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

Abstract

Cytosine methylation, predominantly of the CpG sequence in vertebrates, is one of the major epigenetic modifications crucially involved in the control of gene expression. Due to the difficulty of reconstituting site‐specifically methylated nucleosomal DNA at crystallization quality, most structural analyses of CpG methylation have been performed using chemically synthesized oligonucleotides, There has been just one recent study of nucleosome core particles (NCPs) reconstituted with nonpalindromic human satellite 2‐derived DNAs. Through the preparation of a 146‐bp palindromic α‐satellite‐based nucleosomal DNA containing four CpG dinucleotide sequences and its enzymatic methylation and restriction, we reconstituted a ‘symmetric’ human CpG‐methylated nucleosome core particle (NCP). We solved the crystal structures of the CpG‐methylated and unmodified NCPs at 2.6 and 3.0 Å resolution, respectively. We observed the electron densities of two methyl groups, among the eight 5‐methylcytosines introduced in the CpG‐fully methylated NCP. There were no obvious structural differences between the CpG‐methylated ‘symmetric NCP’ and the unmodified NCP. The preparation of a crystallization‐grade CpG‐methylated NCP provides a platform for the analysis of CpG‐methyl reader and eraser proteins.

Epigenetic Modifications of Cytosine: Biophysical Properties, Regulation, and Function in Mammalian DNA

“…This may overestimate the numbers, as large parts of the genomic DNA are highly condensed and not accessible to these enzymes. On the other hand, the bending associated with nucleosome formation may alter the intrinsic equilibrium significantly . Notably, TDG's analog uracil‐DNA glycosylase (UNG) reportedly utilizes a passive base‐flipping mechanism during its search for cytosine deamination products, trapping the extrahelical substrates upon spontaneous, thermally‐induced base‐pair opening …”

“…The major and minor groove sides are colored in cyan and magenta, respectively, the helix axes are traced in red, and the 5‐position of cytosine is depicted as a green sphere. B‐DNA is the predominant form in solution and in nucleosomes, while the A‐type conformation is adopted by DNA‐RNA duplexes formed during transcription and, though rarely observed in solution, is frequently adopted in DNA crystal structures, due in part to the dehydrating conditions required for crystallization. c) B‐ and A‐DNA models of ideal geometry showing a possible structural reason why methylation at position 5 (shown in green) of cytosine is favored over position 6 (shown in red).…”

Rapid and Definitive Analysis of In Vitro DNA Methylation by Nano-electrospray Ionization Mass Spectrometry

“…The pET15b plasmid containing the DNA region coding M.Sss I methylase was kindly provided by Dr. Takashi Umehara of RIKEN. Recombinant M.Sss I methylase was overexpressed in E. coli and purified according to the previously reported method [21]. Briefly, M.Sss I methylase with an N-terminal His6-tag was overexpressed in E. coli and purified with Ni-NTA column chromatography (Ni Sepharose 6 Fast Flow, GE Bioscience).…”

“…For CpG methylation, the DNA oligomer at a concentration of 0.02 μg/μL was incubated with M.Sss I methylase at a ratio of 15:1 (w/w) at 37 °C for 5 h in 10 mM Tris-HCl buffer (pH 8.0) containing 50 mM NaCl, 2.5 mM EDTA, and 640 μM S-adenosylmethionine (SAM) [21]. The methylation reaction was quenched by heating the solution at 65 °C for 20 min.…”

“…Following the determination of more than 100 structures of NCPs reconstituted with unmodified histone variants originating from various organisms and unmodified ~ 150 bp DNA oligomers, a couple of NCP structures reconstituted with CpG methylated DNA and unmodified histone proteins have been recently reported. Fujii et al designed CpG146, an α-satellite-based DNA of 146 bp containing four CACGTG sequences in each strand and performed CpG methylation with M.Sss I DNA methylase [21]. In CpG146, eight cytosine bases in all of the CpG sequences can theoretically be methylated by M.Sss I methylase.…”