Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays

Abstract

The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.

Crystal structure of human nucleosome core particle containing enzymatically introduced CpG methylation

“… reported the CpG methylation of 5S rDNA in a reconstituted nucleosome by M.Sss I. In addition, a nucleosome containing hemimethyl CpG was reconstituted, using nucleosome array DNA methylated by M.Sss I . Furthermore, Osakabe et al .…”

“…For example, Choy et al [15] reported the CpG methylation of 5S rDNA in a reconstituted nucleosome by M.SssI. In addition, a nucleosome containing hemimethyl CpG was reconstituted, using nucleosome array DNA methylated by M.SssI [16]. Furthermore, Osakabe et al [17] recently reconstituted nucleosome core particles (NCPs) containing M.SssI-methylated CpG dinucleotide sites, using a 160-bp human satellite 2 DNA.…”

RFTS‐dependent negative regulation of Dnmt1 by nucleosome structure and histone tails

“…Repression of the methylation activity of Dnmt1 in the nucleosome core region At single base resolution, analysis of Dnmt1-catalyzed methylation has so far been performed on nucleosomes prepared with unmethylated DNA [17]. To elucidate the effect of nucleosomal structure on mouse Dnmt1 activity on its intrinsic target, we prepared high-purity hemimethylated DNA which can be analyzed by sodium bisulfite sequencing (Fig.…”

“…When mononucleosomes were reconstituted with recombinant histones and unmethylated DNA, Dnmt1 activity toward nucleosomes was slightly reduced compared to naked DNA [15]. Dnmt1 activity toward hemimethylated nucleosomal arrays was also reported to be lower than that toward naked DNA [17]. DNMT1 showed an intrinsic ability to methylate CpG sites packaged into nucleosomes but the activity was DNA sequence dependent [16].…”

“…Analysis of mononucleosomes with unmethylated DNA by bisulfite sequencing showed that Dnmt1 de novo methylation was inhibited in nucleosome cores but not on linker DNA. Dnmt1 activity toward hemimethylated nucleosomal arrays was also reported to be lower than that toward naked DNA [17].…”