WO2017043593A1 - Sugar-sensing epigenome biomarker - Google Patents

Abstract

An antibody of the present invention specifically bonds to a serine 40 residue modified with N-acetylglucosamine (GlcNAc) in a histone H2A protein. The present invention provides a method for detecting GlcNAc-modification of a serine 40 residue in a histone H2A protein, said method having a step for measuring the level of GlcNAc-modification of the serine 40 residue in the histone H2A protein using the antibody. A protein or a peptide marker of the present invention is a marker for monitoring histone modification caused by fluctuations in the concentration of extracellular glucose, said marker comprising an H2A protein, or a peptide fragment thereof, that contains a GlcNAc-modified serine 40 residue.

Description

Sugar-sensing epigenome biomarker

The present invention relates to an antibody that specifically binds to N-acetylglucosamine (GlcNAc) serine 40 residue of histone H2A protein, a method for detecting GlcNAcation of serine 40 residue of histone H2A protein using the antibody, histone A method for screening an inhibitor or activator of GlcNAcylation of the serine 40 residue of H2A protein, a method for detecting a change in GlcNAcation of histone protein that occurs depending on the presence of glucose, a method for evaluating the stability of genomic DNA, And protein markers.
This application claims priority based on Japanese Patent Application No. 2015-177176 filed in Japan on September 9, 2015, the contents of which are incorporated herein by reference.

Chromatin structure is known to be controlled by various modifications in histone proteins to regulate genomic function. In such chemical modifications, post-translational modifications of histone proteins have become important determinants for chromatin reorganization, and specific combinations of reversible histone modifications have an effect on chromatin activity. It is thought to be exerting. This specific combination occurs epigenetically and, despite being stable, some of these are temporarily reconstituted in response to extracellular conditions and cellular activity. Epigenetics systems are responsible for long-term genome control, and epigenetic modification is important as a role in cell genome control and as a basis for development and cell differentiation, and its disruption can cause disease.
Currently, there are many known epigenetic modifications. Among them, histone protein acetylation, methylation, and ubiquitination at specific residues at the histone end are related to gene expression control and genome stability. It has been known.

Recently, N-acetylglucosamine (GlcNAc) conversion as a modification of histone protein as described above has been found to be a new epigenetic phenomenon carved into histone proteins. GlcNAcation is a reversible phenomenon, and the modification reaction is catalyzed by two enzymes, O-linked N-acetylglucosaminyltransferase (OGT) and O-linked N-acetylglucosaminase (OGA). N-acetylglucosamine is obtained from UDP-GlcNAc produced in the cytosol by the hexosamine biosynthetic pathway (HBP). Since the initial source of UDP-GlcNAc is extracellular glucose, the degree of GlcNAcation is thought to vary with extracellular glucose concentration and intracellular HBP activity (see, for example, Non-Patent Document 1). In addition, abnormal blood glucose levels may cause long-term changes in genomic functions of cells and cause various diseases (see, for example, Non-Patent Document 2).

It is known that the amino acid in the histone protein to be GlcNAc is serine or threonine. Among the histone proteins, currently known GlcNAc sites are the 36 th serine, 52 th threonine, 55 th serine, 56 th serine, 64 th position from the N-terminal of the amino acid sequence of histone H2B. Serine, 91st serine, 112th serine and 123th serine, histone H2A amino acid sequence 101st threonine from N-terminal, histone H3 or H3.3 amino acid sequence 10th serine from N-terminal , Threonine at position 32, threonine at position 80, and the 47th serine from the N-terminal among the amino acid sequences of histone H4 (see, for example, Non-Patent Documents 3 and 4).

As described above, GlcNAcation is thought to vary with extracellular glucose concentration, but GlcNAc is low molecular weight and neutral charge, so it is difficult to detect and histone modification caused by fluctuations in extracellular glucose concentration The technology for monitoring has not been established yet (see, for example, Non-Patent Document 5).

The present invention has been made in view of the above circumstances, and provides a method for monitoring histone modification exerted by fluctuations in extracellular glucose concentration.

As a result of intensive studies to achieve the above object, the present inventors have found that, among histone proteins, serine 40 residue of histone H2A protein is converted to GlcNAc in the nucleus, thereby completing the present invention. It came.

The present invention includes the following aspects.
[1] An antibody that specifically binds to N-acetylglucosamine (GlcNAc) serine 40 residue of histone H2A protein.
[2] Detecting GlcNAcation of serine 40 residue of histone H2A protein, comprising measuring the level of GlcNAcation of serine 40 residue of histone H2A protein using the antibody according to [1] Method.
[3] A method for screening an inhibitor or activator of GlcNAcation of serine 40 residue of histone H2A protein, wherein serine 40 residue of histone H2A protein or peptide fragment thereof is present in the presence and absence of a test substance. A step of measuring the GlcNAcation level of the group, and when the GlcNAcation level in the presence of the test substance is lower than the GlcNAcation level in the absence of the test substance, the test substance is Determined to be an inhibitor of GlcNAcylation of serine 40 residue of histone H2A protein, and the GlcNAc level in the presence of the test substance was higher than the phosphorylation level in the absence of the test substance In some cases, the test substance is an activity of GlcNAcization of serine 40 residue of histone H2A protein. Screening method characterized by and a step of determining that the agent.
[4] The screening method according to [3], wherein the measurement step is performed in the presence of glucose.
[5] The screening method according to [3] or [4], wherein the measurement step is performed under conditions in which DNA is damaged.
[6] The level of GlcNAcation of the serine 40 residue of the histone H2A protein or peptide fragment thereof is measured by an antibody that specifically binds to the GlcNAcated serine 40 residue of the histone H2A protein [3] to [5 ] The screening method as described in any one of.
[7] A method for monitoring histone modification exerted by fluctuations in extracellular glucose concentration, comprising the step of measuring the level of GlcNAcation of serine 40 residue of histone H2A protein that occurs depending on the presence of glucose. how to.
[8] The detection method according to [7], wherein the GlcNAc level of the serine 40 residue of the histone H2A protein is measured by an antibody that specifically binds to the GlcNAc-converted serine 40 residue of the histone H2A protein.
[9] A protein or peptide marker, which is a marker for monitoring histone modification exerted by fluctuations in extracellular glucose concentration, comprising an H2A protein or a peptide fragment thereof containing 40 GlcNAcylated serine residues.
[10] A method for evaluating the stability of genomic DNA by epigenetic modification, comprising the step of measuring the level of GlcNAcization of a 40-serine residue of histone H2A protein or a peptide fragment thereof.
[11] The level of GlcNAcation of the serine 40 residue of the histone H2A protein or peptide fragment thereof is measured by an antibody that specifically binds to the GlcNAcated serine 40 residue of the histone H2A protein. Method.
[12] A protein or peptide marker for detecting the stability of genomic DNA by epigenetic modification, comprising a H2A protein or a peptide fragment thereof containing a GlcNAcylated serine 40 residue.

According to the present invention, it is possible to monitor histone modification exerted by fluctuations in extracellular glucose concentration.

FIG. 4 is a view showing differences in amino acid sequences of the 30th to 50th amino acids in histone H2A protein depending on species. It is a figure which shows the difference by the seed | species of the modification of histone protein. It is an image which shows the detection result by the Western blotting method of the graph showing the peak transition of HPLC in Example 1, and each fraction obtained by HPLC. Recombinant proteins of two types of wild-type Flag-H2A1A and Flag-H2A3 in Example 1 and mutant Flag-H2A3-S40A (mutant in which the 40th serine in the amino acid sequence of histone H2A3 protein is changed to alanine) It is an image which shows the detection result by the western blotting method using. 2 is an image showing the results of immunostaining of mouse ES cells using the H2A3-S40-GlcNAc antibody in Example 2. FIG. FIG. 4 is an image showing the results of immunostaining of mouse ES cells using O-GlcNAc (RL2) antibody in Example 2. FIG. It is an image which shows the detection result by the western blotting method using the mouse | mouth ES cell and mouse | mouth TS cell which were culture | cultivated in the culture medium of different glucose concentration in Example 2. FIG. FIG. 4 is an image showing the results of immunostaining of human EAhy cells cultured in media with different glucose concentrations using the H2A3-S40-GlcNAc antibody in Example 3, and a graph measuring changes in the number of GlcNAc dots in the nucleus. FIG. 5 is an image showing the results of immunostaining of human RPTEC cells cultured in media with different glucose concentrations using the H2A3-S40-GlcNAc antibody in Example 3, and a graph measuring changes in the number of GlcNAc dots in the nucleus. FIG. 10 shows the results of ChIP-seq using H2A3-S40-GlcNAc antibody in mouse ES cells cultured in a low sugar and high sugar environment in Example 4. FIG. 6 shows the results of ChIP-seq using H2A3-S40-GlcNAc antibody in human EAhy cells cultured in a low sugar and high sugar environment in Example 5. It is an image which shows the detection result by the western blotting method about the histone variant and protein relevant to DNA damage in Example 6. 6 is a graph showing the results of detecting DNA damage (pyrimidine dimer) induced by ultraviolet irradiation in human EAhy cells that highly expressed wild-type histone protein H2A or mutant histone protein H2A-S40A in Example 7.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as necessary.

<Antibody>
In one embodiment, the present invention provides an antibody that specifically binds to a GlcNAcylated serine 40 residue of a histone H2A protein.

According to the antibody of the present embodiment, it is possible to easily and easily detect a 40-glycine Serine residue of histone H2A protein. Furthermore, by detecting the GlcNAc-modified serine 40 residue of the histone H2A protein using the antibody of this embodiment, abnormalities in epigenetics of various diseases in which metabolic abnormalities such as diabetes and obesity are risk factors are detected. be able to.

In the present specification, the “histone protein” means a core histone protein (H2A, H2B, H3, H4) and a linker histone protein (H1) constituting a nucleosome which is a basic unit of chromatin. Furthermore, in the present specification, “histone protein” includes these variants such as H2AX and H2AZ.
Originally, of the amino acid sequences of histone H2A protein, the 40th amino acid from the N-terminus was alanine (see FIG. 1A). It is known that there is a variant in which the 40th amino acid from the N-terminus is serine (see FIGS. 1A and 1B). The present inventors focused on this histone H2A protein variant, and found that the serine 40 residue of the histone H2A protein is converted to GlcNAc, thereby completing the present invention.
In the present specification and claims, the 40th amino acid from the N-terminal is referred to as “amino acid 40 residue”. For example, when the 40th amino acid from the N-terminal is serine, it is referred to as “serine 40 residue”.

Also, from FIG. 1B, histone modifications such as acetylation, methylation, and GlcNAcation are observed in various organisms, but GlcNAcation of the 40th serine of histone H2A protein is performed only in mammals and later. It is.
In addition, as shown in Examples below, the level of GlcNAcization of serine 40 residue of histone H2A protein increases or decreases in response to changes in extracellular glucose concentration via the hexosamine biosynthetic pathway (HBP). This has been clarified for the first time by the present inventors. Therefore, it is considered that GlcNAcization of serine 40 residue of this histone H2A protein is related to abnormal epigenetics of various diseases in which metabolic abnormalities such as diabetes and obesity are a risk factor in animals after mammals.

The antibody of the present embodiment can specifically bind to the 40-glycine Serine residue of histone H2A protein. The mode of binding of GlcNAc to the serine 40 residue of the histone H2A protein is an O-linked type. That is, the antibody of the present embodiment can specifically bind to the O-linked GlcNAc serine 40 residue of the histone H2A protein. As described above, GlcNAcation of serine 40 residue of this histone H2A protein was newly found, and the antibody of this embodiment is included in the range that specifically binds to this novel antigen.

In this embodiment, the type of “antibody” may be a polyclonal antibody, a monoclonal antibody, or a functional fragment of an antibody. Among these, a monoclonal antibody is preferable. “Antibodies” also includes all classes and subclasses of immunoglobulins.

As used herein, “specifically binding” means that an antibody binds only to a target protein (antigen), for example, in vitro assay, preferably plasmon resonance assay using purified wild-type antigen (For example, BIAcore, GE-Healthcare Uppsala, Sweden, etc.) can be quantified by binding of an antigen to an epitope of an antibody. The affinity of binding can be defined by ka (rate constant for antibody binding from antibody-antigen complex), kD (dissociation constant), and KD (kD / ka). When the antibody specifically binds to the antigen, the binding affinity (KD) is preferably 10 ⁻⁸ mol / L or less, more preferably 10 ⁻⁹ M to 10 ⁻¹³ mol / L. preferable.

In this embodiment, “polyclonal antibody” means an antibody preparation comprising different antibodies against different epitopes. That is, when the antibody of the present embodiment is a polyclonal antibody, it may contain a different antibody that specifically binds to an epitope comprising 40 residues of O-linked GlcNAc serine and having a different amino acid sequence.
The “monoclonal antibody” means an antibody (including an antibody fragment) obtained from a substantially homogeneous antibody population.
Also, in contrast to polyclonal antibodies, monoclonal antibodies mean those that recognize a single determinant on an antigen. That is, when the antibody of this embodiment is a monoclonal antibody, it is an antibody isolated from components of the natural environment.
In the present embodiment, the “functional fragment” of an antibody means a part (partial fragment) of an antibody that specifically recognizes a target protein. Specifically, Fab, Fab ′, F (ab ′) 2, variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc (Fv) 2, diabody, multispecific antibody, And polymers thereof.

When the antibody of the present embodiment is a polyclonal antibody, an antigen (for example, a histone H2A protein containing 40 GlcNAc serine residues, a peptide fragment containing 40 GlcNAc serine residues, or a cell expressing these) The immunized animal can be immunized and purified from the antiserum by conventional means (eg, salting out, centrifugation, dialysis, column chromatography, etc.). Moreover, when the antibody of this embodiment is a monoclonal antibody, it can be produced by a hybridoma method or a recombinant DNA method.

Examples of the hybridoma method include Kohler and Milstein methods (see, for example, Kohler & Milstein, Nature, 256: 495 (1975)). Examples of antibody-producing cells used in the cell fusion step in this method include antigens (for example, histone H2A protein containing 40 GlcNAc-modified serine residues, peptide fragments containing 40 GlcNAc-ized serine residues, or these Spleen cells, lymph node cells, peripheral blood leukocytes, etc. of animals (eg, mice, rats, hamsters, rabbits, monkeys, goats, etc.) immunized with such cells. In addition, antibody-producing cells obtained by allowing an antigen to act in a medium on the cells or lymphocytes previously isolated from an unimmunized animal can also be used. Various known cell lines can be used as the myeloma cells. The antibody-producing cells and myeloma cells may be derived from different animal species as long as they can be fused, but are preferably derived from the same animal species. As a method for obtaining a hybridoma, for example, a spleen cell obtained from a mouse immunized with an antigen and a mouse myeloma cell are produced, and a monoclonal antibody specific to the target protein is obtained by subsequent screening. The method of obtaining the hybridoma to produce is mentioned. Examples of a method for obtaining a monoclonal antibody produced by a hybridoma include a method in which a monoclonal antibody against a target protein is obtained by culturing the hybridoma or from ascites of a mammal to which the hybridoma has been administered.

As a recombinant DNA method, for example, a DNA encoding the antibody or the functional fragment of the antibody of the present embodiment is cloned from a hybridoma, a B cell or the like, and incorporated into an appropriate vector, which is then used as a host cell (for example, a mammalian cell line). , Escherichia coli, yeast cells, insect cells, plant cells, etc.) and producing the antibody of this embodiment as a recombinant antibody (for example, PJ Delves, Antibody Production: Essential Techniques, 1997). WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS, Vandame AM et al., Eur. J. Biochem. 192: 767-775. 1990)).
In the expression of the DNA encoding the antibody of the present embodiment, DNA encoding heavy chain or light chain may be separately incorporated into an expression vector to transform a host cell. DNA encoding heavy chain and light chain May be incorporated into a single expression vector to transform host cells (see, eg, International Patent Application No. 94/11523). The antibody of the present embodiment can be obtained in a substantially pure and uniform form by culturing the above host cell, separating and purifying it from the host cell or culture medium. Separation and purification of the antibody can be performed by a method used in usual polypeptide purification. In the method using the transgenic animal production technique, for example, a transgenic animal (for example, cow, goat, sheep or pig etc.) in which the antibody gene is incorporated is produced, and the antibody gene is derived from the milk of the transgenic animal. And a method for obtaining a large amount of monoclonal antibodies to be used.

The antibody of this embodiment may be an amino acid sequence variant as long as it can specifically bind to GlcNAcylated serine 40 residue of histone H2A protein.
Amino acid sequence variants can be made by introducing mutations into the DNA encoding the antibody chain or by peptide synthesis. The site where the amino acid sequence of the antibody is modified may be the constant region of the heavy chain or light chain of the antibody as long as it has an activity equivalent to that of the antibody before modification, and the variable region (framework region and CDR). In addition, a method of screening an antibody having an improved affinity for an antigen by modifying CDR amino acids may be used (for example, PNAS, 102: 8466-8471 (2005), Protein Engineering, Design & Selection, 21: 485-493 (2008), International Publication No. 2002/051870, J. Biol. Chem., 280: 24880-24888 (2005), Protein Engineering, Design & Selection, 21: 345-351 (2008)).

The number of amino acids to be modified is preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids (eg, within 2 amino acids, 1 amino acid).
The amino acid modification is preferably a conservative substitution.
As used herein, “conservative substitution” means substitution with another amino acid residue having a chemically similar side chain. Groups of amino acid residues having chemically similar amino acid side chains are well known in the technical field to which the present invention belongs. For example, acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), neutral amino acids, amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), hydroxy groups Amino acids with amino acids (serine / threonine), amino acids with sulfur (cysteine / methionine), amino acids with amide groups (asparagine / glutamine), amino acids with imino groups (proline), amino acids with aromatic groups (phenylalanine / tyrosine / And tryptophan). The amino acid sequence variant preferably has a higher binding activity to the antigen than a control antibody (for example, the O-GlcNAc (RL2) antibody described in Examples described later).
The binding activity to the antigen of the antibody of the present embodiment (including the functional fragment of the above-mentioned antibody, amino acid sequence variant, etc.) is evaluated by, for example, ELISA, Western blotting, immunoprecipitation, immunostaining, etc. be able to.

<Method for Detection of GlcNAcation of Serine 40 Residue of Histone H2A Protein by Antibody>
In one embodiment, the present invention provides a method for detecting GlcNAcation of a serine 40 residue of a histone H2A protein, comprising measuring the level of GlcNAcylation of the serine 40 residue of the histone H2A protein using the antibody described above. To do.

According to the detection method of the present embodiment, the 40 residues of GlcNAc serine of the histone H2A protein can be detected easily and simply. Furthermore, abnormalities in epigenetics of various diseases such as metabolic abnormalities such as diabetes and obesity, neurological diseases and cardiovascular diseases can be detected.

In the present embodiment, the method for measuring the GlcNAc level of the serine 40 residue of histone H2A protein using the above-mentioned antibody is not particularly limited. For example, ELISA method, Western blotting method, immunoprecipitation method, immunostaining method Etc.
The detection method of the present embodiment includes a disease associated with GlcNAcylation of serine 40 residue of histone H2A protein associated with a disease, a disease state, a research tool for analysis of a disease state, a tool for monitoring lifestyle, a disease, a disease state, etc. It can be used as a diagnostic tool for predicting onset, sensitivity, and the like, and also as a drug discovery tool using a biomarker described later as an index.

<Screening Method for Inhibitor or Activator of GlcNAcation of Serine 40 Residue of Histone H2A Protein>
In one embodiment, the present invention provides a method for screening an inhibitor or activator of GlcNAcation of serine 40 residue of histone H2A protein, wherein the histone H2A protein or its protein is present in the presence and absence of a test substance. A step of measuring the GlcNAcation level of serine 40 residue of the peptide fragment, and when the GlcNAcation level in the presence of the test substance is lower than the GlcNAcation level in the absence of the test substance The test substance is determined to be an inhibitor of GlcNAcylation of serine 40 residue of histone H2A protein, and the GlcNAc level in the presence of the test substance is the phosphorylation level in the absence of the test substance. The test substance is a histone H2A protein serine 40 Provides a screening method comprising the step of determining that the active agent of the GlcNAc of the group, the.

According to the screening method of the present embodiment, an inhibitor or activator of GlcNAcation of serine 40 residue of histone H2A protein can be obtained easily and simply.

[Step of measuring GlcNAc level]
First, the level of GlcNAcation of serine 40 residue of histone H2A protein or peptide fragment thereof in the presence and absence of the test substance is measured. The histone H2A protein used in the measurement may be a peptide fragment containing 40 residues of GlcNAc serine in the histone H2A protein.

In the screening method of the present embodiment, the “test compound” is not particularly limited. For example, expression products of gene libraries, synthetic low molecular compound libraries, peptide libraries, antibodies, bacterial release substances, cells ( Microorganisms, plant cells, animal cells) extracts and culture supernatants, purified or partially purified polypeptides, marine organisms, plant or animal extracts, soil, random phage peptide display libraries, and the like.

The method for measuring the GlcNAc level is not particularly limited. For example, a method using mass spectrometry, a method using an antibody that specifically binds to the GlcNAcated serine 40 residue of histone H2A protein, and histone H2A protein by autoradiography And a method of detecting serine 40 residues converted to GlcNAc. Among them, the method using an antibody that specifically binds to the GlcNAc-modified serine 40 residue of the histone H2A protein is preferable.
More specifically, examples of the method using an antibody include an ELISA method, a Western blotting method, an immunoprecipitation method, and an immunostaining method. As the antibody that specifically binds to the GlcNAcylated serine 40 residue of the histone H2A protein, the same antibody as the above <antibody> can be used.

For example, the histone H2A protein may be immobilized on a substrate, and the above antibody may be labeled with an enzyme or a fluorescent dye. In this case, after washing the substrate, the level of GlcNAc of the serine 40 residue of the histone H2A protein is measured by reacting the substrate of the enzyme and measuring the coloration or luminescence of the substrate, or by measuring the fluorescence of the fluorescent dye. Can be measured.

Alternatively, when the histone H2A protein is not immobilized on the substrate, for example, the GlcNAc level can be measured by the following method. For example, the histone H2A protein is previously labeled with one of a set of fluorescent dyes that cause FRET (Fluorescence resonance energy transfer). In addition, the antibody is labeled with the other of the set of fluorescent dyes. A group of dye molecules that cause FRET is not particularly limited, and examples thereof include FAM and TAMRA, VIC and TAMRA, and the like.

If this principle is used, it can be performed by a homogeneous assay (Homogeneous assay) in which this step is performed in a solution state throughout. The homogeneous assay has an advantage that the operation is simple because no washing operation is required for the measurement. A detection method in which reaction and washing are performed using an antigen or the like immobilized on a substrate, such as an ELISA method, is called a heterogeneous assay.

In the screening method of the present embodiment, as shown in the examples described later, the level of GlcNAc of the serine 40 residue of histone H2A protein responds to changes in extracellular glucose concentration via the hexosamine biosynthetic pathway (HBP). Therefore, it is preferable to have a measurement step in the presence of glucose.

Further, as shown in the examples described later, the level of GlcNAcization of the serine 40 residue of histone H2A protein increases or decreases in proportion to the repair ability of DNA. Therefore, in the screening method of this embodiment, It is preferable to have a measurement step under conditions that damage By such a process, for example, a drug having a genome repair ability can be screened.

Examples of the method of damaging DNA include ultraviolet (UV) irradiation, radiation irradiation, oxidative stress due to active oxygen, addition of an alkylating agent, addition of a topoisomerase II inhibitor (eg, etoposide, etc.), etc. It is not limited to these.
Further, a method for confirming whether or not DNA has been damaged may be appropriately selected according to the DNA damage method.
For example, when detecting pyrimidine dimer generated by ultraviolet irradiation or the like, a solution using a DNA damaging antibody (for example, anti-cyclobutane type pyrimidine dimer antibody, anti-6-4 type photoproduct antibody, anti-Dewar type photoproduct antibody, etc.) The back surface liquid method etc. are mentioned.
Alternatively, for example, in the case of detecting an abasic site generated by oxidative stress or the like, an ELISA method using ARP (Aldehyde Reactive Probe) can be used.
Alternatively, for example, when detecting double-strand breaks (DSBs) caused by irradiation or the like, immunization using a phosphorylated antibody (anti-γ-H2AX antibody) of a serine 139 residue of histone H2AX Examples thereof include fluorescent staining.

[Determining step]
Subsequently, when the GlcNAcation level in the presence of the test substance is lower than the GlcNAcation level in the absence of the test substance, the test substance has a serine 40 residue of histone H2A protein. It can be determined that it is an inhibitor of GlcNAcation.

On the other hand, when the GlcNAc level in the presence of the test substance is higher than the phosphorylation level in the absence of the test substance, the test substance is a GlcNAc of the serine 40 residue of histone H2A protein. It can be determined that it is a activating agent.

<Method for monitoring histone modification caused by fluctuations in extracellular glucose concentration>
In one embodiment, the present invention is a method for monitoring fluctuations in extracellular glucose concentration, comprising measuring the level of GlcNAcation of serine 40 residue of histone H2A protein that occurs in the presence of glucose. provide.

Conventionally, there is no data indicating that GlcNAcation of histone proteins varies with glucose concentration, and in the present invention, for the first time, it was revealed that GlcNAcation of serine 40 residues of histone H2A protein varies with glucose concentration.

According to the method of the present embodiment, it is possible to detect epigenetic abnormalities of various diseases in which metabolic abnormalities such as diabetes and obesity are risk factors.

In the method of the present embodiment, the method for measuring the GlcNAc level is not particularly limited, and for example, the same method as the above <Method for screening inhibitor or activator of GlcNAc conversion of serine 40 residue of histone H2A protein> Can be mentioned. Among them, the method using an antibody that specifically binds to the GlcNAc-modified serine 40 residue of the histone H2A protein is preferable. More specifically, examples of the method using an antibody include an ELISA method, a Western blotting method, an immunoprecipitation method, and an immunostaining method. As the antibody that specifically binds to the GlcNAcylated serine 40 residue of the histone H2A protein, the same antibody as the above <antibody> can be used.

<Marker for monitoring histone modification caused by fluctuations in extracellular glucose concentration>
In one embodiment, the present invention provides a marker for monitoring histone modification exerted by fluctuations in extracellular glucose concentration, comprising a protein or peptide marker comprising a H2A protein or a peptide fragment thereof comprising 40 GlcNAcylated serine residues. provide.

Conventionally, there is no marker for monitoring histone modification (GlcNAc conversion of serine 40 residue of histone H2A protein) caused by fluctuations in extracellular glucose concentration. In the present invention, serine 40 residue of histone H2A protein converted to GlcNAc is extracellular. It was revealed for the first time that it could be a marker for monitoring glucose concentration.

According to the marker of this embodiment, it is possible to detect epigenetic abnormalities of various diseases in which metabolic abnormalities such as diabetes and obesity are risk factors.
Moreover, since the glucose responsiveness of GlcNAcation of histone H2A protein changes in diabetic patients, the course of treatment can be determined by examining the recovery of responsiveness. Therefore, the marker of the present invention can be applied as a diagnostic marker for metabolic diseases and a follow-up marker for treatment.

The marker of the present embodiment only needs to contain a 40-glycine Serine residue, may be a histone H2A protein, or may be a peptide fragment thereof.

The marker detection method of the present embodiment includes the same method as described above <Screening Method for Inhibitor or Activator of GlcNAcation of Serine 40 Residue of Histone H2A Protein>. Among them, the method using an antibody that specifically binds to the GlcNAc-modified serine 40 residue of the histone H2A protein is preferable. More specifically, examples of the method using an antibody include an ELISA method, a Western blotting method, an immunoprecipitation method, and an immunostaining method. As the antibody that specifically binds to the GlcNAcylated serine 40 residue of the histone H2A protein, the same antibody as the above <antibody> can be used.

<Method for monitoring histone modification caused by DNA damage>
In one embodiment, the present invention is a method for monitoring histone modifications caused by DNA damage, measuring the level of GlcNAcation of serine 40 residues of histone H2A protein or peptide fragments thereof that occurs in the presence of DNA damage. A method comprising the steps of:

According to the method of this embodiment, histone modification caused by DNA damage can be easily monitored.

In the method of the present embodiment, the method for damaging DNA is, for example, the same as the method exemplified in the above <Screening Method for Inhibitor or Activator of GlcNAcation of Serine 40 Residue of Histone H2A Protein> The method is mentioned.
The method for confirming whether or not DNA has been damaged may be appropriately selected according to the method for damaging DNA, and the above <inhibitor or activator of GlcNAcation of serine 40 residue of histone H2A protein Examples thereof include the same methods as those exemplified in Screening Method>.

In the method of the present embodiment, the method for measuring the GlcNAc level is not particularly limited. For example, the method exemplified in <Method for screening inhibitor or activator of GlcNAcation of serine 40 residue of histone H2A protein> described above. The same method is mentioned. Among them, the method using an antibody that specifically binds to the GlcNAc-modified serine 40 residue of the histone H2A protein is preferable.
More specifically, examples of the method using an antibody include an ELISA method, a Western blotting method, an immunoprecipitation method, and an immunostaining method. As an antibody that specifically binds to GlcNAcylated serine 40 residue of histone H2A protein, the same antibodies as those described in <Antibodies> above can be used.

<Marker for monitoring histone modification caused by DNA damage>
In one embodiment, the present invention provides a protein or peptide marker consisting of an H2A protein or a peptide fragment thereof, comprising a GlcNAcylated serine 40 residue, which is a marker for monitoring histone modification caused by DNA damage.

According to the marker of the present embodiment, histone modification caused by DNA damage can be easily monitored.

The marker of the present embodiment only needs to contain a 40-glycine Serine residue, and may be a histone H2A protein or a peptide fragment thereof.

The marker detection method of the present embodiment may be the same as the above <Screening method for inhibitors or activators of GlcNAcation of serine 40 residue of histone H2A protein>. Among them, the method using an antibody that specifically binds to the GlcNAc-modified serine 40 residue of the histone H2A protein is preferable. More specifically, examples of the method using an antibody include an ELISA method, a Western blotting method, an immunoprecipitation method, and an immunostaining method. As the antibody that specifically binds to the GlcNAcylated serine 40 residue of the histone H2A protein, the same antibody as the above <antibody> can be used.

<Method for evaluating the stability of genomic DNA by epigenetic modification>
In one embodiment, the present invention is a method for evaluating the stability of genomic DNA by epigenetic modification, comprising the step of measuring the GlcNAcation level of serine 40 residue of histone H2A protein or peptide fragment thereof I will provide a.

As will be described later in Examples, the present inventors show that the repair ability of genomic DNA is exhibited and the damaged DNA is repaired by converting serine 40 residue of histone H2A protein into GlcNAc. Revealed.
According to the method of the present embodiment, the stability of genomic DNA by epigenetic modification can be easily evaluated.
In the present specification and claims, “stability of genomic DNA” means that genomic DNA is not damaged or is repaired even if genomic DNA is damaged. Means.

In general, epigenetic modification (in this embodiment, mainly GlcNAc conversion of serine 40 residue of H2A protein) is present, past, and future in the expression of genes involved in the epigenetic modification. Status, (1) current transcription program (eg, if gene expression is active, chromatin structure is relaxed), (2) history of gene expression so far in differentiation and development, and (3) It is considered to reflect the state of gene expression in the future (reference: Hammaud SS, “Chromatin and Transcription Transitions of Mammalian Adult Germ Stem Cells and Spermatogen”). m Cell, vol. 15, p239-253, 2014.).
Therefore, for example, when the chromatin structure is relaxed in the current transcription program and gene expression is active, it is considered that the DNA is susceptible to DNA damage. However, the serine 40 residue of histone H2A protein is converted to GlcNAc, and thus genomic DNA Therefore, even if it is damaged, it can be repaired immediately and transfer can be performed without any problem.
For example, when the gene is necessary for future gene expression, the serine 40 residue of the histone H2A protein is converted to GlcNAc in advance, so that the gene can be protected from DNA damage until the expression is increased. Based on this newly discovered function, it is determined whether the serine 40 residue of histone H2A protein is sensitive or resistant to DNA damage in cells, tissues or individuals by analyzing the degree of GlcNAc modification.・ Can be diagnosed.

In general, the DNA repair pathway differs depending on the stage of cell differentiation (eg, stem cells, progenitor cells, mature cells, etc.) and the type of DNA damage, and the stability of the genome is lost due to abnormal DNA repair pathways. It is known that various diseases occur (reference document: McKinnon P. J., “Maintaining genome stability in the nervous system”, nature neuroscience, vol. 16, no. 11, p 15213-1529). .
In the method of the present embodiment, the generation mechanism of epigenetic abnormality in various diseases can be verified by appropriately selecting the stage of cell differentiation and the method of damaging DNA.

In the method of this embodiment, the method for measuring the level of GlcNAc is not particularly limited, and is exemplified in the above-described <Method for screening an inhibitor or activator of GlcNAcation of serine 40 residue of histone H2A protein> described above. The same method as that described above may be used. Among them, the method using an antibody that specifically binds to the GlcNAc-modified serine 40 residue of the histone H2A protein is preferable.
More specifically, examples of the method using an antibody include an ELISA method, a Western blotting method, an immunoprecipitation method, and an immunostaining method. As an antibody that specifically binds to GlcNAcylated serine 40 residue of histone H2A protein, the same antibodies as those described in <Antibodies> above can be used.

<Marker for detecting the stability of genomic DNA by epigenetic modification>
In one embodiment, the present invention provides a marker for detecting the stability of genomic DNA caused by epigenetic modification, comprising a protein or peptide marker comprising a H2A protein or a peptide fragment thereof comprising 40 GlcNAcylated serine residues. provide.

Conventionally, there is no marker for detecting the stability of genomic DNA by epigenetic modification, and in the present invention, the GlcNAc serine 40 residue of histone H2A protein detects the stability of genomic DNA by epigenetic modification. First revealed that it can be a marker.

According to the marker of this embodiment, the stability of genomic DNA by epigenetic modification can be evaluated.
When the marker of the present embodiment is detected, it can be determined that the gene region involved in GlcNAcization of the serine 40 residue of the histone H2A protein is protected from DNA damage, and the genomic DNA is stable.
On the other hand, when the marker of the present embodiment is not detected, it can be determined that the expression in the gene region involved in GlcNAcization of the serine 40 residue of the histone H2A protein is reduced or an epigenetic abnormality has occurred.

The marker of the present embodiment only needs to contain a 40-glycine Serine residue, may be a histone H2A protein, or may be a peptide fragment thereof.

The marker detection method of the present embodiment includes the same method as described above <Screening Method for Inhibitor or Activator of GlcNAcation of Serine 40 Residue of Histone H2A Protein>. Among them, the method using an antibody that specifically binds to the GlcNAc-modified serine 40 residue of the histone H2A protein is preferable. More specifically, examples of the method using an antibody include an ELISA method, a Western blotting method, an immunoprecipitation method, and an immunostaining method. As the antibody that specifically binds to the GlcNAcylated serine 40 residue of the histone H2A protein, the same antibody as the above <antibody> can be used.

<Other uses of antibodies>
Other uses of GlcNAc-specific antibodies for histone proteins include, for example, performing the ChIP-seq method (a technique combining a chromatin immunoprecipitation (ChIP) and a next-generation sequencer) using the antibody. , A region on the genome that is present near the H2A protein containing 40 GlcNAcylated serine residues and protected from DNA damage can be identified.

In addition, as shown in Examples described later, epigenetics modification in different cells (in this embodiment, mainly GlcNAc conversion of serine 40 residue of H2A protein) is involved by the ChIP-seq method using the antibody. It is possible to analyze the difference in the gene group to be analyzed and the difference in the expression pattern of the gene group.

The embodiment of the present invention has been described in detail with reference to the drawings. However, the specific configuration is not limited to this embodiment, and includes a configuration that does not depart from the gist of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples and the like.

[Example 1] Detection of GlcNAcation site of histone protein and preparation of GlcNAcation specific antibody of histone protein (1) Preparation of monoclonal antibody recognizing novel GlcNAcation histone GlcNAcation is performed by serine (S) or threonine (T ). Furthermore, a plurality of histone amino acid sequences ("... YT ..." and "... YS ...") are inferred from the information of GlcNAcated proteins revealed so far, and several types of GlcNAcated peptides Was made. Subsequently, mice were immunized according to a conventional method to obtain antibody-producing myeloma cells using a fusion protein obtained by binding the prepared GlcNAc-modified peptide with bovine serum albumin or GST protein as an antigen. A total of 300 or more clones were screened to obtain an antibody that recognizes GlcNAc-histone.

(2) Histone extraction and fractionation and purification by HPLC (High performance liquid chromatography) Histone was extracted from mouse embryonic stem (ES) cells using Histone purification mini kit (manufactured by Active Motif). Subsequently, the extracted histone was dialyzed with water, and acetonitrile (Solvent A (5) was obtained by HPLC-10Ai (manufactured by Shimadzu Corp.) with a reverse phase chromatography column (Aeris WIDEPORE 3.6 μm XB-C8) set. % Acetonitrile, 0.1% TFA) and Solvent B (90% acetonitrile, 0.1% TFA)) were used for fractionation by a gradient method (flow rate 0.5 ml / min). The result is shown in FIG. 2A.
The fractionated fractions (15 to 72.5 minutes) were collected using a fraction collector FRC-10A (manufactured by Shimadzu Corporation).

(3) Detection of GlcNAcation site of histone For each fraction obtained in (2), Western blotting using the antibody, H2A antibody, H2B antibody, H3 antibody and H4 antibody obtained in (1) according to a conventional method Detection by the method was performed. In addition, silver staining was performed to compare the amount of protein contained in each fraction. The result is shown in FIG. 2A.

From FIG. 2A, it was confirmed that the antibody obtained in (1) recognized the GlcNAc-ized site of H2A.

(4) Examination of antibody specificity According to a conventional method, two types of wild-type Flag-H2A1A and Flag-H2A3, and mutant Flag-H2A3-S40A (among the amino acid sequence of histone H2A3 protein, the 40th from the N-terminus) A recombinant protein in which serine was changed to alanine was prepared. Subsequently, each recombinant protein was detected by Western blotting using the antibody obtained in (1), the H2A antibody, and the Flag antibody. The result is shown in FIG. 2B.

FIG. 2B reveals that the antibody obtained in (1) specifically detects GlcNAcation of the 40th serine in the amino acid sequence of histone H2A3 protein. Hereinafter, the antibody obtained in (1) is referred to as H2A3-S40-GlcNAc antibody.

[Example 2] Detection of GlcNAcation of serine 40 residue of histone H2A3 protein in mouse ES cells and mouse TS cells (1) Culture of mouse ES cells and mouse placental trophoblast stem (TS) cells Mouse ES cells are J1 Strains were used (Li, E., Bestor, T. & Jaenisch, R. Cell 1992, 69, 915-926.). Medium for ES cells (15% FBS (manufactured by Biowest), 2 mM L-glutamine (manufactured by Wako), 1 mM sodium pyruvate (manufactured by Wako), non-essential amino acids, 2-mercaptoethanol, 50 U / mL penicillin (Invitrogen) ), 50 μg / mL streptomycin (manufactured by Invitrogen) added to DMEM (containing 25 mM glucose) (manufactured by Wako)), 1000 U of Leukemia Inhibitory Factor (manufactured by Millipore), and gelatin-coated culture dish Incubated above.

In addition, TS cells were obtained from C57BL / 6N mouse-derived scutellum according to the previous report (Hayakawa K1, et.al., Curr Protoc Stem Cell Biol. Ｆｅ2015 Feb 2; 32: 1E.4.1-1E4.32.). Established from the cell. Subsequently, medium for TS cells (20% FBS (manufactured by Biowest), 2 mM L-glutamine (manufactured by Wako), 1 mM sodium pyruvate (manufactured by Wako), 2-mercaptoethanol, 50 U / mL penicillin (manufactured by Invitrogen) ), 50 μg / mL streptomycin (manufactured by Invitrogen) was added to RPMI-1640 (containing 25 mM glucose), 25 ng / mL FGF4, activin A (manufactured by Wako) and heparin (manufactured by Sigma) were added, Culture was performed.

Subsequently, 1, 5, and 10 mM glucose-containing ES cell culture medium and TS cell culture medium were prepared using glucose-free DMEM (manufactured by Wako) or glucose-free RMPI-1640. Subsequently, ES cells and TS cells were cultured using each prepared culture solution until confluent. Subsequently, the cultured cells were collected by centrifuging the cells into pellets, removing the culture solution. Subsequently, it was immediately frozen in liquid nitrogen and stored at −80 ° C. until use.

(3) Immunostaining of mouse ES cells Mouse ES cells were fixed with 4% paraformaldehyde for 20 minutes. Subsequently, it was treated overnight at 4 ° C. with a blocking solution (PBS (−) containing 5% bovine serum-derived albumin (BSA)), 0.1% Tween 20, 0.2% Triton X-100). Subsequently, the reaction was performed overnight at 4 ° C. using a histone H2A3-S40-GlcNAc antibody or O-GlcNAc (RL2) antibody diluted with PBS (−) containing 5% BSA and 0.1% Tween20. Subsequently, the secondary antibody diluted with PBS (-) was reacted at room temperature for 1 hour. Further, nuclei were stained with 1 μg / mL DAPI (manufactured by Dojindo) for 10 minutes at room temperature. Subsequently, it was sealed with a sealing liquid (manufactured by VECTASHIELD) to prepare a preparation. Observation was performed using an integrated confocal laser microscope (FV10i, manufactured by Olympus Corporation). The results are shown in FIGS. 3A and 3B. FIG. 3A shows the result of staining with H2A3-S40-GlcNAc antibody, and FIG. 3B shows the result of staining with O-GlcNAc (RL2) antibody. In FIG. 3A, “O-GlcNAc peptide absorption” indicates that the GlcNAc-modified peptide prepared in (1) of Example 1 was added and immunostained, and “mouse IgG” represents mouse IgG. It shows what immunostained using.

From FIGS. 3A and 3B, ES cells immunostained with H2A3-S40-GlcNAc antibody differ from ES cells immunostained with O-GlcNAc (RL2) antibody, and a dot-like signal is detected in the nucleus. It was. In addition, no signal for GlcNAc formation was detected in ES cells treated with the GlcNAc-modified peptide prepared in Example 1 (1) and mouse IgG.

(4) Verification of GlcNAc conversion according to change in glucose concentration For mouse ES cells and mouse TS cells cultured at different glucose concentrations prepared in (2), H2A3-S40-GlcNAc antibody, H2A antibody, and As a control, detection by Western blotting using actin β antibody was performed. The results are shown in FIG. 3C.

From FIG. 3C, it became clear that in mouse ES cells, the level of histone GlcNAc increased with decreasing glucose concentration. On the other hand, it was revealed that the level of GlcNAc increased in mouse TS cells with increasing glucose concentration.
From the above, it became clear that the GlcNAcation of the 40th serine from the N-terminal in the amino acid sequence of histone H2A3 protein shows different glucose reactivity depending on the cell type.

[Example 3] Detection of GlcNAcation of serine 40 residue of histone H2A3 protein in human vascular endothelial (EAhy) cells and human renal proximal tubular epithelium (RPTEC) (1) Human vascular endothelial (EAhy) cells and human kidney Culture of Proximal Tubular Epithelial (RPTEC) Cells Human EAhy cells were purchased from American Type Culture Collection (ATCC). Control medium (10% FBS (manufactured by Biowest), 50 U / mL penicillin (manufactured by Invitrogen), 50 μg / mL streptomycin (manufactured by Invitrogen) in DMEM (containing 5 mM glucose) (manufactured by Wako)) The maintained human EAhy cells were seeded in a 4-well dish at 1 × 10 ⁴ cells per well.
Subsequently, control medium (containing 5 mM glucose) or high glucose medium (containing 10 mM glucose or 25 mM glucose) (10% FBS (manufactured by Biowest), 50 U / mL penicillin (manufactured by Invitrogen)), 50 μg / mL streptomycin (Invitrogen) Was added to DMEM (containing 10 mM glucose or 25 mM glucose) (made by Wako)) for 4 days.

(2) Immunostaining of human EAhy cells and human RPTEC cells This was carried out except that human EAhy cells and human RPTEC cells cultured in (1) were used and staining was not performed using O-GlcNAc (RL2) antibody. Immunostaining was performed using the same method as in Example 2 (3). The results are shown on the left side of FIGS. 4A and 4B. FIG. 4A shows the result of immunostaining of human EAhy cells, and FIG. 4B shows the result of immunostaining of human RPTEC cells.
4A and 4B, the number of dots of the GlcNAc-ized signal in the nucleus was measured with image analysis software Cell Profiler (www.cellprofiler.org). Statistical processing was performed by Wilcoxon rank sum test. The results are shown on the right side of FIGS. 4A and 4B.

From FIG. 4A and FIG. 4B, it was revealed that the level of GlcNAcation increases with increasing glucose concentration also in human EAhy cells and human RPTEC cells.

[Example 4] ChIP-seq using H2A3-S40-GlcNAc antibody in mouse ES cells under low and high sugar environments
(1) Chromatin immunoprecipitation (ChIP)
The ChIP assay was performed using ChIP-IT (registered trademark) Express Kit (manufactured by Active Motif) according to the manufacturer's instructions.
First, a chromatin fragmentation reagent is added to mouse ES cells cultured in a low sugar (containing 1 mM glucose) and high sugar (containing 25 mM glucose) environment, incubated at 37 ° C. for 10 minutes, the cells are disrupted, and chromatin is fragmented. did. Subsequently, in a solution containing 100 μL of fragmented chromatin, 3 μg of H2A3-S40-GlcNAc antibody and 40 μL of magnetic beads (sheep anti-mouse IgG antibody-conjugated Dynabead M-280 or protein G-binding Dynabead (Invitrogen) ) And mixed overnight at 4 ° C. with stirring. After immunoprecipitation, DNA was eluted by incubation at 65 ° C. for 6 hours using elution buffer (10% SDS, 300 mM NaCl, 10 mM Tris-HCl, and 5 mM EDTA, pH 8.0). Subsequently, the obtained DNA was purified using ChIP DNA Clean and Concentration Kit (manufactured by Zymo Research) and used for the subsequent sequence.

(2) Sequencing A library for high-throughput DNA sequencing was prepared according to the manufacturer's instructions using TruSeq DNA Sample Preparation v2 Kit, Set A / B (Illumina, FC-121-2001 / -2002). did.
First, 50 ng of the DNA obtained in (1) or the adenylation of the 3 ′ end of the input DNA was repaired, and the adapter oligo was ligated. Subsequently, a DNA fragment of 120 to 500 bp was purified using AMpure XP beads (A63880, manufactured by Beckman Coulter, Inc.), and further amplified using PCR. Subsequently, the library DNA was electrophoresed on a 2% agarose gel, and then purified using Zymoclean Gel DNA Recovery Kit (Dymo Research, D4007). The purified library DNA was quantified using BioAnalyzer 2000 (manufactured by Agilent). The sequence was Illumina HiSeq 2000 systems.

Subsequently, pre-filtering processing is performed on the raw data of ChIP to delete the sequence adapter sequence and low-quality read information, and the tag is mapped from the mouse genome (mm9) using Bowtie software, and the Galaxy browser (www.galaxy) is used. Peak was detected using MACS software at .psu.edu). The Venn diagram and gene ontology analysis were performed using BioVenn (http://www.cmbi.ru.nl/cdd/bioven/) and DAVID program (https://david.ncifcrf.gov/), respectively. These analysis results are all shown in FIG.

FIG. 5 shows that the mouse ES cells cultured in a low sugar (1 mM glucose) environment (58.9%) are more genomic (58.9%) than the mouse ES cells cultured in a high sugar (25 mM glucose) environment. It was revealed that there is a high proportion of histone H2A3 protein in which the serine 40 residue is converted to GlcNAc in the coding region.
In addition, a mouse ES cell cultured in a low sugar (1 mM glucose) environment was obtained by immunoprecipitation with the H2A3-S40-GlcNAc antibody rather than a mouse ES cell cultured in a high sugar (25 mM glucose) environment. There were many.
Furthermore, in mouse ES cells cultured in a low sugar (1 mM glucose) environment, it was revealed that many histone H2A3 proteins in which serine 40 residues were converted to GlcNAc were present at the transcription start site (8.1%).

In addition, gene ontology analysis shows that in a low sugar (1 mM glucose) environment and a high sugar (25 mM glucose) environment, a gene group controlled by a histone H2A3 protein in which a 40-serine residue is GlcNAc-converted is partially common (for example, There are also gene groups involved in the protein modification process, gene groups involved in the Wnt signal pathway, etc.), but it has been clarified that there are a plurality of different ones.

[Example 5] ChIP-seq using H2A3-S40-GlcNAc antibody in human vascular endothelial (EAhy) cells
(1) Chromatin immunoprecipitation (ChIP)
A ChIP assay was performed using the same method as that described in (1) of Example 4 except that human vascular endothelial (EAhy) cells were used instead of mouse ES cells to obtain DNA fragments.

(2) Sequencing Using the human genome instead of the mouse genome, the DNA fragment obtained in (1) was sequenced using the same method as in (2) of Example 4. The analysis results are shown in FIG.

FIG. 6 shows that the transcription start point, the coding region of the genome, and the serine in the non-coding region between human EAhy cells cultured in a high sugar (25 mM glucose) environment and human EAhy cells cultured in a low sugar (1 mM glucose) environment. There was no significant difference in the abundance of histone H2A3 protein in which 40 residues were GlcNAcated.
In addition, the amount of DNA fragments obtained by immunoprecipitation of human EAhy cells cultured in a high sugar (25 mM glucose) environment and human EAhy cells cultured in a low sugar (1 mM glucose) environment with an H2A3-S40-GlcNAc antibody. There was no difference.
Furthermore, in human EAhy cells cultured in a high sugar (25 mM glucose) environment, it was revealed that many histone H2A3 proteins in which serine 40 residues were converted to GlcNAc were present at the transcription start site.

From gene ontology analysis, the gene group controlled by histone H2A3 protein in which serine 40 residue is converted to GlcNAc in a high sugar (25 mM glucose) environment is five kinds of gene groups such as genes involved in purine metabolism. It became clear.

[Example 6] Detection of DNA repair-related proteins by Western blotting in mouse ES cells under low and high sugar environments (1) Recovery of nuclear proteins First, low sugar (containing 1 mM glucose) and high sugar (containing 25 mM glucose) ) Using LysoPure Nuclear and Cytoplasmic Extract from mouse ES cells cultured under conditions of addition of camptothecin (CPT), etoposide (ETP), or CPT and ETP (non-treated). Then, according to the manufacturer's instructions, the nuclear protein was recovered.
CPT is a quinoline alkaloid that inhibits the action of type I topoisomerase (topoI) of the DNA enzyme, and ETP forms a complex with topoisomerase II after cleaving DNA and inhibits recombination of DNA. Both are tumor agents and compounds that cause DNA damage.

(2) Detection of DNA repair-related protein by Western blotting Next, using the collected nuclear protein, γH2AX antibody (an antibody that specifically binds to H2AX phosphorylated at serine 139 residue) according to a conventional method , H2AX antibody, AcH2AZ antibody (an antibody that specifically binds to H2AZ in which lysine 4 residue, lysine 7 residue, and lysine 11 residue are acetylated), H2AZ antibody, H2A3-S40-GlcNAc antibody, H2A antibody, p53-binding protein 1 (p53-binding protein 1; 53BP1) antibody (an antibody that binds to p53 and specifically binds to 53BP1, a protein that enhances the transcriptional activity of p53), and a Rad51 antibody (a second DNA in eukaryotes) Rad51, a protein involved in repair of heavy chain breaks Detection was by Western blotting using an antibody) that specifically binds. The results are shown in FIG.

FIG. 7 shows that serine 139 residue is phosphorylated in mouse ES cells cultured under low-sugar (containing 1 mM glucose) and high-sugar (containing 25 mM glucose) conditions without CPT and ETP added (non-treated). The detected histone H2AX protein was not detected.
On the other hand, in an ES cell under a low sugar (containing 1 mM glucose) and high sugar (containing 25 mM glucose) environment and having undergone CTP or ETP treatment to artificially cause DNA damage, serine 139 residue in histone H2AX protein Phosphorylation level of 53, and 53BP1 and Rad51 were further induced, while the acetylation level of histone H2AZ protein in the nucleus decreased.

In addition, in mouse ES cells cultured in a low sugar (containing 1 mM glucose) environment, the GlcNAc level of histone H2A3 protein serine 40 residue was high regardless of the presence or absence of CTP or ETP treatment. On the other hand, in mouse ES cells cultured in a high sugar (containing 25 mM glucose) environment, the GlcNAcation level of serine 40 residue of histone H2A3 protein was remarkably increased by CTP or ETP treatment.

[Example 7] DNA repair ability test by difference in UV irradiation time in human vascular endothelial (EAhy) cells that highly expressed wild type histone H2A3 protein (WT) or mutant H2A3 protein (H2A-S40A) (1) wild type Preparation of human vascular endothelial (EAhy) cells highly expressing histone H2A3 protein (WT) or mutant H2A3 protein (H2A-S40A) High expression of wild-type histone H2A3 protein (WT) or mutant H2A3 protein (H2A-S40A) The human vascular endothelial (EAhy) cells were seeded in 3 × 10 ⁵ cells in a 10 cm dish and cultured for 96 hours under DMEM (5 mM glucose) containing 10% FBS.

(2) Ultraviolet irradiation Subsequently, the cells previously cultured in (1) were irradiated with ultraviolet rays (UV-C, 254 nm) for 0 minutes, 1 minute, or 5 minutes.

(3) ELISA method using anti-cyclobutane-type pyrimidine dimer antibody Subsequently, the cells collected immediately after the ultraviolet irradiation treatment in (2) were treated with Lysis buffer [100 mM Tris-HCl (pH 8.0), 5 mM EDTA, 0.2% It was suspended in SDS, 200 mM NaCl, 100 ug / ml proteinase K] and incubated overnight at 55 ° C. with shaking. Thereafter, extraction was performed once with phenol / chloroform / isoamyl alcohol (hereinafter sometimes referred to as “PCI”), and RNase (manufactured by Roche) was treated at 37 ° C. for 30 minutes to degrade RNA. PCI extraction was performed once again, and genomic DNA was precipitated with ethanol. After removing the ethanol, it was dissolved in TE buffer [10 mM Tris-HCl, 1 mM EDTA (pH 8.0)]. Genomic DNA was stored at 4 ° C. until use.
Subsequently, 10 ng of genomic DNA was added to a 96 well protamine surface coated plate and incubated overnight at 37 ° C. Subsequently, blocking was performed by incubating at 37 ° C. for 30 minutes using PBS containing 2% fetal bovine serum and 0.1% Tween20. Subsequently, an anti-cyclobutane-type pyrimidine dimer (CPD) antibody (manufactured by Cosmo Bio, NM-DND-001) (diluted 1: 1000 with PBS) was used as a primary antibody, and 37 Incubated for 1 hour at ° C. Subsequently, the plate was washed with PBS, and HRP-conjugated anti-mouse IgG antibody (manufactured by Jackson ImmunoResearch) (diluted 1: 2000 using PBS)) was used as a secondary antibody at 37 ° C. Incubated for 30 minutes. After washing 3 times with PBS, 1 × stabilized peroxide substrate buffer (Terumo) containing OPD substrate was added, and absorbance at 490 nm was measured using iMark microplate reader (Bio-Rad). . The results are shown in FIG.

FIG. 7 shows that human vascular endothelial (EAhy) cells highly expressing wild-type histone H2A3 protein (WT) are pyrimidine dimers more than human vascular endothelial (EAhy) cells highly expressing mutant H2A3 protein (H2A-S40A). The production amount of was suppressed. This was presumed to be due to the fact that the serine 40 residue of the histone H2A3 protein was converted to GlcNAc, whereby DNA repairing ability was exhibited and a part of the primidine dimer generated by ultraviolet irradiation was repaired.

ADVANTAGE OF THE INVENTION According to this invention, the method of monitoring the histone modification which the fluctuation | variation of extracellular glucose concentration exerts can be provided.
Moreover, according to the present invention, the stability of genomic DNA can be easily evaluated.

Claims (12)

1. An antibody characterized in that it specifically binds to N-acetylglucosamine (GlcNAc) serine 40 residue of histone H2A protein.
2. A method for detecting GlcNAcation of a serine 40 residue of a histone H2A protein, comprising the step of measuring the GlcNAcation level of a serine 40 residue of the histone H2A protein using the antibody according to claim 1.
3. A method for screening an inhibitor or activator of GlcNAcation of serine 40 residue of histone H2A protein, comprising:
   Measuring the level of GlcNAcation of serine 40 residue of histone H2A protein or peptide fragment thereof in the presence and absence of a test substance;
   When the level of GlcNAcation in the presence of the test substance is lower than the level of GlcNAcation in the absence of the test substance, the test substance is converted to GlcNAc of serine 40 residue of histone H2A protein. When the GlcNAc level in the presence of the test substance is higher than the phosphorylation level in the absence of the test substance, the test substance is a histone H2A protein. Determining that it is an activator of GlcNAcation of serine 40 residues of
   A screening method comprising:
4. The screening method according to claim 3, wherein the measurement step is performed in the presence of glucose.
5. The screening method according to claim 3 or 4, wherein the measurement step is performed under conditions in which DNA is damaged.
6. The GlcNAc level of serine 40 residue of the histone H2A protein or peptide fragment thereof is measured by an antibody that specifically binds to the GlcNAc-converted serine 40 residue of histone H2A protein. The screening method according to item.
7. A method for monitoring histone modifications caused by fluctuations in extracellular glucose concentration comprising:
   A method comprising measuring the level of GlcNAcylation of a serine 40 residue of a histone H2A protein that occurs depending on the presence of glucose.
8. The method according to claim 7, wherein the GlcNAcation level of the serine 40 residue of the histone H2A protein is measured by an antibody that specifically binds to the GlcNAcated serine 40 residue of the histone H2A protein.
9. A marker for monitoring histone modifications caused by fluctuations in extracellular glucose concentration,
   A protein or peptide marker comprising a H2A protein or a peptide fragment thereof containing 40 residues of GlcNAc serine.
10. A method for evaluating the stability of genomic DNA by epigenetic modification comprising:
    A method comprising the step of measuring the GlcNAcation level of a serine 40 residue of a histone H2A protein or a peptide fragment thereof.
11. The method according to claim 10, wherein the GlcNAc level of serine 40 residue of the histone H2A protein or a peptide fragment thereof is measured by an antibody that specifically binds to the GlcNAc-converted serine 40 residue of the histone H2A protein.
12. A marker for detecting the stability of genomic DNA by epigenetic modification,
    A protein or peptide marker comprising a H2A protein or a peptide fragment thereof containing 40 residues of GlcNAc serine.

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