DNA transmethylase

Abstract

Provided is a DNA transmethylase or the like that can be methylated by recognizing a sequence context other than a CG dinucleotide sequence in which endogenous methylation occurs, said sequence context being equivalent to that of the CG dinucleotide sequence. The present invention relates to a protein or the like that has obtained an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating a cytosine residue on the 5'side in the sequence by deleting a portion of amino acid residues including the N-terminus of a DNA methyltransferase of M.CviQIX or M.CviPII.

Description

Technical Field

[0001] The present invention relates to a novel DNA methyltransferase. Background Art

[0002] DNA methyltransferase (MTase) is an enzyme that induces methylation of DNA, one of the representative epigenetic modifications, and is also a useful tool for epigenomic analysis. DNA methylation at the 5th position of cytosine (DNA cytosine 5-methylation (5mC)) is widely observed in the genomic DNA of organisms.

[0003] However, regarding DNA cytosine 5-methylation, the MTases identified so far (non-patent documents 1 to 4) have the disadvantage that their recognition sequences overlap with the CG dinucleotide sequence, which is the recognition sequence for DNA methylation inherent in mammalian cells (endogenous methylation), or the number of bases in the recognition sequence is too long for detailed epigenetic mapping.

[0004] Prior art literature

[0005] Non-patent document 1: Renbaum, P. et al., Cloning, characterization, and expression in Escherichia coli of the gene coding for the CpG DNA methylase from Spiroplasma sp. strain MQ1 (M.SssI). Nucleic acids research, 1990. 18(5): p.1145-1152.

[0006] Non-patent document 2: Xu, M. et al., Cloning, characterization and expression of the gene coding for a cytosine-5-DNA methyltransferase recognizing GpC. Nucleic Acids Res, 1998. 26(17): p. 3961-3966. (same as reference 21 described below)

[0007] Non-patent document 3: Zhang, B. et al., The M.AluI DNA-(cytosine C5)-methyltransferase has an unusually large, partially dispensable, variable region. Nucleic Acids Res, 1993. 21(4): p. 905-911.

[0008] Non-patent document 4: Vladimir SDedkov et al., Cloning and Study of New DNAMethyltransferase M. FatI Modifying Cytosine in a Recognition SiteCATG. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 2015.6(6): p.1341-1348. Summary of the invention

[0009] Problems to be solved by the invention

[0010] Under such circumstances, it is desirable to develop a DNA methyltransferase (MTase) or the like that can introduce methylation that can be distinguished from endogenous methylation, that is, can recognize and carry out methylation in a sequence context other than the CG dinucleotide sequence that undergoes endogenous methylation and that is as short as the sequence.

[0011] The present invention has been made in view of the above-mentioned circumstances, and provides the following protein (MTase) and a gene encoding the same.

[0012] [1] A protein as defined below (a), (b) or (c).

[0013] (a) A portion of amino acid residues including the N-terminus of the DNA methyltransferase of M.CviQIX or M.CviPII was deleted to obtain a protein having the activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue on the 5' side of the sequence.

[0014] (b) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence of the protein of (a) above, and having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue at the 5' side of the sequence.

[0015] (c) A protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence of the protein of (a) above, and having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue at the 5' side of the sequence.

[0016] [2] A protein as defined below (a), (b) or (c).

[0017] (a) A protein having the amino acid sequence shown in SEQ ID NO: 6 or 8.

[0018] (b) A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence of the protein of (a) above, and having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue at the 5' side of the sequence.

[0019] (c) A protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence of the protein of (a) above, and having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue at the 5' side of the sequence.

[0020] [3] A gene encoding the protein described in [1] or [2] above.

[0021] [4] A gene comprising DNA of (a), (b) or (c) below.

[0022] (a) A DNA comprising the base sequence shown in SEQ ID NO: 5 or 7.

[0023] (b) A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA of (a) above, and that encodes a protein having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating a cytosine residue at the 5' side of the sequence.

[0024] (c) A DNA having 80% or more identity with the DNA of (a) above, and encoding a protein having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue at the 5' side of the sequence.

[0025] [5] A recombinant vector comprising the gene described in [3] or [4] above.

[0026] [6] A transformant comprising the recombinant vector described in [5] above.

[0027] [7] A method for producing a protein having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating a cytosine residue on the 5' side of the sequence,

[0028] The manufacturing method comprises:

[0029] A step of culturing the transformant described in [6] above; and

[0030] The step of collecting the protein from the obtained culture.

[0031] [8] An epigenome analysis method comprising using the protein described in [1] or [2] above, or a protein produced by the method described in [7] above.

[0032] Effects of the Invention

[0033] According to the present invention, a protein (MTase) and a gene encoding the same can be provided, which can introduce methylation that can be distinguished from DNA methylation (endogenous methylation) inherent in mammalian cells (more specifically, can specifically recognize a "CC dinucleotide sequence" and 5-methylate the cytosine residue on the 5' side of the sequence). The protein (MTase) of the present invention is useful in, for example, implementing an epigenome analysis method (epigenome mapping) that can theoretically simultaneously perform profiling of endogenous methylation and epigenomes other than endogenous methylation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] [ Figure 1 ] is a diagram showing the scheme of the plasmid vector pCZS.

[0035] [ Figure 2 ] is a diagram showing the dual affinity purification strategy used to purify candidate MTases.

[0036] A. Structure of expressed protein.

[0037] B. Proteins purified by HisTrap purification and dual affinity purification combining HisTrap and StrepTrap purification. Here, M.CviPI is shown as an example of MTase.

[0038] C. The DNA methylation activity of purified M.CviPI was measured using the methylation-sensitive restriction endonuclease HaeIII. As substrate DNA, non-methylated λ DNA (Promega) was used. As a control, a lane using M.CviPI purchased from NEB was set up.

[0039] [ Figure 3 ] is a diagram showing that the N-terminal extension of M.CviQIX has an inhibitory effect on DNA methylation.

[0040] A. Diagram showing the recombinant protein structure of M.CviQIX.

[0041] B. Recognition sequence of restriction endonuclease HaeIII. The recognition sequence contains CC dinucleotides. If the first C is 5-methylated, HaeIII cannot cut the substrate DNA.

[0042] C. A diagram showing the results of digesting genomic DNA extracted from bacterial cells expressing the various M.CviQIX proteins shown in A with HaeIII and analyzing the result with E-Gel Ex.

[0043] D. Scheme showing the structure of various M.CviQIX protein constructs. Tag refers to a sequence derived from the vector.

[0044] E. Digestion of genomic DNA from bacterial cells using the construct from D.

[0045] [ Figure 4 ] is a diagram showing the experimental strategy for clarifying the recognition sequence of MTase candidates.

[0046] A. The scheme used.

[0047] B. Examples of identified sequence motifs.

[0048] C. Draw a phylogenetic tree based on the similarity of amino acid sequences. The identified recognition sequences are arranged according to the gene names. For the common recognition sequences of each clade, annotations are attached to the top of the clade.

[0049] [ Figure 5 ] is a diagram showing the results of NOMe-Seq of fixed yeast nuclei.

[0050] A. Comparison of methylation levels between CCMT-treated and GCMT-treated nuclei. The bin size is 10 bp.

[0051] B. Yeast genes (Track 1), DNA methylation levels at GC sites (Track 2, NOMe-Seq using GCMT), and CC sites (Track 3, NOMe-Seq using CCMT), read coverage of ATAC-Seq (Track 4, RR12926697) (Reference 25), and read coverage of MNase-Seq (Track 5, RR6729489) (Reference 26).

[0052] CE. Aggregation plot near the start codon of yeast genes. Shown is an example of plotting based on DNA methylation signals at CC (C) sites and GC (D) sites, and MNase-Seq (SRR6729489).

[0053] [ Figure 6 ] is a diagram showing the results of NOMe-Seq of IMR-90 cells.

[0054] AD. Comparison of DNA methylation levels between CCMT-treated and untreated nuclei. Endogenous DNA methylation at CG sites (A and B), and methylation at CC sites (C and D) introduced by CCMT in vitro.

[0055] E. Screenshot of the genome browser (track 8) showing human genes (track 1), intrinsic methylation levels (tracks 2, 3, and 4 for untreated, CCMT-treated, and GCMT-treated, respectively), MTase-induced methylation (tracks 5, 6, and 7 for untreated, CCMT-treated, and GCMT-treated, respectively), and read coverage of ATAC-Seq (SRR7765313). The figure on the right is an enlarged view of the position indicated in the figure on the left. For the tracks showing MTase-induced methylation patterns (tracks 5, 6, and 7), peaks detected based on macs2 are shown at the bottom of each track.

[0056] [ Figure 7 ] is a diagram showing attempts to express M.CviQIX and M.CviPII based on several expression vectors.

[0057] A. Scheme of constructs used for expression of M.CviQIX.

[0058] B. Recognition sequence of restriction endonuclease used to study the methylation status of genomic DNA. If the underlined C is methylated, the activity of the enzyme is inhibited.

[0059] C. Analysis of methylation status of genomic DNA extracted from E. coli cells in which expression of M.CviQIX shown in A was induced

[0060] Scheme for the N-terminal deletion mutants of DMCviQIX. A cold shock promoter-based construct was used.

[0061] E. Restriction digestion assay of genomic DNA extracted from E. coli cells harboring the construct shown in D.

[0062] F. Comparison of deletion mutants of M.CviPII.

[0063] G. Restriction digestion test of genomic DNA extracted from Escherichia coli cells containing the construct with F.

[0064] Figure 8 is a figure showing the purification of MTase and its activity.

[0065] A. Representative SDS-PAGE gel image for analyzing protein purification.

[0066] W: Whole cell extract;

[0067] 3 and 4: The 3rd and 4th fractions purified by HisTrap column, respectively.

[0068] I: Input of StrepTrap purification.

[0069] S: Purified protein.

[0070] B. Analysis of MTase activity of the purified protein. The unmethylated λDNA was treated with the purified MTase, digested with the methylation-sensitive restriction endonuclease HpaII, and analyzed with E-Gel Ex.

[0071] Fig. 9 is a figure showing the research results of the optimal salt concentration of CCMT.

[0072] A. Strategy used in the analysis.

[0073] B. Methylation levels observed under the salt conditions used.

[0074] Fig.10 is a figure showing the analysis results of using GCMT- and CCMT-based NOMe-Seq for fixed budding yeast nuclei.

[0075] A. Aggregate plot of methylation levels for GCMT, CCMT-based NOMe-Seq, and MNase-seq.

[0076] B. Venn diagram showing overlapping peaks identified by GCMT-based, CCMT-based NOMe-Seq, ATAC-seq, and MNase-seq. Detailed Description of the Invention

[0077] Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these descriptions, and it can also be appropriately modified and implemented within the scope that does not damage the gist of the present invention, other than the following examples.

[0078] ​​​This specification includes the entire contents of the specification of Japanese Patent Application No. 2022-087994 (filed on May 30, 2022) on which the priority right of the present application is claimed.

[0079] All publications cited in this specification, such as prior art documents, open publications, patent publications and other patent documents, are incorporated into this specification by reference.

[0080] In the present specification, "DNA methyltransferase" has the same meaning as "DNA methyltransferase" and "MTase".

[0081] It should be noted that the details of each reference cited in the following description are listed at the end of this specification.

[0082] 1. Summary of the invention and technical background

[0083] DNA cytosine 5-methylation (5mC) is widely observed in the genomic DNA of organisms, forming one of the major epigenetic modifications. Traditionally, it is believed that the increase in 5mC levels in mammalian cells is related to gene silencing, but it has been revealed that the methylation level of the gene itself is positively correlated with the expression level of the gene (references 1 to 4). Therefore, the distribution pattern of 5mC is cell type specific, and the genome-wide distribution of 5mC or methylome has attracted the attention of many researchers.

[0084] 5-methylation of cytosine in mammalian cells depends on three DNA methyltransferases (MTases), namely DNMT1, DNMT3A, and DNMT3B (references 5 to 7). DNMT1 is a maintenance DNA methyltransferase that introduces methylation to hemimethylated CG dinucleotides. On the other hand, DNMT3A and DNMT3B are de novo DNA methyltransferases that introduce DNA methylation at unmethylated CG sites. Although some exceptions are known in pluripotent stem cells, oocytes, neurons, and glial cells, DNA methylation at sites other than CG rarely occurs in normal somatic tissues (references 8, 9). Therefore, it is generally believed that 5mC is almost exclusively introduced into CG dinucleotides, and cytosines in situations other than CG dinucleotides are not methylated in most mammalian cells.

[0085] The configuration of 5mC in the genome is closely related to other epigenetic modifications. For example, in gene promoters, 5mC is scarce and trimethylation of lysine 4 of histone H3 (H3K4me3) is concentrated (reference 10). Another example is the dimethylation and trimethylation of histone H3 lysine 36 (H3K36me2 and H3K36me3), whose configuration in the genome is positively correlated with transcriptional activity. H3K36me2 and H3K36me3 are enriched in gene bodies and recruit DNMT3A / B to introduce 5mC. Therefore, these modifications support gene body methylation (references 11, 12). Most epigenetic modifications are interrelated, so the detection of DNA methylation simultaneously with other epigenetic markers on the same DNA molecule will stimulate the determination of enthusiasm, and it can be considered that the realization of such a determination will take epigenetic determination to the next stage. However, most existing methods for detecting the epigenome are not suitable for simultaneous detection.

[0086] For example, when using chromatin immunoprecipitation and subsequent base sequencing (ChIP-Seq) (References 13, 14), which are the most commonly used techniques for detecting epigenomes, in ChIP-Seq, the chromatin is first fragmented, and then the target epigenome on the fragmented chromatin is immunoprecipitated, and the DNA bound to the epigenome is sequenced. ChIP-Seq is based on chromatin fragmentation, so the proximal epigenomes located on different nucleosomes will be separated at this initial fragmentation stage. Therefore, ChIP-Seq is not practical for measuring the relationship between two epigenetic modifications on a single DNA molecule. Therefore, the fragmentation-based step is not suitable for simultaneously measuring multiple epigenetic modifications on a single DNA molecule.

[0087] However, there are several steps in epigenomic detection that are not fragmented. For example, DNA methyltransferase (MTase) can be used as a probe to measure the ease of access to chromatin (chromatin accessibility).

[0088] Nucleosome occupancy and methylome sequencing (NOMe-Seq) is one of the earliest methods to measure chromatin accessibility using MTase (reference 15), and it has recently been combined with nanopore sequencing (reference 16). The same principle has recently been applied to Fiber-seq (reference 17), which is a combination of the nonspecific adenine N-6 MTase M.EcoGII and nanopore sequencing. Chromatin accessibility measures the DNA-protein interaction of an unidentified protein, but MTase can also be used to detect the interaction between DNA and a specific protein. One of the representative technologies is DamID (reference 18). In DamID, the dam methyltransferase fused to the target protein can be expressed in the target cell, and the DNA methylation introduced at the N-6 position of adenosine can be detected to measure the specific interaction between the fusion protein and DNA. In the original DamID report (reference 18), a methylation-sensitive restriction endonuclease was used to detect the methylation signal, but this is not an absolute condition for detecting DNA methylation signals. In fact, there are also examples of DNA methylation introduced with DamID being detected using single-molecule sequencers. In addition, DiMeLo-Seq, which introduces N-6 methylation of adenine near specific epigenomic modifications, has also been achieved, and the demand for (epigenomic) detection technology using MTase is further increasing.

[0089] Therefore, MTase enables the simultaneous detection of multiple epigenomes on one DNA molecule without fragmentation.

[0090] In MTase-based epigenetic analysis, the resolution mainly depends on the frequency of the sequence recognized in the genome, and the length of the recognized sequence determines the frequency of the sequence. One of the most basic epigenomic units, the nucleosome, binds to approximately 150 base pairs (bp) of genomic DNA, so the resolution required for epigenome mapping must be higher than 150bp. Therefore, in order to obtain sufficient resolution for epigenetic determination, the recognition sequence must be less than 3bp (64bp resolution). Many MTases have been identified from bacteria and viruses, and non-specific adenine N-6MTases such as M.EcoGII have achieved fine mapping of chromatin accessibility (reference 17). However, when focusing on DNA cytosine C-5MTase, the MTases that can be used as probes for epigenome mapping are limited to three types: M.SssI ( C G), M.CviPI (G C )、M.CviPII( CCD) (target cytosine is underlined) (References 19, 20). In addition, there is original DNA methylation (endogenous methylation) of CG dinucleotides in mammalian cells, so the presence of sequences that completely repeat with CG dinucleotides (M.SssI) or sequences that partially repeat with CG dinucleotides (M.CviPI) is a problem. Therefore, it is desirable to develop and identify a cytosine C-5MTase that never repeats with CG dinucleotides and has a short recognition sequence.

[0091] The inventors of the present application have discovered a systematic strategy for identifying cytosine C-5MTases in short recognition sequences, and have been able to identify 6 new MTases that recognize sequences. Furthermore, focusing on the MTases that can recognize CC dinucleotides among these MTases (whose non-repetitive specificity with CG dinucleotides is believed to be beneficial for epigenomic mapping of mammalian cells), a new MTase has been developed. More specifically, the new MTase is an MTase that can specifically recognize CC dinucleotide sequences and 5-methylate the cytosine residues on the 5' side of the sequence. To date, there has been no report or realization of a new MTase that can recognize and methylate a sequence context (context) of only 2 bases such as CC dinucleotides and non-repetitive with CG that undergoes endogenous methylation.

[0092] 2. Protein

[0093] The protein involved in the present invention is a protein having a function as a DNA methyltransferase (MTase). Specifically, it is a protein having the activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue on the 5' side of the sequence. More specifically, it is a protein having the above-mentioned activity (the activity of 5-methylating the cytosine residue on the 5' side of the CC dinucleotide sequence) (obtaining the activity) by deleting a part of the amino acid residues including the N-terminus of the MTase of M.CviQIX or M.CviPII.

[0094] Here, M.CviQIX is a MTase reported to be derived from Paramecium bursaria Chlorella virus NY2A, and its amino acid sequence is the amino acid sequence shown in SEQ ID NO: 2. It should be noted that the base sequence encoding the amino acid sequence is not particularly limited, and examples thereof include the base sequence shown in SEQ ID NO: 1.

[0095] M.CviPII is a MTase derived from Chlorella virus, and its amino acid sequence is the amino acid sequence shown in SEQ ID NO: 4. It should be noted that the base sequence encoding the amino acid sequence is not particularly limited, and an example thereof is the base sequence shown in SEQ ID NO: 3.

[0096] It should be noted that the MTase of M.CviQIX and M.CviPII are one of the various MTases known in the past, and are known as enzymes that can specifically recognize "CCD trinucleotide sequences" (where D is A, G or C) and 5-methylate the cytosine residues on the 5' side. Generally, for those skilled in the art, there is no reasonable reason to further modify such MTases that can recognize and methylate the relatively short sequence of CCD trinucleotides. Moreover, even if it is assumed that the MTase is to be modified into an MTase that can recognize and methylate a shorter dinucleotide sequence, there is no reasonable reason to think of a means to delete the amino acid residues comprising its N-terminus.

[0097] The protein obtained by deleting a part of amino acid residues including the N-terminus of the MTase of M.CviQIX is not limited, and may be, for example, a protein obtained by deleting 11 amino acid residues from the N-terminus of the MTase, a protein obtained by deleting 13 amino acid residues, or a protein obtained by deleting 15 amino acid residues, among which a protein obtained by deleting 15 amino acid residues is preferred. The amino acid sequence of the protein obtained by deleting 15 amino acid residues from the N-terminus of M.CviQIX is the amino acid sequence shown in SEQ ID NO: 6 (it should be noted that the base sequence encoding the amino acid sequence is the base sequence shown in SEQ ID NO: 5). Therefore, as the protein involved in the present invention, a protein having the amino acid sequence shown in SEQ ID NO: 6 is also preferably used.

[0098] The so-called protein obtained by deleting a part of the amino acid residues including the N-terminus of the MTase as M.CviPII is not limited, and for example, it can be a protein obtained by deleting 11 amino acid residues from the N-terminus of the MTase, a protein obtained by deleting 13 amino acid residues, or a protein obtained by deleting 15 amino acid residues, wherein a protein obtained by deleting 15 amino acid residues is preferably cited. The amino acid sequence of the protein obtained by deleting 15 amino acid residues from the N-terminus of M.CviPII is the amino acid sequence shown in sequence number 8 (it should be noted that the base sequence encoding the amino acid sequence is the base sequence shown in sequence number 7). Therefore, as the protein involved in the present invention, a protein having the amino acid sequence shown in sequence number 8 is also preferably cited.

[0099] The protein involved in the present invention is not limited, and for example, preferably includes an amino acid sequence in which one or several amino acids are deleted, substituted or added to the amino acid sequence of the various proteins mentioned above in this item, and has the activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue on the 5' side of the sequence (so-called mutant protein).

[0100] Here, the "amino acid sequence obtained by deleting, substituting or adding one or several amino acids" is preferably an amino acid sequence obtained by deleting, substituting or adding about 1 to 10 amino acids, preferably 1 to several, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid. The introduction of mutations such as deletion, substitution or addition can be carried out using a mutation introduction kit using site-directed mutagenesis, such as GeneTailor TM Site-directed mutagenesis system (Invitrogen), and TaKaRa site-directed mutagenesis system (Prime STAR (registered trademark) Mutagenesis Basalkit, Mutan (registered trademark)-Super Express Km, etc.: manufactured by Takara Bio), etc. In addition, whether the above-mentioned deletion, substitution or addition mutations are introduced can be confirmed using various amino acid sequencing methods, and structural analysis methods based on X-rays and NMR, etc.

[0101] In addition, the protein involved in the present invention is not limited, and for example, preferably includes an amino acid sequence having an identity (homology) of more than 80% with the amino acid sequence of the various proteins mentioned above in this item, and a protein (so-called mutant protein) having the activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue on the 5' side of the sequence.

[0102] Here, the identity is more preferably 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0103] The mutant protein can be produced by genetic engineering using a gene encoding the amino acid sequence of the protein.

[0104] In the present invention, the so-called "specific recognition of CC dinucleotide sequence" refers to the ability to recognize only a dinucleotide formed by the connection of two cytosines (C). The so-called "5-methylation of the cytosine residue on the 5' side of the CC dinucleotide sequence" refers to methylation of the carbon at the 5-position of the cytosine residue on the 5' side of the aforementioned recognized CC, and the methylation results in 5-methylcytosine or 5-methylcytidine (5mC). As for such methylation activity, for example, it can be detected, measured, and evaluated by investigating the presence or absence of DNA cleavage methods based on HaeIII (recognizing GGCC) and HpaII (recognizing CCGG) whose cleavage activity is known to be inhibited by the presence of 5mC, bisulfite sequencing, and the like.

[0105] The protein involved in the present invention may be a protein obtained by combining and linking peptides derived from natural substances or a protein obtained by artificial chemical synthesis, and is not limited thereto.

[0106] Proteins derived from natural substances can be directly obtained from natural substances using known recovery and purification methods; or they can be obtained by integrating the gene encoding the protein into various expression vectors and the like using known gene recombination techniques, and then introducing the gene into cells to express the protein, and then using known recovery and purification methods. Alternatively, by using a commercially available kit, such as a reagent kit (reagent kit) PROTEIOS TM (Toyobo), TNT TM System (Promega), PG-Mate as a synthesis device TM The protein can be produced by a cell-free protein synthesis system such as TRANSPORT (Toyobo) and RTS (Roche Diagnostics), and obtained by a known recovery method and purification method, but is not limited thereto.

[0107] In addition, chemically synthesized protein can be obtained using known protein synthesis methods. As the synthesis method, for example, an azide method, an acyl chloride method, an anhydride method, a mixed anhydride method, a DCC method, an activated ester method, a carbonyl diimidazole method, and a redox method can be cited. In addition, the synthesis can also be applied to any of solid phase synthesis and liquid phase synthesis. Commercially available protein synthesis devices can also be used. After the synthesis reaction, known purification methods such as chromatography can also be combined to purify the protein.

[0108] In the present invention, derivatives of the proteins can be included together with the various proteins of the present invention, or can be included instead of the proteins. The derivatives include all substances prepared from the proteins, such as substances in which a part of the constituent amino acids is substituted with non-natural amino acids, substances in which a part of the constituent amino acids (mainly the side chains) is chemically modified, etc.

[0109] In addition, in the present invention, salts of the proteins and / or the derivatives thereof may be included together with the various proteins and / or the derivatives of the proteins described above, or may be included instead of the proteins and / or the derivatives thereof. As the salt, a physiologically acceptable acid addition salt or alkaline salt is preferred. As acid addition salts, for example, salts formed with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, and sulfuric acid; or salts formed with organic acids such as acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid. As alkaline salts, for example, salts formed with inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, and magnesium hydroxide; or salts formed with organic bases such as caffeine, piperidine, trimethylamine, and pyridine.

[0110] The salt can be prepared using a suitable acid such as hydrochloric acid or a suitable base such as sodium hydroxide, for example by treatment in water or a liquid containing an inert water-miscible organic solvent such as methanol, ethanol or dioxane using standard protocols.

[0111] 2. Recombinant genes

[0112] The gene encoding the protein of the present invention is not particularly limited, and examples thereof include genes comprising the following DNAs (a), (b) or (c). The gene comprising these DNAs may be a gene consisting only of these DNAs, or may be a gene comprising these DNAs in part, as well as known base sequences (transcription promoter, SD sequence, Kozak sequence, terminator, etc.) required for gene expression, and recognition sequences of various restriction endonuclease sites, without limitation.

[0113] (a) A DNA comprising the base sequence shown in SEQ ID NO: 5 or 7.

[0114] (b) A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA of (a) above, and that encodes a protein having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating a cytosine residue at the 5' side of the sequence.

[0115] (c) A DNA having 80% or more identity with the DNA of (a) above, and encoding a protein having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue at the 5' side of the sequence.

[0116] In the DNA of (a) above, as described above, the base sequence shown in sequence number 5 is a base sequence encoding an amino acid sequence obtained by deleting 15 residues from the N-terminus in the amino acid sequence of MTase of M.CviQIX (sequence number 2). In addition, the base sequence shown in sequence number 7 is a base sequence encoding an amino acid sequence obtained by deleting 15 residues from the N-terminus in the amino acid sequence of MTase of M.CviPII (sequence number 4).

[0117] The DNA of (b) can be obtained from a cDNA library or a genomic library by using the DNA of (a) or a DNA consisting of a base sequence complementary thereto, or a DNA obtained by fragmenting them as a probe, and performing known hybridization methods such as colony hybridization, plaque hybridization, and Southern blot hybridization. The library can be a library prepared by a known method, or a commercially available cDNA library or genomic library, without limitation.

[0118] For detailed procedures of the hybridization method, reference may be made to Molecular Cloning, A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (1989) and the like as appropriate.

[0119] The so-called "stringent conditions" when performing hybridization methods refer to the conditions for washing after hybridization, such as the conditions of a buffer solution having a salt concentration of 15 to 330 mM and a temperature of 25 to 65° C., preferably a salt concentration of 15 to 150 mM and a temperature of 45 to 55° C. Specifically, for example, conditions such as 80 mM and 50° C. can be cited. In addition to such conditions such as salt concentration and temperature, various conditions such as probe concentration, probe length, and reaction time can be considered to appropriately set the conditions for obtaining the DNA of (b) above.

[0120] The DNA of (c) is a DNA having an identity (homology) of 80% or more with the DNA of (a), and more preferably a DNA having an identity of 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. This DNA is also preferred as the DNA to be hybridized in (b).

[0121] Such mutant DNAs as the DNA of (c) can be prepared, for example, by the site-directed mutagenesis method described in Molecular Cloning, A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), etc. Specifically, the mutant DNAs can be prepared by known methods such as the Kunkel method and the gapped duplex method, using a kit for introducing mutations using the site-directed mutagenesis method. As such a kit, for example, the QuickChange TM Site-directed mutagenesis kit (Stratagene), GeneTailor TM Site-directed mutagenesis system (manufactured by Invitrogen), TaKaRa site-directed mutagenesis system (Mutan-K, Mutan-Super Express Km, etc.: manufactured by TakaraBio), etc.

[0122] Alternatively, the PCR primers designed to introduce a missense mutation to the bases that represent the codons for the desired amino acids can be used, and a DNA containing a base sequence encoding wild-type MTG is used as a template, and PCR is performed under appropriate conditions to prepare the PCR primers. The DNA polymerase used in the PCR is not particularly limited, and is preferably a DNA polymerase with high fidelity, such as Pwo DNA (Roche Diagnostics), Pfu DNA polymerase (Promega), Platinum Pfx DNA polymerase (Invitrogen), KOD DNA polymerase (Toyobo), KOD-plus-polymerase (Toyobo), etc. The reaction conditions of PCR can be appropriately designed according to the optimal temperature of the DNA polymerase used, the length and type of the DNA to be synthesized, etc. For example, in the case of cycle conditions, it is preferred to use "90-98°C, 5-30 seconds (thermal denaturation·dissociation)→50-65°C, 5-30 seconds (annealing)→65-80°C, 30-1200 seconds (synthesis·extension)" as one cycle and perform a total of 20 to 200 cycles.

[0123] As the DNAs (b) and (c) mentioned above, for example, a DNA composed of a base sequence that is not completely identical to the base sequence of the DNA (a) mentioned above, but whose amino acid sequence after translation becomes completely identical (i.e., a DNA obtained by applying a silent mutation to the DNA (a) mentioned above) is particularly preferred.

[0124] Regarding the description of the "activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue at the 5' side of the sequence" of the DNA of (b) and (c), the description in the previous item can be appropriately applied.

[0125] As a gene encoding the protein involved in the present invention, the codons corresponding to each amino acid after translation are not particularly limited, and therefore it can be a gene containing DNA that displays codons commonly used in mammals such as humans (preferably codons with high usage frequency) after transcription. In addition, it can also be a gene containing DNA that displays codons commonly used in microorganisms such as Escherichia coli and yeast, plants, etc. (preferably codons with high usage frequency).

[0126] Furthermore, when the protein according to the present invention is a protein comprising the aforementioned linker sequence, the gene encoding the protein may be a gene comprising a DNA encoding the amino acid sequence of the linker sequence.

[0127] 3. Recombinant vectors and transformants

[0128] In order to express the protein involved in the present invention, first, it is necessary to integrate the gene involved in the present invention into an expression vector to construct a recombinant vector. At this time, with respect to the gene integrated into the expression vector, a transcription promoter, SD sequence (when the host is a prokaryotic cell) and a Kozak sequence (when the host is a eukaryotic cell) can be connected upstream in advance as needed, a terminator can be connected downstream, and an enhancer, a splicing signal, a poly A tailing signal (polyA addition signal), a screening marker, etc. can also be connected in advance. It should be noted that each element required for gene expression such as the above-mentioned transcription promoter can be initially contained in the gene, or the element can be utilized when originally contained in the expression vector, and the use of each element is not particularly limited.

[0129] As a method for integrating the gene into the expression vector, various methods using known gene recombination techniques such as a method using restriction endonucleases and a method using topoisomerases can be used. In addition, the expression vector is not limited as long as it is a vector such as plasmid DNA, phage DNA, retrotransposon DNA, retroviral vector, artificial chromosome DNA, etc. that can hold the gene encoding the protein of the present invention, and a vector suitable for the host cell used can be appropriately selected and used.

[0130] Next, the constructed recombinant vector is introduced into a host to obtain a transformant, which is then cultured to express the protein of the present invention. It should be noted that the "transformant" referred to in the present invention refers to a cell into which a foreign gene has been introduced into a host, including, for example, a cell into which a foreign gene has been introduced by introducing plasmid DNA (transformation) into a host, and a cell into which a foreign gene has been introduced by infecting a host with various viruses and bacteriophages (transduction).

[0131] The host is not limited as long as it is a cell capable of expressing the protein of the present invention after the above-mentioned recombinant vector is introduced, and can be appropriately selected. Examples thereof include known hosts such as human and mouse cells of various animals, various plant cells, bacteria, yeast, and plant cells.

[0132] When animal cells are used as hosts, for example, human fibroblasts, CHO cells, monkey COS-7 cells, Vero cells, mouse L cells, rat GH3, human FL cells, etc. Insect cells such as Sf9 cells and Sf21 cells can also be used.

[0133] When bacteria are used as the host, for example, Escherichia coli, Bacillus subtilis, etc. can be used.

[0134] When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc. can be used.

[0135] When plant cells are used as hosts, for example, tobacco BY-2 cells can be used.

[0136] The method for obtaining the transformant is not limited and can be appropriately selected in consideration of the combination of the host and the type of expression vector. For example, preferably mentioned are electroporation method, liposome transfection method, heat shock method, PEG method, calcium phosphate method, DEAE dextran method, and methods of infecting the transformant with various viruses such as DNA virus and RNA virus.

[0137] In the obtained transformant, the codon type of the gene contained in the recombinant vector may be identical to or different from the codon type of the host actually used, and there is no limitation.

[0138] 4. Protein production method

[0139] The production of the protein involved in the present invention can be specifically implemented by a method comprising the following steps: a step of culturing the aforementioned transformant; and a step of collecting a protein having the activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue on the 5' side of the sequence from the obtained culture. Here, the so-called "culture" refers to any of the culture supernatant, cultured cells, cultured bacteria, or the fragments of cells or bacteria. The culture of the aforementioned transformant can be carried out according to the conventional method for host culture. The target protein accumulates in the aforementioned culture. In the present invention, the aforementioned collection step may also include a protein purification step.

[0140] As the medium used for the above-mentioned culture, any of various known natural media and synthetic media can be used as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host and can culture the transformant efficiently.

[0141] During the culture, in order to prevent the recombinant vector contained in the transformant from falling off and the gene encoding the target protein from falling off, the culture may be carried out under a state of applying a screening pressure. That is, when the screening marker is a drug resistance gene, the corresponding drug may be added to the culture medium, and when the screening marker is an auxotrophic complementation gene, the corresponding nutritional factor may be removed from the culture medium.

[0142] When culturing a transformant transformed with an expression vector using an inducible promoter as a promoter, an appropriate inducer (eg, IPTG, etc.) may be added to the medium as necessary.

[0143] The culture conditions of the transformant are not particularly limited as long as they do not hinder the productivity of the target protein and the growth of the host, and are usually carried out at 10°C to 40°C, preferably at 20°C to 37°C for 5 to 100 hours. The pH can be adjusted using an inorganic acid or an organic acid, an alkaline solution, etc. Examples of the culture method include solid culture, static culture, shaking culture, and aeration stirring culture.

[0144] After cultivation, when the target protein is produced in the bacterium or in the cell, the target protein can be collected by crushing the bacterium or the cell. As a method for crushing the bacterium or the cell, high pressure treatment based on a French press or a homogenizer, ultrasonic treatment, grinding treatment based on glass beads, etc., enzyme treatment using lysozyme, cellulase or pectinase, freeze-thaw treatment, hypotonic solution treatment, lysis-induced treatment based on phage, etc. can be utilized. After crushing, the crushed residue of the bacterium or the cell (including the insoluble fraction of the cell extract) can be removed as needed. As a method for removing the residue, for example, centrifugation, filtration, etc. can be cited, and flocculants, filter aids, etc. can also be used as needed to improve the efficiency of residue removal. The supernatant obtained after removing the residue is a soluble fraction of the cell extract, which can be used as a crudely purified protein solution.

[0145] Furthermore, when the target protein is produced in bacteria or cells, the bacteria or cells themselves can be recovered by centrifugation, membrane separation, or the like and used in an undisrupted state.

[0146] On the other hand, when the target protein is produced outside the bacteria or cells, the culture solution is used directly, or the bacteria or cells are removed by centrifugation, filtration, etc. Then, the target protein can be collected from the culture by extraction based on ammonium sulfate precipitation, etc. as needed, and further separated and purified by dialysis, various chromatography methods (gel filtration, ion exchange chromatography, affinity chromatography, etc.) as needed.

[0147] The production yield of the protein obtained by culturing the transformant etc. can be confirmed by SDS-PAGE (polyacrylamide gel electrophoresis) etc., for example, per unit of culture solution, per unit of wet weight or dry weight of bacterial cells, per unit of protein in crude enzyme solution, etc.

[0148] In addition to the above-mentioned protein synthesis system using a transformant, the target protein can also be produced using a cell-free protein synthesis system that does not use any living cells.

[0149] The so-called cell-free protein synthesis system is a system that uses a cell extract to synthesize a target protein in an artificial container such as a test tube. In addition, the cell-free protein synthesis system that can be used also includes a cell-free transcription system that synthesizes RNA using DNA as a template.

[0150] At this time, the cell extract used is preferably derived from the aforementioned host cells. As the cell extract, for example, an extract derived from eukaryotic cells or prokaryotic cells can be used, and more specifically, an extract of CHO cells, rabbit reticulocytes, mouse L-cells, HeLa cells, wheat germ, budding yeast, Escherichia coli, etc. can be used. It should be noted that these cell extracts can be concentrated or diluted before use, or can be used directly, without limitation.

[0151] The cell extract can be obtained by, for example, ultrafiltration, dialysis, polyethylene glycol (PEG) precipitation, or the like.

[0152] Such cell-free protein synthesis can also be performed using commercially available kits. For example, the reagent kit PROTEIOS TM (Toyobo), TNT TM System (Promega), PG-Mate as a synthesis device TM (Toyobo), RTS (Roche Diagnostics), etc.

[0153] The target protein produced by cell-free protein synthesis can be purified by appropriately selecting a means such as chromatography as described above.

[0154] 5. Uses of protein

[0155] The present invention also includes an epigenome analysis method (epigenome mapping) using the protein of the present invention (including the protein obtained by the aforementioned production method).

[0156] Although the demand for MTase as a tool for epigenome mapping is increasing, the enzymes that can be used are limited, especially cytosine C-5MTase that recognizes short sequences. The protein involved in the present invention is a protein that can recognize and methylate a CC dinucleotide sequence that is different from the CG dinucleotide sequence that is endogenously methylated in mammalian cells (not repeated with the CG dinucleotide sequence). Therefore, the protein involved in the present invention is useful as a new probe for epigenome analysis methods (epigenome mapping).

[0157] Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these.

[0158] [Example 1]

[0159] 1. Materials and Methods

[0160] Sequence analysis

[0161] The amino acid sequence of M.CviQIX was obtained from REBASE (http: / / rebase.neb.com / rebase / rebase.html) (references 19 and 20), and the amino acid sequence of M.CviPII was obtained from reference 21.

[0162] Artificial gene synthesis and subcloning into expression vectors

[0163] The genes encoding M.CviQIX and M.CviPII were synthesized by Eurofin Genomics Inc. based on the codon optimization of E. coli. Genes that do not have BamHI and EcoRI recognition sequences within the gene but have BamHI and EcoRI recognition sequences at the 5' and 3' ends, respectively, were synthesized (sequence numbers 9 and 10 (sequence numbers 9 and 10 are sequences obtained by adding the above recognition sequences to the 5' and 3' ends of the base sequences of sequence numbers 5 and 7, respectively.)). The gene fragment was subcloned into pCZS ( Figure 1 ) BamHI-EcoRI site. The N-terminal deletion mutant genes of M.CviQIX and M.CviPII were prepared by circularizing the expression vectors containing the respective full-length forms by seamless cloning (Takara Bio) after inverse PCR.

[0164] Small-scale induction of MTase and extraction of bacterial genomic DNA

[0165] The bacterial expression vector pCZS containing genes encoding M.CviQIX and M.CviPII was introduced into T7Express (New England Biolabs, Ipswich, MA). The cells were inoculated into 3 mL of LB medium (1% [w / v] tryptone, 0.5% [w / v] yeast extract, and 0.5% [w / v] sodium chloride) (100 μg / mL of carbenicillin (Nacalai Tesque, Kyoto, Japan)), and allowed to proliferate overnight at 37°C while vigorously shaking and mixing. 50 μL of each cell suspension was diluted with 3 mL of LB medium supplemented with carbenicillin, and the cells were cultured at 37°C for 3 hours while shaking. The cell suspension was cooled in ice water for 30 minutes, and 3 μL of 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG, Nacalai Tesque) was added to induce protein expression. Cells were collected by centrifugation and used for genomic DNA purification based on DNeasy Blood & Tissue Kit (Qiagen). Genomic DNA concentration was measured using Qubit dsDNA BR Assay Kit and Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).

[0166] Analysis of DNA methylation based on restriction enzyme digestion

[0167] Genomic DNA (100 ng) was added to a 10 μL reaction containing 1×CutSmart buffer and restriction enzymes HaeIII or HpaII. The reaction was incubated at 37°C for 1 hour and the enzyme was heat-inactivated at 70°C for 10 minutes. After treatment with any of the restriction enzymes mentioned above, DNA analysis was performed using the E-Gel Power SNAP system (manufactured by Thermo Fisher Scientific) on a 2% E-Gel Ex gel.

[0168] Purification of recombinant MTase

[0169] The bacterial cells transformed with pCZS expressing the gene encoding MTase were inoculated into 30 mL of LB medium containing 100 μg / mL of carbenicillin and cultured at 37°C with vigorous shaking overnight. For seed culture, transfer to a 2L conical flask with a baffle containing 1L of LB medium supplemented with carbenicillin, and further cultured at 37°C with shaking for 4 hours. The flask was cooled in ice water for 30 minutes, and then 230 mg of IPTG was added to induce protein expression. The flask was incubated while being vigorously mixed and shaken at 16°C overnight. The cells were collected by centrifugation at 2500×g for 15 minutes, and the collected cells were frozen at -80°C until use.

[0170] Bacterial cells were resuspended in 20 mL of HisTrap buffer A (20 mM sodium phosphate, pH 7.5, 800 mM NaCl, 20 mM imidazole, 1 mM DTT, and 10% glycerol) containing a protease inhibitor cocktail (Nacalai Tesque) and disrupted by sonication. Lysates were removed by centrifugation at 15,000 × g for 15 minutes and filtered using a 0.45 μm syringe filter. The first affinity purification was performed using a 5 mL HisTrap HP column (Cytiva, Marlborough, MA) using HisTrap buffer A and HisTrap buffer B (20 mM sodium phosphate, pH 7.5, 800 mM NaCl, 200 mM imidazole, 1 mM DTT, 10% glycerol). The fractions containing the target protein were further purified using StrepTrap buffer A (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) and StrepTrap buffer B (00 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, and 2.5 mM d-dethiobiotin) using 1 mL StrepTrap HP (Cytiva). Chromatographic purification was performed using an AKTA Start System (Cytiva). The affinity purified protein was concentrated by ultrafiltration using an Amicon Ultra-4 30K device (Millipore), and bovine serum albumin (BSA) and glycerol were added to obtain final concentrations of 100 μg / mL and 50% (v / v), respectively, and then stored at -20°C until use.

[0171] Budding yeast NOMe-Seq

[0172] S288C cells (National Institute of Technology and Evaluation, Biological Resource Center of NBRC1136) were cultured in 50 ml of YPD medium (1% yeast extract, 2% bacterial peptone, 2% glucose (dextrose)) and shake-cultured at 30°C overnight. The cell suspension was diluted with 1 L of YPD and further shake-cultured at 30°C for 4 hours. The cells were collected by centrifugation at 2000×g for 15 minutes, washed twice with 50 mL of water, and resuspended in 20 mL of 1 M sorbitol. 20 μL of β-mercaptoethanol and 20 mg of Zymolyase 100T (Seikagaku Kogyo Co., Ltd., Japan) were added to the cell suspension, and incubated at room temperature for 15 minutes to digest and remove the cell wall to obtain spheroplasts. Next, the spheroplasts were washed twice with 20 mL of 1 M sorbitol and resuspended in 20 mL of 1 M sorbitol. 240 μL of 38% formaldehyde (Wako Chemical, Osaka, Japan) was added to the resuspended spheroplasts and incubated at room temperature for 5 minutes to fix. The fixed spheroplasts were washed twice with 20 mL of 1 M sorbitol and centrifuged at 5000 × g for 5 minutes to form a pellet. Next, the spheroplasts were resuspended in 10 mL of nuclear preparation solution (10 mM HEPES-KOH, pH 7.5, 10 mM NaCl, 3 mM MgCl2, and 0.5% [v / v] Nonidet P 40 substitute (Nonidet P-40substitue)) and incubated at room temperature for 5 minutes. The nuclei were collected by centrifugation at 5000×g for 5 minutes and washed twice with 10 mL of nuclear washing solution (10 mM HEPES-KOH, pH 7.5, 10 mM NaCl, 3 mM MgCl2). The nuclei were resuspended in 10 mL of nuclear washing solution, and 500 μL of the solution was dispensed into 20 tubes and stored at -80°C until use.

[0173] The frozen nuclear suspension was thawed and resuspended in 500 μL of 1× methylation buffer (50 mM Tris-HCl, pH 8.5, 50 mM NaCl, 1 mM DTT, 1.6 mM SAM). Next, an appropriate amount of the purified protein with 15 amino acid residues deleted from the N-terminus of M.CviQIX (called CCMT, refer to the results section below) was added. After incubation at 37°C for 1 hour, the nuclei were centrifuged at 5000×g for 1 minute to separate the precipitate, and resuspended in 40 μL of 10 mM Tris-HCl, pH 8.0. 20 mg / ml proteinase K and 5 μL of 10% (w / v) SDS were added to the nuclear suspension and incubated at 50°C for 1 hour. Next, 150 μL of 1M TrisHCl, pH 8.0 was added to the reaction, multilayered in white mineral oil, and incubated at 80°C for 24 hours. Transfer the aqueous phase to a new test tube and mix with Buffer AL and 200 μL of isopropanol. Load the mixture onto a DNeasy column and centrifuge at 10,000 × g for 1 minute, then wash with 500 μL of Buffer W1 and Buffer W2, respectively. Finally, elute the purified DNA with 200 μL of Buffer AE and use the tPBAT protocol for WGBS analysis. Buffer AL, Buffer W1, Buffer W2, and Buffer AE are reagents included in the DNeasy Blood & Tissue Kit.

[0174] NOMe-Seq in mammalian cells

[0175] IMR-90 cells (JCRB cell bank #JCRB9054) were cultured at 37°C in an atmosphere of 100% humidity and 5% CO2 using four T75 flasks containing 30 mL of minimum essential medium (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 50 U / mL of penicillin-streptomycin (Thermo Fisher Scientific). The proliferated cells were treated with trypsin and collected by centrifugation at 2000×g for 5 minutes. The cells were washed twice with 20 mL of D-PBS (Nacalai Tesque) and centrifuged at 1×10 7 The cells were resuspended in CELLBANKER 1 (Takara Bio) at 100 cells / ml. The cell suspension was aliquoted into 500 μL portions and stored at -80°C until use.

[0176] Thaw the frozen cell suspension before use and wash twice with PBS. Then, resuspend the cells in nuclear preparation buffer (10mM Tris-HCl, pH 8.0, 10mM NaCl, 3mM MgCl2, 0.1mM EDTA), and 0.5% (w / v) NP-40, and incubate on ice for 10 minutes. Then, collect the nuclei by centrifugation at 2000×g for 5 minutes, resuspend the optimal amount of CCMT in 500μL of 1× methylation buffer, and incubate at 37°C for 30 minutes. Then, centrifuge at 2000×g for 5 minutes to collect the nuclei and remove the supernatant. Dissolve the nuclei in 20μL of 20mg / ml proteinase K and add 200μL of buffer ATL. After incubating the reaction solution at 56°C for 1 hour, add 200μL of buffer AL and 200μL of isopropanol. The mixture was loaded onto a DNeasy column and washed with 500 μL of buffer W1 or buffer W2. The purified DNA was eluted with 200 μL of buffer AE for WGBS analysis.

[0177] 2. Results

[0178] N-terminal deletion enhances the MTase activity of M.CviQIX and M.CviPII

[0179] The inventors of the present application attempted to produce an active protein of M.CviQIX, which is recorded in the database as having MTase activity for the first C of CCD trinucleotide. However, in most bacterial cells with expression vectors in which the gene was subcloned, no DNA methylation activity was detected ( Figure 3 A~ Figure 3 C). However, in cells expressing M.CviQIX subcloned with expression vectors pCTF and pCNS, strong MTase activity was confirmed. That is, the genomic DNA of cells with these two expression vectors showed resistance to both HaeIII digestion and HpaII digestion ( Figure 3 C, and Figure 7 A. Figure 7 B). pCTF and pCNS were designed to produce proteins in which a trigger factor (TF) and SUMO were fused to the N-terminus of M.CviQIX ( Figure 3 A). TF and SUMO can be expected to have effects on protein solubilization and folding (Reference 22). In addition, genomic DNA extracted from bacterial cells with expression vectors tagged with SUMO at the N-terminus showed stronger properties to treatment with methylation-sensitive restriction endonucleases than genomic DNA extracted from bacterial cells with expression vectors tagged with SUMO at the C-terminus ( Figure 3C). Therefore, the purification of the products of these N-terminal TF and SUMO tag constructs was attempted. However, after affinity purification using an N-terminal His6 tag, the two proteins showed almost no MTase activity. These results reveal that there may be certain factors at the N-terminus that are different from the solubility improvement and folding of the protein, which regulate the DNA methylation activity of M.CviQIX. Therefore, the hypothesis that the N-terminal sequence may be the cause of the low methylation activity of purified M.CviQIX was established. Therefore, it was decided to perform genomic DNA analysis of bacterial cells with an expression vector containing an N-terminal deletion protein of M.CviQIX. There are three methionine residues at the N-terminus of M.CviQIX. Therefore, it is believed that any of these three residues may be used for translation initiation in the cells of the organism that originally possessed the gene. Therefore, three mutants were made that were missing until the amino acid residue of the residue before each methionine, and the methylation status of the host genome was analyzed. As a result, it was found that the deletion of 11, 13, or 15 amino acids at the N-terminus of M.CviQIX enhanced the methylation of the host genome ( Figure 3 D. Figure 3 E, and Figure 7 That is, any of the three methionine residues can be used as a start codon for producing active M.CviQIX. In addition, the same results were obtained for M.CviPII ( Figure 7 F. Figure 7 That is, it is shown that the N-terminal amino acid sequences of M.CviQIX and M.CviPII show an inhibitory effect on DNA methylation activity.

[0180] Determination of recognition sequences of M.CviQIX and M.CviPII based on WGBS

[0181] The inventors of the present application induced the expression of the N-terminal 15 amino acid deletion (NDEL15) mutants of M.CviQIX and M.CviPII in E. coli cells, extracted bacterial genomic DNA, and performed whole genome bisulfite sequencing analysis (WGBS) ( Figure 4 The methylation level of each cytosine residue was measured, and the frequency of occurrence of the surrounding base sequences that contributed to the methylation level was indicated by a logo. The results showed that M.CviQIX and M.CviPII introduced methylation to the first C in the CC dinucleotide.

[0182] Purification of MTase and confirmation of its activity in vitro

[0183] Next, the inventors of the present application attempted to purify active proteins using the protein genes of M.CviQIX and M.CviPII with 15 amino acid residues deleted at the N-terminus. At this time, as a control, the purification of the active protein of M.CviPI, whose purification conditions had been established, was attempted at the same time. Bacterial cells with expression vectors cloned with the genes were cultured to induce protein expression, and then these MTases were purified using a double affinity purification method using HisTag and StrepII-tag ( Figure 2 ). The protein was stored in the presence of 100 μg / ml BSA, 10 mM DTT, and 50% glycerol, and the enzyme activity was preserved during storage at -20°C. The purified protein was found to be highly homogeneous in SDS-PAGE. The restriction endonucleases and WGBS were tested using λ phage DNA as a substrate. As a result, these MTases caused DNA cleavage inhibition, thus confirming that they showed methylation activity ( Figure 8 B).

[0184] NOMe-Seq with CC-recognizing MTase generates the same methylation pattern as GCMT

[0185] The recognition sequence of CC dinucleotide is short, and it can achieve fine epigenome mapping with a resolution of about 10bp. Therefore, the inventors of the present application tried to establish MTase that recognizes CC dinucleotide, namely M.CviQIX or M.CviPII, as a probe for epigenome mapping. The combinations of expression vectors and genes were compared, and as a result, M.CviQIX expressed under the regulation of a cold shock promoter showed the highest yield and activity, so the protein was named CCMT. The salt concentration in the methylation reaction of CCMT was studied, and it was found that 50mM sodium chloride is the optimal condition for strict DNA methylation activity ( Fig. 9 ).

[0186] Next, CCMT was used in NOMe-Seq analysis of formaldehyde-crosslinked budding yeast nuclei. Formalin-fixed yeast spheroplasts were permeabilized in a buffer containing NP-40 to remove the cell membrane, and then a CCMT-based methylation reaction was applied in the presence of S-adenosylmethionine to perform a methylation reaction on DNA on chromatin. After genomic DNA was purified, the methylation status of the DNA was analyzed using WGBS.

[0187] The methylation level of 10-bp bins was calculated, and the methylation status of the DNA of nuclei treated with the commercially available M.CviPI (GCMT) that recognizes GC dinucleotides and nuclei treated with CCMT were compared. As a result, the two data sets showed the same distribution pattern of 5mC on the genome. It should be noted that there is no endogenous DNA methylation in budding yeast, so the detected D5mC can be interpreted as all introduced by GCMT or CCMT. Figure 5 A. Figure 5 As shown in Figure B, the same trend is also observed in the genomic DNA methylation pattern. In the aggregation diagram of CCMT signals and GCMT signals, several similar genomic features are centered ( Figure 5 C. Figure 5 D, and Fig.10 A). A sharp DNA methylation peak was observed in the promoter region of the gene ( Figure 5 B). These strong DNA methylation signals colocalize well with the signals detected using ATAC-Seq. Therefore, it is believed that these signals represent open chromatin regions. The peaks detected by NOMe-Seq and ATAC-Seq based on CCMT and GCMT colocalize well ( Fig.10 B).

[0188] DNA methylation levels in CC and GC dinucleotides are well concentrated in the promoter proximal region ( Figure 5 C. Figure 5 D). Undoubtedly, these signals are nucleosome-free regions that are frequently observed in gene promoters as regions where nucleosomes do not bind or bind weakly. The promoter-proximal DNA methylation signals in the region from -100 to -250 of the transcription start site are negatively correlated with the low signal region detected by MNase-Seq ( Figure 5 E). The DNA methylation signal observed at the transcription start site showed a periodicity of 160 to 170 bp and was negatively correlated with the signal observed by MNase-Seq ( Figure 5 B. Figure 5 C~ Figure 5 These observations recapitulate the properties reported for NOMe-Seq and MNase-seq (ref. 23).

[0189] CCMT-based NOMe-Seq achieves fine mapping of the mammalian epigenome

[0190] Next, NOMe-Seq based on CCMT was applied to IMR-90 cells, which are cultured cell lines derived from humans. After permeabilization of the cell membrane by treatment in a buffer containing NP-40, CCMT treatment was performed, and genomic DNA was purified and applied to WGBS analysis. In addition, samples without CCMT treatment were prepared as controls. High-quality methylation group data with 25.7-fold and 24.5-fold coverage were obtained using samples with and without CCMT treatment, respectively (Table 1).

[0191] [Table 1]

[0192] Basic statistics of methylome data from NOMe-Seq analysis of IMR90 cells

[0193]

[0194] As expected, the intrinsic DNA methylation levels at the CG sites were almost the same between CCMT-treated and untreated cells (Figure 6A, Figure 6B). On the other hand, the DNA methylation levels at the CC sites were increased only in CCMT-treated nuclei ( Figure 6 C. Figure 6 D). These results show that endogenous CG methylation at CC dinucleotides can be clearly separated from CCMT-induced methylation.

[0195] Next, use a genome browser to visually compare the two datasets. Figure 6 As shown in E, several large domains with different intrinsic DNA methylation levels were observed in the two datasets. There is no doubt that these domains are highly methylated regions and moderately methylated domains, which have been documented to exist in IMR-90 cells (reference 24). It was shown that for the comparison between NOMe-Seq based on CCMT and the untreated control, it was observed that the endogenous methylation status of mammalian cells for CG dinucleotides in these two datasets was almost the same. On the other hand, a strong and sharp methylation peak of CC methylation was observed in the samples treated with CCMT, while this methylation peak was not observed in the untreated control ( Figure 6 E). In addition, these peaks are largely co-localized with the peaks identified using ATAC-Seq. Therefore, the peaks observed in the CCMT-induced methylation signal indicate highly accessible chromatin regions ( Figure 6 These results strongly support the ability of CCMT-based NOMe-Seq to simultaneously measure both the intrinsic methylome and chromatin accessibility of mammalian cells.

[0196] 3. Investigation

[0197] Although the demand for MTase as a tool for epigenome mapping is increasing, the enzymes that can be used are limited, especially the cytosine C-5MTase with short recognition sequences is restricted. In the present embodiment, it is clear that the methylation activity of M.CviQIX and M.CviPII is inhibited by the amino acid sequence of several residues on its N-terminal side, and it is revealed that its N-terminal deletion strain has high DNA methylation activity instead. These artificial mutant proteins can be purified, and the activity is also maintained in vitro. In addition, it is also clear that as cytosine C-5MTase, CC dinucleotide as a novel recognition sequence is the target.

[0198] This CC dinucleotide never overlaps with the CG dinucleotide, which is the recognition sequence of mammalian endogenous MTase, and is therefore considered to have a great advantage as a probe for simultaneously detecting mammalian endogenous MTase and additional epigenetic mapping of any epigenomic state. This was confirmed by the implementation of NOMe-Seq in this example.

[0199] The following are the references cited in this specification.

[0200] 1.Greenberg,MVC and Bourc'his,D.(2019)The diverse roles of DNAmethylation in mammalian development and disease.Nature Reviews MolecularCell Biology,20,590-607.

[0201] 2.Edwards,JR,Yarychkivska,O.,Boulard,M.and Bestor,TH(2017)DNAmethylation and DNA methyltransferases.Epigenetics&Chromatin,10,23.

[0202] 3. Jones, PA (2012) Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nature Reviews Genetics, 13, 484-492.

[0203] 4.Petryk,N.,Bultmann,S.,Bartke,T.and Defossez,P.-A.(2021)Staying trueto yourself:mechanisms of DNA methylation maintenance in mammals.NucleicAcids Res.,49,3020-3032.

[0204] 5.Jones,P.A.and Liang,G.(2009)Rethinking how DNA methylation patternsare maintained.Nat Rev Genet,10,805-811.

[0205] 6.Edwards,J.R.,Yarychkivska,O.,Boulard,M.and Bestor,T.H.(2017)DNAmethylation and DNA methyltransferases.Epigenetics Chromatin,10,23.

[0206] 7.Tajima,S.,Suetake,I.,Takeshita,K.,Nakagawa,A.and Kimura,H.(2016)Domain Structure of the Dnmt1,Dnmt3a,and Dnmt3b DNA Methyltransferases.Adv.Exp.Med.Biol.,945,63-86.

[0207] 8.Jang,H.S.,Shin,W.J.,Lee,J.E.and Do,J.T.(2017)CpG and Non-CpGMethylation in Epigenetic Gene Regulation and Brain Function.Genes,8,148.

[0208] 9.Patil,V.,Ward,R.L.and Hesson,L.B.(2014)The evidence for functionalnon-CpG methylation in mammalian cells.Epigenetics,9,823-828.

[0209] 10.van de Lagemaat,L.N.,Flenley,M.,Lynch,M.D.,Garrick,D.,Tomlinson,S.R.,Kranc,K.R.and Vernimmen,D.(2018)CpG binding protein(CFP1)occupies openchromatin regions of active genes,including enhancers and non-CpGislands.Epigenetics&Chromatin,11,59.

[0210] 11.Teissandier,A.and Bourc'his,D.(2017)Gene body DNA methylationconspires with H3K36me3 to preclude aberrant transcription.The EMBO Journal,36,1471-1473.

[0211] 12.Dhayalan,A.,Rajavelu,A.,Rathert,P.,Tamas,R.,Jurkowska,R.Z.,Ragozin,S.and Jeltsch,A.(2010)The Dnmt3a PWWP domain reads histone 3 lysine36 trimethylation and guides DNA methylation.The Journal of biologicalchemistry,285,26114-26120.

[0212] 13.Solomon,M.J.,Larsen,P.L.and Varshavsky,A.(1988)Mapping protein-DNAinteractions in vivo with formaldehyde:evidence that histone H4 is retainedon a highly transcribed gene.Cell,53,937-947.

[0213] 14.Johnson,D.S.,Mortazavi,A.,Myers,R.M.and Wold,B.(2007)Genome-widemapping of in vivo protein-DNA interactions.Science,316,1497-1502.

[0214] 15.Kelly,T.K.,Liu,Y.,Lay,F.D.,Liang,G.,Berman,B.P.and Jones,P.A.(2012)Genome-wide mapping of nucleosome positioning and DNA methylationwithin individual DNA molecules.Genome Res.,22,2497-2506.

[0215] 16.Lee,I.,Razaghi,R.,Gilpatrick,T.,Molnar,M.,Gershman,A.,Sadowski,N.,Sedlazeck,F.J.,Hansen,K.D.,Simpson,J.T.and Timp,W.(2020)Simultaneousprofiling of chromatin accessibility and methylation on human cell lines withnanopore sequencing.Nat Methods,17,1191-1199.

[0216] 17.Stergachis,A.B.,Debo,B.M.,Haugen,E.,Churchman,L.S.andStamatoyannopoulos,J.A.(2020)Single-molecule regulatory architecturescaptured by chromatin fiber sequencing.Science,368,1449-1454.

[0217] 18.Steensel,B.v.and Henikoff,S.(2000)Identification of in vivo DNAtargets of chromatin proteins using tethered Dam methyltransferase.Nat.Biotechnol.,18,424-428.

[0218] 19.Roberts,R.J.and Macelis,D.(1997)REBASE-restriction enzymes andmethylases.Nucleic Acids Res.,25,248-262.

[0219] 20.Roberts,R.J.,Vincze,T.,Posfai,J.and Macelis,D.(2015)REBASE--adatabase for DNA restriction and modification:enzymes,genes andgenomes.Nucleic Acids Res.,43,D298-D299.

[0220] 21.Xu,M.,Kladde,M.P.,Van Etten,J.L.and Simpson,R.T.(1998)Cloning,characterization and expression of the gene coding for a cytosine-5-DNAmethyltransferase recognizing GpC.Nucleic Acids Res.,26,3961-3966.

[0221] 22.Malakhov,M.P.,Mattern,M.R.,Malakhova,O.A.,Drinker,M.,Weeks,S.D.andButt,T.R.(2004)SUMO fusions and SUMO-specific protease for efficientexpression and purification of proteins.Journal of Structural and FunctionalGenomics,5,75-86.

[0222] 23.Klemm,SL,Shipony,Z.and Greenleaf,WJ(2019)Chromatin accessibility and the regulatory epigenome.Nature Reviews Genetics,20,207-220.

[0223] 24.Lister,R.,Pelizzola,M.,Dowen,RH,Hawkins,RD,Hon,G.,Tonti-Philippines,J.,Nery,JR,Lee,L.,Ye,Z.,Ngo,QMet al.(2009)Human DNA methylomesat base resolution show widespread epigenomic differences.Nature,462,315-322.

[0224] 25.Braberg, H., Echeverria, I., Bohn, S., Cimermancic, P., Shiver, A., Alexander, R., Xu, J., Shales, M., Dronamraju, R., Jiang, S. et al proteincomplexes.Science,370.

[0225] http: / / dx.doi.org / 10.1037 / 0033-295X.112.1.27 Miura, F., Fujino, T., Kogashi, K., Shibata, Y., Miura, M., Isobe, H. and Ito, T. (2018)Triazole linking for preparation of a next-generation sequencinglibrary from single-stranded DNA.Nucleic Acids Res.

[0226] One of the most expensive characters

[0227] According to the present invention, a protein (MTase) and a gene encoding the same can be provided, which can introduce methylation that can be distinguished from DNA methylation (endogenous methylation) inherent in mammalian cells (more specifically, can specifically recognize a "CC dinucleotide sequence" and 5-methylate the cytosine residue on the 5' side of the sequence). The protein (MTase) of the present invention is useful in, for example, implementing an epigenome analysis method (epigenome mapping) that can theoretically simultaneously analyze endogenous methylation and epigenomes other than endogenous methylation.

[0228] Sequence Listing Free Text

[0229] SEQ ID NO: 5: Recombinant DNA

[0230] SEQ ID NO. 6: Recombinant protein

[0231] SEQ ID NO. 7: Recombinant DNA

[0232] SEQ ID NO. 8: Recombinant protein

[0233] SEQ ID NO. 9: Synthetic DNA

[0234] SEQ ID NO. 10: Synthetic DNA

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Claims

1. Proteins of (a), (b) or (c) below: (a) A portion of amino acid residues including the N-terminus of the DNA methyltransferase of M.CviQIX or M.CviPII is deleted to obtain a protein having the activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue on the 5' side of the sequence; (b) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence of the protein of (a) above, and having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue on the 5' side of the sequence; (c) A protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence of the protein of (a) above, and having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue at the 5' side of the sequence.

2. Proteins of (a), (b) or (c) below: (a) a protein having the amino acid sequence shown in SEQ ID NO: 6 or 8; (b) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence of the protein of (a) above, and having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue on the 5' side of the sequence; (c) A protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence of the protein of (a) above, and having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue at the 5' side of the sequence.

3. A gene encoding the protein according to claim 1 or 2.

4. A gene comprising DNA of (a), (b) or (c) below: (a) a DNA comprising the base sequence shown in SEQ ID NO: 5 or 7; (b) a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA of (a) above, and the DNA encodes a protein having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating a cytosine residue on the 5' side of the sequence; (c) A DNA having 80% or more identity with the DNA of (a) above, and encoding a protein having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating the cytosine residue at the 5' side of the sequence. A recombinant vector comprising the gene according to claim 3 or 4. A transformant comprising the recombinant vector according to claim 5.

7. A method for producing a protein having an activity of specifically recognizing a CC dinucleotide sequence and 5-methylating a cytosine residue on the 5' side of the sequence, The manufacturing method comprises: A step of culturing the transformant according to claim 6; and The step of collecting the protein from the obtained culture.

8. An epigenome analysis method, comprising using the protein according to claim 1 or 2, or the protein produced by the method according to claim 7.

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