JP2004339109A - MONOCLONAL ANTIBODY AGAINST METHYLATED (di-CH3) LYSINE 9 OF HISTONE H3 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a monoclonal antibody specifically recognizing histone H3 methylated at Lys 9 or its fragment. <P>SOLUTION: This monoclonal antibody is prepared from a hybridoma produced by bonding an amino acid of 11 residues composed of Lys 4 to Lys 14 containing methylated Lys 9 of histone H3 through cysteine to KLH (keyhole limpet hemocyanine), injecting the bonded material as an antigen to a mouse and using a spleen cell obtained from the mouse to give the hybridoma. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００４９】
【発明の効果】
本発明のモノクローナル抗体は、Ｌｙｓ９がメチル化されたヒストンＨ３に対して、特異性が高く、ウェスタンブロッティング、免疫蛍光染色、免疫組織化学的染色などに使用可能であり、さらに、免疫沈降法、クロマチン免疫沈降法などによる解析に使用可能である。
従って、ヒストンＨ３のＬｙｓ９のメチル化が重要な要因と考えられるインプリンティング遺伝子、及び該インプリンティングの異常による先天性疾患などの解析に有用である。
さらに、最近インプリンティング遺伝子の異常と特定の腫瘍との関連性が明らかとなっているため、Ｈ３のＬｙｓ９のメチル化の有無を確認することで、かかる腫瘍の発生の可能性等の診断にも有効に用いることができる。
【００５０】
参考文献
佐々木裕之：「現代以外の基礎第５巻、生殖と発生」（岩波書店）
中山潤一、実験医学 第２０巻、第１１号（増刊）：８２−８８， ２００２．
Ａａｇａａｒｄ等， ＥＭＢＯ Ｊ．， １８：１９２３−１９３８， １９９９．
Ｂａｎｎｉｓｔｅｒ等， Ｎａｔｕｒｅ， ４１０：１２０−１２４， ２００１．
Ｇｏｄｉｎｇ等， Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ， Ｓａｎ Ｄｉｅｇｏ． ４９２ ｐｐ． １９９６．
Ｈａｒｌｏｗ及びＬａｎｅ， Ａｎｔｉｂｏｄｉｅｓ： Ａ ｌａｂｏｒａｔｏｒｙ ｍａｎｕａｌ． Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ， Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ． ７２６ ｐｐ， １９８８．
Ｈａｒｌｏｗ及びＬａｎｅ， Ｕｓｉｎｇ ａｎｔｉｂｏｄｉｅｓ： Ａ ｌａｂｏｒａｔｏｒｙ ｍａｎｕａｌ． Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ ＰＲｅｓｓ， Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ， Ｎｅｗ Ｙｏｒｋ． １９９９．
Ｈｅａｒｄ等， Ｃｅｌｌ， １０７：７２７−７３８， ２００１．
Ｋｏｚｂｏｒ等， Ｊ Ｉｍｍｕｎｏｌ． １３３：３００１−５， １９８４．
Ｌａｃｈｎｅｒ等， Ｎａｔｕｒｅ， ４１０：１１６−１２０， ２００１．
Ｍｉｌｓｔｅｉｎ及びＣｕｅｌｌｏ， Ｎａｔｕｒｅ． ３０５：５３７−４０， １９８３．
Ｎａｋａｙａｍａ等， Ｓｃｉｅｎｃｅ， ２９２：１１０−１１３， ２００１．
Ｎｉｅｌｓｅｎ等， Ｎａｔｕｒｅ， ４１２：５６１−５６５， ２００１．
Ｎｉｓｈｉｏｋａ等， Ｇｅｎｅｓ ＆ Ｄｅｖ．， １６：４７９−４８９， ２００２
Ｒｅａ等， Ｎａｔｕｒｅ， ４０６：５９３−５９９， ２０００．
Ｓｃｈｏｏｋ， Ｍｏｎｏｃｌｏｎａｌ ａｎｔｉｂｏｄｙ ｐｒｏｄｕｃｔｉｏｎ ｔｅｃｈｎｉｑｕｅｓ ａｎｄ ａｐｐｌｉｃａｔｉｏｎｓ． Ｍａｒｃｅｌ Ｄｅｋｋｅｒ， Ｉｎｃ．， Ｎｅｗ Ｙｏｒｋ． ３３６ ｐｐ． １９８７．
Ｔａｃｈｉｂａｎａ等， Ｊ． Ｂｉｏｌ． Ｃｈｅｍ．， ２７６：２５３０９−２５３１７， ２００１．
Ｔｓｃｈｉｅｒｓｃｈ等， ＥＭＢＯ Ｊ．， １３：３８２２−３８３１， １９９４．
Ｕｒａｎｏ等， Ｏｎｃｏｇｅｎｅ， ８：１３７１−１３７６， １９９３．
Ｗａｌｔｅｒ等， Ｓｅｍｉｎ Ｃｅｌｌ Ｄｅｖ Ｂｉｏｌ．， １４：１０１−１１０， ２００３．
Ｗａｎｇ等， Ｍｏｌ． Ｃｅｌｌ， ８：１２０７−１２１７， ２００１．
Ｚｈａｎｇ等， Ｇｅｎｅｓ ＆ Ｄｅｖ．， １５：２３４３−２３６０， ２００１．
Ｚｈｅｎｈａｎ等， Ａｍ Ｊ Ｈｕｍ Ｇｅｎｅｔ．， ６９：１３８９−１３９４， ２００１．
【００５１】
【配列表】

【図面の簡単な説明】
【図１】図１は、Ｈ３−ｍＫ９に対するモノクローナル抗体のスクリーニングの経過を示すイムノブロットの結果である。ＳＡＭは、Ｓ−アデノシルメチオニンのことであり、ＩＢは、イムノブロットのことを意味する。
【図２】図２は、Ｈ３−ｍＫ９ ５．１．１の抗原特異性を示した図である。Ｃｏｏｍａｓｓｉｅとは、クマジーブリリアントブルーのことであり、タンパク染色したことを意味する。
【図３】図３は、Ｈ３−ｍＫ９ ５．１．１の抗原特異性についてビアコアシステム（Ｂｉａｃｏｒｅ社）を用いて検討した結果を示す。
【図４】図４は、ＨｅＬａ細胞から酸抽出を行ったヒストン画分に対するＨ３−ｍＫ９ ５．１．１の特異性を確認した結果、及びＨｅＬａ細胞抽出液から、Ｈ３−ｍＫ９ ５．１．１によって、Ｌｙｓ９がメチル化されたヒストンＨ３が特異的に免疫沈降されることを示す図である。ＩｇＬは、イムノグロブリン軽鎖の位置を示す。ｌｙｓａｔｅは、抽出液そのものをイムノブロットしたこと示す。また、矢印はヒストンＨ３の位置を示す。
【図５】図５は、細胞内タンパク質とＤＮＡをクロスリンクした細胞内抽出液を用いて、Ｈ３−ｍＫ９ ５．１．１により免疫沈降を行った結果を示す。
【図６】図６は、Ｈ３−ｍＫ９ ５．１．１を用いて細胞の免疫染色を行った結果を示す。Ｈ３−ｍＫ９（ｄｉ−）及びＨ３−ｍＫ４（ｄｉ−）は、それぞれＨ３−ｍＫ９ ５．１．１及びＨ３−ｍＫ４に対する抗体（ＵＢＩ社）を用いて染色をした結果を示し、ｍｅｒｇｅは、両染色像を重ね合わせた結果を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a monoclonal antibody that specifically binds to histone H3 in which the lysine ninth is methylated and does not bind to histone H3 in which the lysine ninth is not methylated, and uses thereof.
[0002]
[Prior art]
Eukaryotic chromosomal DNA is highly folded and present in the nucleus by adopting a nucleosome structure. In order to promote or suppress the expression of a gene encoded on chromosomal DNA having such a structure, it is necessary to locally relax the structure of chromatin or, on the contrary, to maintain a rigid structure. Therefore, local changes in chromatin structure and its control have a great influence on gene expression regulation, and are phenomena deeply involved in gene function expression.
[0003]
Factors that induce changes in chromatin structure include those that directly change the nucleosome structure itself using ATP, such as chromatin remodeling factors such as Swi / Snf and Iswi, and those such as histone acetylase (HAT). In some cases, post-transcriptional modification of histones alters the structure of nucleosomes. As post-transcriptional modifications of histones, acetylation, methylation, phosphorylation, ubiquitination and the like are known. Among them, histone methylation has been rapidly studied in recent years, and it has been revealed that it plays an important role in controlling epigenetic gene expression together with DNA methylation (for example, Zhang et al., Genes & Dev., 15). : 2343-2360, 2001.). Here, it is understood that “epigenetics” refers to changing gene expression without changing the nucleotide sequence in the process of ontogenesis and further stably maintaining this change. Genetic phenomena include genomic imprinting and X chromosome inactivation, suggesting the involvement of DNA methylation and histone methylation.
[0004]
Genomic imprinting is a phenomenon that is observed in mammals including humans. In the process of gametogenesis, "signs" or "memory" that are derived from sperm or eggs are modified into chromosomes. Is to imprint on the gene. This “sign” or “memory” is maintained even after the process of fertilization or somatic cell division, and gene expression is performed according to the information.
Genomic imprinting is thought to be a factor in lethal parthenogenesis in mammals. A mammalian pronucleus and a female pronucleus are present in mammalian spermatozoa and ova, respectively, and normal ontogenesis is possible only when these two pronuclei are aligned, but in the case of only one of them, Fatal during embryonic development. Therefore, in mammals, malformation during the developmental process may occur due to the improper functioning of genome imprinting (Hiroyuki Sasaki: "Basics other than modern times, reproduction and development" (Iwanami Shoten) Vol. 9 chapter).
[0005]
Prader-Willi syndrome, Angelman syndrome, Beckwith-Wiedemann syndrome and the like are known as congenital diseases caused by abnormal genome imprinting. Prader-Willi Syndrome is a disease characterized by obesity, short stature, short-term muscle weakness and the like, in part due to deletion of a specific region of chromosome 15 from the father, It is clear that both chromosome 15 derived from the mother have been inherited in place of chromosome 15. On the other hand, Angelman syndrome is a disease characterized by symptoms such as ataxic walking and paroxysmal laughter, and it is thought that a partial deletion of chromosome 15 derived from a mother may contribute to this. Also, Beckwith-Wiedemann syndrome is a disease characterized by symptoms such as knee hernia, macrotongue, megalopathy, visceral hypertrophy, and complication of malignant tumor, and it is suspected that imprinting on chromosome 11 is involved. Furthermore, it is thought that imprinting may be involved in cancers such as Wilms tumor (Walter et al., 2003).
Thus, genomic imprinting is thought to play an important role in the development of congenital diseases and certain types of tumors, and the mechanism of histone methylation and DNA methylation during imprinting has been elucidated. It can be expected that this will contribute to the elucidation, prevention and treatment of further causes of the above-mentioned diseases and the like.
[0006]
Regarding the methylation of histone H3, it is known that lysine at positions 4, 9, 27, 36, and 79, and arginine at positions 2, 17, and 26 undergo methylation (Zhang et al., 2001). Recent studies have revealed the role of histone H3 lysine methylation in relation to chromatin structure transformation.
It has been reported that the histone methyltransferase SUV39H1 present in mammalian cells is localized in the heterochromatin region where transcription is inactivated, and interacts with the heterochromatin protein HP1 (Aagaard). Et al., 1999). In addition, it is apparent that the SUV39H1 protein has an activity as an enzyme (HMTase; histone methyltransferase) that specifically introduces a methyl group into ninth lysine of histone H3 (hereinafter, referred to as H3-Lys9) in vitro. Was done. Furthermore, the existence of Clr4 of fission yeast, SU (VAR) 3-9 of Drosophila, and the like as homologues of SUV39H1 have also been clarified. In addition, G9a that transfers a methyl group to lysine 9 and lysine 27 has been identified in humans, and the presence of E (Z) as a Drosophila homolog has been reported. Many HMTases retain a conserved region called the SET sequence (Tschiersch et al., 1994), and the vicinity of this region is considered to be a region important for the enzymatic activity to introduce a methyl group (Rea et al. 2000).
[0007]
Histone H3 in which Lys9 is methylated (hereinafter referred to as H3-mK9) is abundant in the heterochromatin region (Nakayama et al., 2001), and HP1 recognizes and binds to the methylation modification of H3-Lys9. It is thought to form a higher-order chromatin structure characteristic of heterochromatin (Lachner et al., 2001; Bannister et al., 2001). In addition, phosphorylation of Ser10 of histone H3, which is known to be involved in chromosome aggregation and transient gene activation, inhibits methylation transfer activity by SUV39H1 in vivo, and conversely, Lys9 Since the phosphorylation of Ser at position 10 is inhibited when the methyl group is transferred, modifications of adjacent Lys9 and Ser10 affect the structure of neighboring chromatin by acting antagonistically to each other, It is presumed that the expression of the gene present in the chromatin region is controlled.
In addition, since the methylation activity of H3-Lys9 is abolished by immunoprecipitation of Rb, which is an inhibitory oncogene product, it is suggested that Rb may interact with a specific methylase (eg, SUV39H1). Was. Furthermore, since the HP1 protein is recruited near the promoter together with H3-Lys9 methylation on the cyclin E promoter whose transcription is repressed by Rb, the methylation of H3-Lys9 is dynamically controlled and It has been suggested that it is involved in the regulation of expression, in particular, repressive regulation (Nielsen et al., 2001).
[0008]
In contrast to H3-mK9, which is distributed in heterochromatin, H3-mK4 is distributed on euchromatin, and is expected to be involved in gene transcription activation.
It has been reported that an HDAC (histone deacetylase) complex, a transcription corepressor, binds to the tail at the N-terminal side of histone H3 and suppresses transcription. However, Lys4 is methylated. It has been suggested that the binding of the tail of histone H3 is greatly inhibited and the transcriptional activation state is maintained (Nishioka et al., 2002). In addition, since the methylation of H3-Lys4 suppresses the methylation reaction of H3-Lys9 in vitro by SUV39H1 (Wang et al., 2001; Nishioka et al., 2002), methylation of H3-Lys4 and H3-lys9 is suppressed. It is thought that they act antagonistically to control the structure of chromatin, thereby controlling gene expression. Also, unlike methylation by SUV39H1, methylation of H3-Lys9 by G9a does not inhibit the reaction by methylation of H3-Lys4 (Nishioka et al., 2002). Therefore, considering that G9a is localized in the transcription activation region, it is possible that G9a-induced Lys9 methylation may be involved in the transient control of transcription in the euchromatin region.
[0009]
In addition, the methylation status of histone H3 in the 15q11-q13 region of chromosome 15 that was thought to be involved in the onset of Prader-Willi syndrome and Angelman syndrome has recently been reported in detail (Zhenhan et al., 2001). In the paternal copy of this region, H3-Lys4 is methylated, and in the maternal copy, H3-Lys9 is methylated, indicating that the methylation of Lys9 and Lys4 is It was suggested that it was important for the onset.
Furthermore, two X chromosomes are present in female mammalian cells, one of which is inactivated. It is known that H3-lys9 methylation is involved in this inactivation. Have been. The inactivation of the X chromosome is thought to be caused by the localization of the Xist RNA that does not encode a protein on the X chromosome, but H3-Lys9 is methylated immediately after the localization of the Xist RNA. (Heard et al., 2001).
[0010]
As described above, methylation of histone H3 plays an important role in controlling gene expression through structural transformation of chromatin. To date, the analysis of the methylation status on histone H3 including Lys9 has been carried out by using an antibody specific to a methylated amino acid to confirm the presence or absence of histone H3 methylation in a specific gene region. Thus, the state of transcription activation in the region was monitored (for example, see Non-Patent Document 1). However, conventionally used antibodies have a problem in specificity for methylated amino acid residues, and have not shown sufficient binding specificity for analysis by immunoprecipitation. This has hindered detailed analysis.
[0011]
[Non-patent document 1]
Nakayama et al., Science, 292: 110-113, 2001.
[0012]
[Problems to be solved by the invention]
In view of the above circumstances, the present inventors have conducted intensive studies to produce an antibody that specifically binds to histone H3 in which Lys9 is methylated, and as a result, selected those in which Lys9 of histone H3 is methylated. Thus, a monoclonal antibody that specifically recognized and bound and did not bind to histone H3 in which Lys9 was not methylated could be obtained.
Therefore, an object of the present invention is to provide a monoclonal antibody or a fragment thereof that selectively recognizes and binds to histone H3 in which Lys9 is methylated and does not bind to histone H3 in which Lys9 is not methylated.
Another object of the present invention is to provide a hybridoma that produces the monoclonal antibody.
Another object of the present invention is to provide a method for preparing the monoclonal antibody.
Still further, the present invention provides a kit comprising the monoclonal antibody or a fragment thereof, a reagent for detecting the monoclonal antibody or a fragment thereof, or a carrier necessary for precipitating histone H3 bound to the monoclonal antibody or a fragment thereof. The purpose is to provide.
[0013]
[Means for Solving the Problems]
Thus, the present invention provides the following (1) to (13).
(1) A monoclonal antibody or a fragment thereof that selectively binds to methylated histone H3 and discriminates between methylated histone H3 and unmethylated histone H3.
(2) The monoclonal antibody or the fragment thereof according to (1), wherein the methylated site of the methylated histone H3 is ninth lysine of histone H3.
(3) The monoclonal antibody or a fragment thereof according to the above (2), which binds to the ninth lysine of methylated histone H3.
(4) The monoclonal antibody or a fragment thereof according to any one of (1) to (3), which is obtained from a hybridoma deposited at the National Institute of Advanced Industrial Science and Technology under the Accession No. FERM P-19106 under the depository number.
(5) The monoclonal antibody or a fragment thereof according to any one of the above (1) to (4), which selectively binds to the methylated histone H3 in an ELISA method or a blot method.
(6) The monoclonal antibody or a fragment thereof according to any one of (1) to (4), which selectively binds to the methylated histone H3 in immunofluorescence or immunohistochemistry.
(7) The monoclonal antibody or a fragment thereof according to any one of (1) to (4), wherein in the immunoprecipitation method, the antibody selectively binds to the methylated histone H3 to immunoprecipitate the methylated histone H3. .
(8) The monoclonal antibody or the fragment thereof according to the above (7), wherein the immunoprecipitation method is a chromatin immunoprecipitation method.
(9) A mammal immunized with a KLH (keyhole limpet hemocyanin) cross-linked to a partial peptide of histone H3 (amino acid residue 4-14, SEQ ID NO: 1) in which the ninth lysine is dimethylated. Any one of the above (1) to (8), obtained by fusing spleen cells with cells capable of self-proliferation derived from an animal of the same or different species as the mammal, and cloning the fused cells. A hybridoma producing the described monoclonal antibody.
(10) The hybridoma according to the above (9), wherein the hybridoma is deposited at the National Institute of Advanced Industrial Science and Technology Biological Depositary under the accession number FERM P-19106.
(11) A method for producing an antibody, comprising culturing the hybridoma according to (9) or (10) and preparing the monoclonal antibody according to any one of claims 1 to 8 from a culture supernatant. .
(12) A kit comprising the monoclonal antibody or a fragment thereof according to any of the above (1) to (8), and a reagent for detecting the monoclonal antibody or a fragment thereof.
(13) A kit comprising the monoclonal antibody or a fragment thereof according to any of the above (1) to (8), and a carrier necessary for precipitating histone H3 bound to the monoclonal antibody or a fragment thereof.
[0014]
“Methylation” in the present specification includes monomethylation, dimethylation, and trimethylation, and is preferably dimethylation.
The “histone H3” in the present specification includes all histones derived from eukaryotes, but is preferably derived from plants or animals, more preferably derived from mammals, and most preferably derived from mammals. Preferably it is of human origin.
The amino acid numbers of histone H3 referred to in the present specification are those in which the N-terminal alanine is the first and the C-terminal is sequentially numbered. Therefore, for example, the “ninth lysine” is a lysine corresponding to the ninth when the N-terminal alanine of histone H3 is 1.
The “ELISA method” in the present specification includes all types of techniques that can be performed by those skilled in the art using ordinary knowledge. Examples include, but are not limited to, direct or indirect ELISAs and capture ELISAs. Details of the ELIAS method are described in Harlow and Lane, Antibodies: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 726 pp, 1988. And Harlow and Lane, Using antibodies: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 1999. See etc.
As used herein, the term "blot method" includes any type of technique that can be performed by those skilled in the art using ordinary knowledge. For example, but not limited to, a dot blot method in which an antigen is directly spotted on a nylon membrane or a nitrocellulose membrane, or a Western blot method in which an antigen is electrophoretically transferred.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention detects a monoclonal antibody that specifically binds to a methylated histone H3, particularly a dimethylated histone H3 at lysine 9 (Lys9), a hybridoma producing the monoclonal antibody, and the monoclonal antibody. A kit comprising a reagent or a carrier necessary for precipitating histone H3 bound to the monoclonal antibody.
Therefore, the monoclonal antibody of the present invention and a method for preparing a hybridoma producing the monoclonal antibody will be described in detail below.
[0016]
1. Preparation of monoclonal antibodies
(1) Preparation and immunization of immunogen
The monoclonal antibodies of the present invention are prepared using the hybridoma method (Milstein and Cuello, 1983).
The method includes the following four steps;
(A) immunizing a host animal or a lymphocyte derived from the host animal;
(B) recovering monoclonal antibody secreting (or potentially secreting) lymphocytes,
(C) fusing lymphocytes to immortalized cells,
(D) Select cells that secrete the desired monoclonal antibody.
Histone H3 in which only Lys9 is methylated may be used as the immunogen, but a peptide consisting of about 20 amino acid residues including Lys9, preferably a peptide in which only Lys9 is methylated is used. And most preferably a peptide consisting of the 4th to 14th amino acids of histone H3, wherein only Lys9 is methylated. When a peptide is used as an antigen, it is preferable to use a peptide obtained by binding the peptide to a carrier protein in order to enhance the immunity of the peptide for actual immunization. As the carrier protein, any protein that can be selected by those skilled in the art can be used. For example, BSA (Bovine Serum Album), OVA (Ovalbumin), KLH (Keyhole Limpet Hemocyanin) and the like can be used. , KLH are most preferred.
As a method for binding the peptide to the carrier protein, a carbodiimide method, a glutaraldehyde method, a diazo condensation method, a maleimidobenzoyloxysuccinimide (MBS) method and the like can be used, and the MBS method is most preferable. In this method, Cys is previously bound to the C-terminal portion of a peptide used as an immunogen, and the amino group of KLH is linked to the SH group of Cys bound to the peptide.
[0017]
As a mammal for immunizing the immunogen, for example, a mouse, rat, guinea pig, hamster, or other suitable host animal can be selected as the immunized animal. When performing immunization, the injection route of the immunogen can be easily selected by those skilled in the art, and is not particularly limited. For example, any route such as subcutaneous, intraperitoneal, intravenous, intramuscular, etc. Is also good. The number of immunizations is preferably several times every several days to several weeks. The amount of the immunogen used for one immunization is preferably 1 to 1000 μg per animal.
Alternatively, lymphocytes obtained from an immunized animal may be immunized in vitro. If human cells are desired, peripheral blood lymphocytes (PBLs) are generally used. However, spleen cells or lymphocytes from other mammals are more common and preferred.
[0018]
(2) Preparation of hybridoma
After immunization, antibody-producing cells (spleen cells) obtained from the host animal are fused with an immortalized cell line using a fusion agent such as polyethylene glycol to establish hybridoma cells having both antibody-producing ability and self-proliferation ability ( Goding, 1996). As an immortalized cell line used as a fusion cell, a human, rat or mouse myeloma cell line immortalized by transformation is used. After performing the cell fusion, the cells are grown in a suitable medium containing factors that allow such inhibition to inhibit the growth or survival of the unfused lymphocytes and immortalized cell lines. Usually, parental cells lacking hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT) are used as immortalized cell lines. In this case, hypoxanthine, aminopterin and thymidine inhibit the growth of HGPRT-deficient cells and are added to a medium that allows hybridoma growth (HAT medium).
[0019]
(3) Selection of antibody-producing hybridoma
In preparing a monoclonal antibody, a preferred immortalized cell line is a mouse myeloma cell line, which is available from American Type Culture Collection (Manassas, VA) and the like. For production of human monoclonal antibodies by human myeloma and mouse-human heteromyeloma cell lines, see Kozbor et al., 1984; School, 1987.
Since hybridoma cells secrete antibodies extracellularly, the presence or absence of production of monoclonal antibodies can be confirmed using a culture solution. The binding specificity of the produced monoclonal antibodies can be assessed by immunoprecipitation, such as radioimmunoassay (RIA), or by in vitro binding assays, such as enzyme-linked immunosorbent assay (ELISA) (Harlow and Lane, 1988; Harlow and Lane, 1999).
Hybridoma cells secreting a monoclonal antibody against histone H3 in which Lys9 has been methylated can be isolated as a single clone by limiting dilution and subculture (Goding, 1996). Suitable media include Dulbecco's Modified Eagle's Medium, RPMI-1640, and optionally, protein-free or serum-free media (eg, Ultra DOMA PF or HL-1; Biowhittaker; Walkersville, MD). The hybridoma cells may also be grown in ascites of a suitable host animal. The hybridoma strain producing a monoclonal antibody against the ninth lysine of histone H3 (H3-mK9 5.1.1) was obtained from the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary (Higashi 1 Tsukuba, Ibaraki Prefecture). No. 1-3) and deposited under the accession number FERM P-19106 on November 14, 2002.
[0020]
(4) Preparation of monoclonal antibody
The monoclonal antibody of the present invention can be obtained from a medium or ascites by protein A agarose (Sepharose), protein G agarose (Sepharose), hydroxyapatite chromatography, gel electrophoresis, dialysis, ammonium sulfate precipitation or affinity chromatography (Harlow and Lane, 1988; Harlow). And Lane, 1999).
In addition, a monoclonal antibody can also be prepared by a genetic recombination technique if a desired hybridoma can be obtained (US Pat. No. 4,166,452, 1979). To identify a gene encoding the monoclonal antibody polypeptide from a hybridoma cell line that secretes a monoclonal antibody recognizing H3-Lys9, for example, an oligonucleotide probe that specifically binds to mouse heavy and light chain antibody genes Can be used for screening. When the antibody heavy chain and light chain genes are obtained as a result of the screening, the target antibody gene can be identified by determining the sequences of the genes. The isolated DNA fragment is prepared by introducing an antibody gene into an appropriate expression vector in order to express a monoclonal antibody, and converting the vector into a simian COS-7 cell or a Chinese hamster ovary (CHO) cell that does not produce other Ig proteins. Or into a host cell, such as a myeloma cell.
[0021]
(5) Active fragment of monoclonal antibody
The monoclonal antibody of the present invention can be used as it is, or can be used after being fragmented as an active fragment. The active fragment may be any fragment as long as the specific binding activity to methylated histone H3 is not lost. The preparation of these active fragments can be performed by applying a known method such as papain, pepsin, or trypsin treatment to the purified monoclonal antibody (see “Immunobiochemical Research Method (Seismic Chemistry Experimental Course 5)”). , Edited by The Biochemical Society of Japan, p. 89 (1986)).
[0022]
2. Immunoprecipitation
(1) Immunoprecipitation
The monoclonal antibody of the present invention can also be used for immunoprecipitation. The immunoprecipitation method is a method that can be performed within the ordinary skill in the art.
Precipitated from the cell or tissue homogenate as a complex containing an antigen protein (histone H3 in the present invention) and a conjugate of a primary antibody and agarose-bound proteins A, G, and the like. The agarose conjugate may be selected depending on the origin and isotype of the species of the antibody used.
First, the agarose conjugate is washed several times with a washing buffer (for example, PBS or RIPA buffer) (for example, after adding the washing buffer, centrifuging at 12,000 × g for 2 minutes to remove the washing solution). This is repeated several times (three times or more), for example, centrifuged at 12,000 × g for 10 minutes, and collected in a precipitate. Add the antibody diluted to the appropriate concentration to the washed agarose conjugate and incubate at room temperature or 4 ° C for 60 minutes to several hours with gentle shaking. After the agarose conjugate-antibody complex is collected by centrifugation (for example, 12,000 × g, 2 minutes), it is washed several times (three times or more) with a washing buffer (described above). After washing, an appropriate amount of a sample containing an antigen (such as a tissue or cell homogenate) is added, and the mixture is incubated at 4 ° C for several hours to overnight with gentle shaking and stirring. After the incubation, the agarose conjugate-antibody complex is recovered by centrifugation (for example, 12,000 × g, 2 minutes), and then washed several times (3 times or more) with a washing buffer (described above).
[0023]
(2) SDS-PAGE
The pellet of the agarose conjugate-antibody complex is subjected to a sample treatment according to a standard method, centrifuged at room temperature (for example, 12,000 × g, 30 seconds), and the supernatant is subjected to SDS-PAGE. If necessary, western blotting or the like may be subsequently performed.
[0024]
2. Chromatin immunoprecipitation
The monoclonal antibody of the present invention includes an antibody (H3-mK9 5.1.1) which specifically binds to dimethylated Lys9 of histone H3. The monoclonal antibodies and fragments thereof can also be used in chromatin immunoprecipitation, which is a powerful means for analyzing the mechanism of transcriptional control through chromatin structure. Chromatin immunoprecipitation can be carried out within the ordinary skill in the art. Specifically, the method comprises the following steps: (1) fixation of cells with formalin, (2) immunoprecipitation, (3) release of fixation and recovery of DNA, and (4) detection of amplification of DNA by PCR.
[0025]
(1) Cell fixation with formalin
The chromatin immunoprecipitation method may be performed using any method that can be performed by those skilled in the art. For example, the chromatin immunoprecipitation method can be performed using Chromatin Immunorecipitation (ChIP) Assay Kit of Upstate biotechnology.
Specifically, the target cells are cultured in an appropriate medium (for example, DMEM, 5% FBS) at 10 cm dish for 2 days, and then cultured for 1 day in a serum-free medium (DMEM, 0% FBS). After the culture, 1/10 volume of 10% formaldehyde / PBS is added to the medium, and the mixture is incubated at a final concentration of 1% at 37 ° C. for 10 minutes to crosslink intracellular proteins and DNA.
After 10 minutes, the medium is aspirated and washed with ice-cold PBS containing protease inhibitors (eg, 1 mM PMSF, 1 μg / ml aprotinin). All the subsequent operations are performed on ice, and PBS and buffer are prepared by adding a protease inhibitor having the same concentration at the time of use and adding it.
[0026]
(2) Immunoprecipitation
1 ml PBS is added to the dish, the cells are scraped, transferred to a tube, and centrifuged at 700 × g, 4 ° C. for 3 minutes. The supernatant was removed, 150 μl of Lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, 1 mM PMSF, 1 μg / ml aprotinin) was added, and the mixture was stirred. Let stand for minutes.
Next, after sonicating the sample in ice for 10 seconds, the sample is sonicated about 4 times at intervals of about 20 seconds. Avoid raising sample temperature and bubbling during sonication. By this operation, the cross-linked DNA fragment is fragmented. The length of the DNA fragment is preferably 200 to 1000 bp in consideration of the amplification efficiency by the subsequent PCR. In order to obtain this DNA fragment length, various strengths and times are tested, and the optimal conditions are determined.
[0027]
A sample was prepared using a ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, 1 mM PMSF, 1 μg / g). Dilute 10-fold with ml aprotinin). To this, 40 μl protein A or G agarose (1.5 ml protein A or G agarose beads, 600 μg sonicated salmon sperm DNA, 1.5 mg BSA, 4.5 mg recombinant protein A or G / 1.5 ml buffer; 10 mM Tris -HCl, pH 8.0, 1 mM EDTA, 0.05% sodium azide) and incubate at 4 ° C for 30 minutes.
The supernatant is removed by centrifugation at 1500 rpm for 15 seconds at 4 ° C., an appropriate amount of antibody (for example, 1 μg) is added, and the mixture is incubated at 4 ° C. for 6 hours to overnight. After incubation, add 50 μl of protein A or G agarose and incubate at 4 ° C. for 1 hour.
Next, protein A or G agarose is washed under four-step washing conditions. Each is incubated at 4 ° C. for 3 minutes;
(A) Low Salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl),
(B) High Salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl),
(C) LiCl buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1),
(D) TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) × 2 times,
After washing, 50 μl Elution buffer (10 mM DTT, 1% SDS, 0.1 M NaHCO 3) ₃ ) Is added and elution is carried out at room temperature for 15 minutes. The supernatant is removed by centrifugation, 50 μl of Elution buffer is added again to the resin, and the above-mentioned elution is repeated again.
[0028]
(3) Cross-link release and DNA recovery
NaCl is added to the sample to 0.2 M and incubated at 65 ° C. for 6 hours. By this operation, the crosslink of the protein / DNA complex is released.
Add 2 μl of 0.5 M EDTA, 4 μl of 1 M Tris-HCl, and 0.6 μl of pH 6.5, 6 mg / ml Proteinase K, and incubate at 45 ° C. for 1 hour.
DNA is extracted by a DNA purification kit (for example, QIGEN) or the phenol-chloroform method, and eluted with 50 μl of purified water. 5 μl or 10 μl of this is used for PCR.
[0029]
(4) Detection of DNA amplification by PCR
Using the DNA obtained by the method described in the above (3) as a template, a PCR reaction is performed using appropriate primers to amplify a specific region on the genome to be analyzed. The reaction conditions and the like need to be individually examined depending on the region to be amplified and the length to be amplified. For example, a reaction mixture (5 μl of DNA, 5 μl of buffer for PCR, 4 μl of NTPs, primers (100 pmol each), 36 μl of distilled water, 0.5 μl of rTaq / 50 μl), 1 cycle of 96 ° C. for 5 minutes, 96 ° C. 30 minutes The reaction is carried out at 25 ° C. for 30 minutes and at 56 ° C. for 30 minutes and at 72 ° C. for 30 minutes for 25 to 40 cycles at 72 ° C. for 3 minutes to amplify the target DNA fragment.
In the course of the reaction, an appropriate amount (10 μl) is sampled, and amplification products (amplified DNA fragments) at three points, for example, 25 cycles, 33 cycles, and 40 cycles are checked by agarose electrophoresis to determine the optimal number of cycles for amplification. . By determining the DNA sequence and the like of the obtained DNA fragment, it becomes possible to specify a gene that is expected to be transcriptionally regulated by methylation of the 9th lysine of histone H3.
[0030]
8. Immunofluorescence and immunohistochemistry
(1) Immunofluorescence
When fluorescent staining of a frozen tissue section is performed, it can be performed based on ordinary knowledge of those skilled in the art. Although not limited, it can be carried out using the following method. For example, a tissue section to be observed is immersed in isopentane or the like cooled with liquid nitrogen and frozen. Next, the frozen tissue is transferred to a cryostat chamber and allowed to stand for about 30 minutes until the temperature reaches equilibrium. After mounting the tissue block on the stub using an embedding agent, the surface of the block is trimmed at a temperature suitable for the target tissue to prepare a 5.3 to 5 μm section. The prepared section is placed on a clean slide glass and dried at room temperature for 5 to 16 hours.
After diluting the monoclonal antibody of the present invention to an optimal concentration, the diluted solution is layered on an appropriate amount (50 to 70 μl) of the section, the chamber is covered, and the mixture is incubated at 37 ° C. After the incubation, the slide glass is washed about 2 to 3 times with PBS.
Next, a fluorescent-labeled (for example, fluorescein, rhodamine, carbocyanine (Cy3, Cy5, etc.), Texas Red, etc.)-Labeled secondary antibody is diluted to an optimal concentration, and the diluted solution is applied to an appropriate amount (50-70 μl) on a section. After overlaying and capping the chamber, incubate at 37 ° C. After the incubation, the slide glass is washed about 2 to 3 times with PBS in a light-shielded state. After staining, observation is performed with a fluorescence microscope.
[0031]
When staining cells, target cells are grown on a slide glass or cover glass in an attached state, or in the case of floating cells, attached to a slide glass by centrifugation. The slide glass or cover glass to which the cells are adhered fixes the intracellular protein using, for example, alcohol, acetone, formaldehyde, paraformaldehyde, or the like. Thereafter, if necessary, the permeability of the cell membrane and / or the nuclear membrane may be increased by treatment with a suitable surfactant (for example, Triton X-100 or the like). After diluting the monoclonal antibody of the present invention (in the case of the direct method, the monoclonal antibody is labeled with fluorescence) to an optimal concentration, dilute the diluted solution to an appropriate amount (50 to 70 μl) on a slide glass or cover glass. Overlay cells and incubate at room temperature for 60 minutes. After the incubation, the slide glass is washed about 2 to 3 times with PBS.
Next, in the case of the indirect method, after diluting the labeled secondary antibody to an optimal concentration, the diluted solution is overlaid on an appropriate amount (50 to 70 μl) of cells, and incubated at room temperature. After the incubation, the slide glass is washed about 2 to 3 times with PBS. After staining, observation is performed with a fluorescence microscope.
[0032]
(2) Immunohistochemistry
Slides of formalin-fixed, paraffin-embedded tissue sections are placed in a 56-60 ° C. oven for 15 minutes. Thereafter, slides are transferred to a xylene bath and xylene is changed twice every 5 minutes. After removing excess liquid, slides are rehydrated twice in fresh absolute ethanol for 3 minutes each. Then, after removing the excess liquid, the slide is placed in fresh 90% ethanol for 3 minutes. Then, after removing the excess liquid, the slide is allowed to stand in fresh 80% ethanol for 3 minutes, and then rinsed with weak running water for 30 seconds. Next, it is left to stand in PBS at room temperature for 30 minutes to further rehydrate. In order to inactivate endogenous peroxidase, a sufficient amount of 3% hydrogen peroxide solution is dripped over the entire section to overlay the entire section, and then incubated at room temperature for 5 minutes. After that, it is rinsed with PBS and left still in PBS for 2 minutes.
[0033]
After diluting the monoclonal antibody of the present invention to an optimal concentration on a section of a slide from which PBS has been sufficiently removed, an appropriate amount (100 μl) of the diluted solution is evenly layered, and then overlaid in a humidified chamber at 37 ° C. for 60 minutes or more Incubate. After the incubation, the slide glass is washed about 2 to 3 times with PBS.
After removing the excess liquid from the slide reacted with the primary antibody, for example, a biotin-labeled secondary antibody is diluted to an optimal concentration, and an appropriate amount of the secondary antibody is evenly layered on a section. Incubate for at least 30 minutes at room temperature in a humidified chamber. After that, it is rinsed with PBS and left still in PBS for 2 minutes.
After removing excess liquid from the slide reacted with the secondary antibody, for example, after diluting, for example, avidin conjugated with alkaline phosphatase or horseradish peroxidase to an optimum concentration, an appropriate amount of the slide is uniformly layered on the section. Incubate for at least 20 minutes at room temperature in a humidified chamber. After that, it is rinsed with PBS and left still in PBS for 5 minutes.
Next, a sufficient amount of a substrate mixture (for example, containing diaminobenzidine) is added to the section, and the mixture is allowed to react for about 5 to 10 minutes. Before the background rises, the reaction mixture is gently rinsed with distilled water to stop the reaction. Terminate.
[0034]
9. kit
(1) Reagents, etc.
The subject of the present invention also includes kits. Generally, the kit of the present invention includes a monoclonal antibody or a fragment thereof that discriminates between methylated histone H3 and unmethylated histone H3, and a reagent for detecting the monoclonal antibody. The monoclonal antibody preferably binds selectively to histone H3 methylated at Lys9, and is most preferably a monoclonal antibody that binds to methylated Lys9.
The kit of the present invention may contain a reagent for making the monoclonal antibody of the present invention accessible to histone H3 in the cell nucleus, but may include, but is not limited to, for example, a surfactant such as Triton X-100.
[0035]
The kit of the present invention also includes an immunofluorescent reagent, for example, an immunoglobulin (secondary antibody) that recognizes the monoclonal antibody of the present invention, such as fluorescein, rhodamine, carbocyanine (Cy3, Cy5, etc.), Texas Red, etc. Or a biotinylated immunoglobulin antibody in combination with avidin or streptavidin. In addition, the kit of the present invention includes an immunohistochemical or immunocytochemical detection reagent, such as, but not limited to, an immunoglobulin (secondary antibody) recognizing the monoclonal antibody of the present invention, comprising alkaline phosphatase or Biotinylated anti-immunoglobulin antibodies in combination with avidin or streptavidin derivatized with horseradish peroxidase may be included.
The kits of the present invention may also include one or more immunohistochemical reagents, including, but not limited to, anti-immunoglobulin antibodies derivatized with horseradish peroxidase, or avidin derivatized with horseradish peroxidase. And a biotinylated anti-immunoglobulin antibody in combination with streptavidin, and a chromogenic substrate for the immunoperoxidase reagent, such as diaminobenzidine.
[0036]
In addition, the kit of the present invention may include a carrier necessary for immunoprecipitating histone H3. Suitable carriers include protein A or G agarose conjugates. Further, carrier DNA (eg, salmon sperm DNA, etc.), other necessary buffers (eg, a buffer required for denaturing a protein, a buffer necessary for washing an antigen-antibody complex, etc.), and salts (eg, an appropriate concentration of NaCl solution).
[0037]
(2) Containers, etc.
The reagent contained in the kit is made of any material that can store individual components for a long period of time and does not absorb the reagent contained in the kit and does not change the characteristics of the substance contained in the container. Supplied in a container. For example, but not limited to, organic polymers such as polycarbonate, polystyrene, or any other suitable material typically used to hold reagents.
(3) Instruction manual
Instructions for use are also supplied with the kit. Instructions for use are printed on paper or other material and / or electrically or electromagnetically readable such as a floppy disk, CD-ROM, DVD-ROM, Zip disk, video tape, audio tape, etc. It may be supplied as a simple medium. Also, the detailed instructions may not physically accompany the kit, in which case the user will use the website specified by the kit manufacturer or provider or provided by email Thus, an appropriate instruction can be obtained.
[0038]
【Example】
Examples are shown below, but the present invention is not limited thereto.
[0039]
Example 1: Preparation of monoclonal antibody of the present invention
(1) Preparation and immunization of immunogen
The immunogen includes a partial peptide of histone H3 in which the 9th lysine is dimethylated (residues 4-14, SEQ ID NO: 1 (synthesized by Michael Berne; Tufts University, USA); KQRTAR [dimethylated K] Cys was added to the C-terminal of STGGK, and a cross-linked amino group of KLH (keyhole limpet hemocyanin, Pierce) and an SH group of Cys was used. After the prepared antigen was mixed with an adjuvant (TiterMax), the mice were immunized. After several immunizations, when the antibody titer reached a preferable value, the spleen was removed from the immunized mouse and fused with myeloma cells (NS-1 cell line) according to a standard method.
(2) Antibody screening
Next, an antigen for screening a target monoclonal antibody was prepared. GST-fused histone H3 partial peptide (GST-H3 (1-57)) as a substrate, SAM (S-adenosylmethionine) as a methyl group donor, GST-G9a (C-terminal from amino acid 706 of G9a) Fusion between a polypeptide containing GST and GST) as a methylating enzyme, 50 mM Tris, pH 8.5, 20 mM KCl, 10 mM MgCl ₂ , 10 mM β-mercaptoethanol, and 250 mM sucrose in 30 μl of a reaction solution at 37 ° C. for 60 minutes to carry out the methylation reaction of Lys9 of histone H3 (Tachibana et al., 2001.).
Using a methylated or unmethylated GST-H3 (1-57) prepared in the presence or absence of SAM, an increase in the antibody titer of an antibody that specifically recognizes H3-mK9 by immunoblotting. After confirming the above, the target clone was screened by ELISA (Urano et al., 1993). Furthermore, for the clones obtained by the ELISA method, a polyclonal antibody against H3-mK9 (UBI) (FIG. 1, αH3-mK9 (mouse polyclonal)) was used as a positive control, and an anti-EGFP antibody (αEGFP) was used as a negative control. Screening by immunoblot was performed based on the difference in the binding to the antigen depending on the presence or absence of methylation (FIG. 1). As a result, clone 5.1.1 was selected as an antibody that specifically binds to H3-mK9 (hereinafter, referred to as H3-mK9 5.1.1) (FIGS. 1, 5.1.1).
[0040]
(3) Antigen specificity of H3-mK9 5.1.1
FIG. 2 shows the specificity of the obtained monoclonal antibody. H3-mK9 5.1.1 did not recognize any peptide that had undergone a methylation reaction in the absence of SAM, which is a methyl group donor (FIG. 2, IB: -lane), and did not methylate in the presence of SAM. Only the peptide which was made to recognize was recognized (FIG. 2, IB: + lane). In addition, several bands to which H3-mK9 5.1.1 reacts are confirmed because a protease degradation product generated during the preparation of GST-H3 (1-54) was recognized. It is not related to the antigen specificity of 1.1.
Furthermore, the specificity of H3-mK9 5.1.1 for histone H3 in which Lys9 was methylated was examined.
Peptides in which the lysines at positions 4, 9, and 27 of histone H3 have been dimethylated (H3-m2K4; ART [dimethylated K] QTARK; SEQ ID NOs: 2, H3-m2K9; KQTAR, respectively) [Dimethylated K] STGGK; SEQ ID NO: 1, H3-m2K27; TKAAR [Dimethylated K] SAPAT; SEQ ID NO: 3, H4-m2K20; AKRHR [Dimethylated K] VLRDN; SEQ ID NO: 4 (The peptide used here is The specificity of H3-mK9 5.1.1 was analyzed using a Biacore system (BIAcore3000, Biacore) using a synthesis by Michael Berne (Tufts University, USA)). Peptides were diluted in 10 mM sodium acetate, pH 4.0, coupled to a CM5 sensor chip, and the remaining binding sites were blocked with 1 M ethanolamine, pH 8.5. As a control, the same operation without peptide was used. The binding between the peptide and H3-mK9 5.1.1 was determined by 10 mM HEPES, pH 7.4, 0.15 M NaCl, 1 mM CaCl. ₂ , 0.005% surfactant 20 (v / v).
As a result of the analysis, H3-mK9 5.1.1 reacted only with H3-m2K9, and did not react with H3-m2K4, H3-m2K27, or H4-m2K20 (FIG. 3). The methylation of the peptide used here is all di-CH3 (dimethylated), but it is weaker than the unmethylated peptide and tri-CH3 (trimethylated) peptide by H3-mK9. 5.1.1 reacted. However, this reaction is due to the high sensitivity of the Biacore system and does not affect the intrinsic antigen specificity of H3-mK9 5.1.1.
In addition, immunoblotting was performed on histone H3 prepared by acid extraction of HeLa cells using H3-mK9 5.1.1.
As a result, H3-mK9 5.1.1 specifically recognized endogenous histone H3 (FIG. 4, lane 2) and did not recognize other histones H2A, H2B, H4 (FIG. 4, lane 1). And 2).
[0041]
Example 2: Immunoprecipitation with H3-mK9 5.1.1
It was examined whether H3-mK9 5.1.1 can specifically immunoprecipitate histone H3 in which Lys9 present in cells is methylated.
H3-mK9 5.1.1 (2 μl) was added to protein G agarose (20 μl of a 1: 1 suspension) and incubated at 4 ° C. for 75 minutes to add H3-mK9 5.1.1 to protein G agarose. Join.
The extract of HeLa cells was prepared by adding an appropriate amount of RIPA buffer (0.1% SDS, 0.5% DOC, 1% NP-40, 150 mM NaCl, 50 mM Tris-Cl pH 8.0) to an appropriate amount of cell pellet. And homogenized (if necessary, sonication or nuclease treatment was used to cut the DNA), followed by centrifugation at 100,000 g for 60 minutes to collect the supernatant.
HeLa cell extract was added to the complex of protein G agarose and H3-mK9 5.1.1, incubated at 4 ° C. for 75 minutes, and then centrifuged (1,200 g for 1 minute). Was collected in the precipitate. The immunoprecipitate was sample-treated according to a standard method, and then subjected to electrophoresis on a polyacrylamide gel (15% concentration, gel pH: 8.8) without SDS.
After completion of the electrophoresis, the electrophoresed protein was transferred to a nylon membrane, and immunoblotting was performed using H3-mK9 5.1.1 (FIG. 4, lane 4).
As a result, only when immunoprecipitation was performed with H3-mK9 5.1.1 (FIG. 4, lane 4), histone H3 was recovered in the precipitate (FIG. 4, arrow). Therefore, it is clear that H3-mK9 5.1.1 specifically recognizes histone H3 mK9 (where Lys9 is methylated) present in cells and can be used for immunoprecipitation. Was.
[0042]
In addition, the immunoprecipitation method using H3-mK9 5.1.1 can be carried out even when the intracellular protein and DNA are cross-linked in advance by formaldehyde (FIG. 5). Before preparing the cell extract, the cells were treated in 1% formaldehyde, and then H3-mK9 5.1.1 (FIG. 5, IP; 5.1.1) and only protein G beads as negative control ( Immunoprecipitation was performed using FIG. 5, IP; Protein G beads). The method described in the above item [0041] was followed except that the cells were treated with 1% formaldehyde before preparing the extract of HeLa cells. As a result, it was revealed that H3-mK9 5.1.1 specifically recognized histone H3 in which Lys9 was methylated and immunoprecipitated even when the cell extract after cross-linking was used.
[0043]
Example 3: Chromatin immunoprecipitation (ChIP)
Since H3-mK9 5.1.1 of the present invention can be used for analysis by ChIP, it is a very useful tool in the analysis of genes that are transcriptionally regulated by the methylation of Lys9 of histone H3. Can be used.
The analysis by ChIP in this example was performed according to the protocol attached to Chromatin Immunorecipitation (ChIP) Assay Kit sold by Upstate biotechnology.
HeLa cells were cultured in a DMEM medium (5% FBS) at 10 cm dish, and then further changed to a serum-free medium (DMEM, 0% FBS). After culturing in a serum-free medium, formaldehyde was added to the medium to a final concentration of 1%, and the mixture was incubated at 37 ° C for 10 minutes.
Thereafter, the medium was removed, and the cells were washed with ice-cold PBS containing a protease inhibitor (1 mM PMSF, 1 μg / ml aprotinin).
Next, 1 ml PBS was added to the dish, the cells were transferred to a scraping tube, and then centrifuged at 700 × g at 4 ° C. for 3 minutes to collect the cells. The supernatant is discarded, and an appropriate amount of Lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, 1 mM PMSF, 1 μg / ml aprotinin) is added to the collected cells, and the mixture is stirred on a vortex mixer and then mixed on ice. Let stand for minutes.
Thereafter, the sample was subjected to ultrasonic treatment for 10 seconds at intervals of 20 seconds in ice for a total of four times. The obtained DNA fragment was checked by electrophoresis or the like so as to have a length of 200 to 1000 bp.
[0044]
A sample was prepared using a ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, 1 mM PMSF, 1 μg / ml). Aprotinin). To this, 40 μl of protein G beads (Amersham Bioscience) (1.5 ml beads, 600 μg sonicated salmon sperm DNA, 1.5 mg BSA, 4.5 mg recombinant protein G / 1.5 ml buffer; 10 mM Tris- HCl, pH 8.0, 1 mM EDTA, 0.05% sodium azide) was added and incubated at 4 ° C. for 30 minutes.
The supernatant was removed by centrifugation at 1,500 rpm for 15 seconds at 4 ° C., 1 μg of H3-mK9 5.1.1 was added, and the mixture was incubated at 4 ° C. overnight. After incubation, 50 μl of Protein G beads were added and incubated at 4 ° C. for 1 hour.
The Protein G beads were then washed at 4 ° C. for 3 minutes each under four washing conditions;
(A) Low Salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl),
(B) High Salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl),
(C) LiCl buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1),
(D) TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) × 2 Wash
After washing, 50 μl Elution buffer (10 mM DTT, 1% SDS, 0.1 M NaHCO 3) ₃ ), Elution was performed at room temperature for 15 minutes.
[0045]
4 μl of 5M NaCl was added to the eluted sample, and the mixture was incubated at 65 ° C. for 6 hours to remove crosslinks of the protein / DNA complex. Further, 2 µl of 0.5 M EDTA, 4 µl of 1 M Tris-HCl, pH 6.5, and 0.6 µl of 6 mg / ml Proteinase K were added, and the mixture was incubated at 45 ° C for 1 hour to digest proteins.
The DNA was extracted according to a standard method and dissolved in 50 μl of purified water.
Using the obtained DNA as a template, a PCR reaction was performed using the following primers (100 pmol each) for amplifying the Lite 1 region as an imprinting gene.
Forward direction (Lit1 CHIP-F); 5′-GCC GAG TCA GAA CGC ACT GG-3 ′ (SEQ ID NO: 5)
Reverse direction (Lit1 CHIP-R); 5′-TTC CCA ATC CCC CAC ACC TG-3 ′ (SEQ ID NO: 6)
As the reaction conditions, the amplification reaction was performed at 96 ° C. for 5 minutes for 1 cycle, 96 ° C. for 30 minutes, 56 ° C. for 30 minutes, 72 ° C. for 30 minutes from 25 cycles to 40 cycles, and 72 ° C. for 3 minutes. When the sequence of the amplified DNA fragment was confirmed, it was confirmed that the Lite 1 gene region was amplified, and it was apparent that H3-mK9 5.1.1 also had high specificity in the ChIP method. became.
[0046]
(4) Example 4: Immunofluorescence staining
The intracellular localization of Histone H3 in which Lys9 was methylated by the monoclonal antibody of the present invention was observed by immunofluorescence staining. HeLa cells were treated with 100% methanol stored at −20 ° C. for 15 seconds for fixation, washed three times with PBS, and then treated with 0.05% Triton X-100 for 2 minutes to remove cell membranes and nuclear membranes. Permeability treatment was performed. Antibodies to rabbits H3-mK9 5.1.1 and H3-mK4 (rabbit polyclonal antibody, UBI) diluted 1000-fold with PBS are each overlaid on the cells, and at 37 ° C. for 60 minutes while preventing the cells from drying. And reacted. Next, after the antibody reaction, the cells were washed four times with PBS, and 250-fold diluted FITC-labeled anti-mouse-goat antibody (Sigma) and TRITC-labeled anti-rabbit-goat antibody (Sigma) were each added. , H3-mK9 5.1.1 and H3-mK4 were overlaid on the reacted cells, and reacted at 37 ° C. for 45 minutes while preventing the cells from drying. After the reaction, the cells were washed four times with PBS, mounted using a fluorescent preservative, and then observed with a fluorescent microscope.
[0047]
As a result of microscopic observation, the heterochromatin region was stained with H3-mK9 5.1.1 (FIG. 6, H3-mK9 (di-)), and the euchromatin region was stained with the antibody against H3-mK4 (UBI). (FIG. 6, H3-mK4 (di-)). This result was consistent with the previous observation that Lys9 methylation of histone H3 is frequently observed in the heterochromatin region (Nakayama et al., 2001).
[0048]

[0049]
【The invention's effect】
The monoclonal antibody of the present invention has high specificity for histone H3 in which Lys9 is methylated, and can be used for Western blotting, immunofluorescence staining, immunohistochemical staining, and the like. Further, the immunoprecipitation method, chromatin It can be used for analysis by immunoprecipitation.
Therefore, the present invention is useful for analysis of imprinting genes in which the methylation of Lys9 of histone H3 is considered to be an important factor, and congenital diseases caused by abnormal imprinting.
Furthermore, since the relationship between abnormalities in the imprinting gene and specific tumors has recently been elucidated, by confirming the presence or absence of H3 Lys9 methylation, it is possible to diagnose the possibility of the occurrence of such tumors. It can be used effectively.
[0050]
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[0051]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows the results of an immunoblot showing the progress of screening for a monoclonal antibody against H3-mK9. SAM refers to S-adenosylmethionine and IB refers to immunoblot.
FIG. 2 is a diagram showing the antigen specificity of H3-mK9 5.1.1. Coomassie is Coomassie brilliant blue, which means that protein staining was performed.
FIG. 3 shows the results of examining the antigen specificity of H3-mK9 5.1.1 using a Biacore system (Biacore).
FIG. 4 shows the results of confirming the specificity of H3-mK9 5.1.1 for the histone fraction obtained by acid extraction from HeLa cells, and the results of H3-mK9 5.1. FIG. 1 shows that 1 specifically immunoprecipitates histone H3 in which Lys9 has been methylated. IgL indicates the position of the immunoglobulin light chain. Lysate indicates that the extract itself was immunoblotted. The arrow indicates the position of histone H3.
FIG. 5 shows the results of immunoprecipitation with H3-mK9 5.1.1 using an intracellular extract obtained by cross-linking intracellular proteins and DNA.
FIG. 6 shows the results of immunostaining of cells using H3-mK9 5.1.1. H3-mK9 (di-) and H3-mK4 (di-) show the results of staining using antibodies (UBI) against H3-mK9 5.1.1 and H3-mK4, respectively. The result of superimposing the stained images is shown.

Claims (13)

A monoclonal antibody or a fragment thereof that selectively binds to methylated histone H3 and discriminates between methylated histone H3 and unmethylated histone H3. The monoclonal antibody or a fragment thereof according to claim 1, wherein the methylated site of the methylated histone H3 is lysine 9 of histone H3. 3. The monoclonal antibody or a fragment thereof according to claim 2, which binds to a ninth lysine methylated in histone H3. 4. The monoclonal antibody or a fragment thereof according to any one of claims 1 to 3, which is obtained from a hybridoma deposited at the National Institute of Advanced Industrial Science and Technology under the deposit number of deposit number FERM P-19106. The monoclonal antibody or a fragment thereof according to any one of claims 1 to 4, which selectively binds to the methylated histone H3 in an ELISA method or a blot method. The monoclonal antibody or a fragment thereof according to any one of claims 1 to 4, which selectively binds to the methylated histone H3 in immunofluorescence or immunohistochemistry. The monoclonal antibody or a fragment thereof according to any one of claims 1 to 4, wherein the antibody selectively binds to the methylated histone H3 to immunoprecipitate the methylated histone H3 in an immunoprecipitation method. The monoclonal antibody or a fragment thereof according to claim 7, wherein the immunoprecipitation method is a chromatin immunoprecipitation method. A spleen cell of a mammal immunized with a KLH (keyhole limpet hemocyanin) cross-linked to a partial peptide of histone H3 in which ninth lysine is dimethylated (amino acid residue 4-14, SEQ ID NO: 1); The monoclonal antibody according to any one of claims 1 to 8, which is obtained by fusing the mammal with a cell capable of self-proliferation derived from the same or different animal and cloning the fused cell. A hybridoma that produces The hybridoma according to claim 9, wherein the hybridoma has been deposited with the National Institute of Advanced Industrial Science and Technology Biological Depositary under the accession number FERM P-19106. A method for producing an antibody, comprising culturing the hybridoma according to claim 9 or 10, and preparing the monoclonal antibody according to any one of claims 1 to 8 from a culture supernatant. A kit comprising the monoclonal antibody or a fragment thereof according to any one of claims 1 to 8, and a reagent for detecting the monoclonal antibody or a fragment thereof. A kit comprising the monoclonal antibody or a fragment thereof according to any one of claims 1 to 8, and a carrier necessary for precipitating histone H3 bound to the monoclonal antibody or a fragment thereof.

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