in humans, is primarily responsible for maintenance of the DNA methylation pattern and is considered an important cancer drug target. Recently, laccaic acid A (LCA), a highly substituted anthraquinone natural product, was identified as a direct, DNA-competitive inhibitor of DNMT1. Here, we have successfully screened a small library of simplified anthraquinone compounds for DNMT1 inhibition. Using an endonuclease-coupled DNA methylation assay, we identified two anthraquinone compounds, each containing an aromatic substituent, that act as direct DNMT1 inhibitors. These simplified anthraquinone compounds retain the DNA-competitive mechanism of action of LCA and exhibit some selectivity for DNMT1 over DNMT3a. The newly identified compounds are at least 40-fold less potent than LCA, but have significantly less complex structures. Collectively, this data indicates that substituted anthraquinone compounds could serve as a novel scaffold for developing DNMT1-specific inhibitors.