WO2008138572A1

WO2008138572A1 - Method and reagents for analyzing nucleic acid methylation reactions

Info

Publication number: WO2008138572A1
Application number: PCT/EP2008/003779
Authority: WO
Inventors: Albert Jeltsch; Sergey Ragozine; Rachna Goyal
Original assignee: Jacobs University Bremen Gmbh
Priority date: 2007-05-09
Filing date: 2008-05-09
Publication date: 2008-11-20
Also published as: DE112008001210A5; DE102007022274A1

Abstract

The invention relates to the area of screenings for and assessment of inhibitors of eukaryotic nucleic acid methylation reactions as well as reporter plasmids and methylation inhibitor standards useful for said screenings and assessment.

Description

Methods and reagents for the study of nucleic acid methylation reactions

The invention relates to the field of screening for and evaluation of inhibitors of eukaryotic nucleic acid methylation reactions, as well as useful reporter plasmids and methylation inhibitor standards.

Nucleic acid methylation, and particularly DNA methylation, is described in eukaryotes, particularly in mammalian cells, and particularly in humans, by the DNA methyltransferases Dnmtl (NCBI accession numbers: P26358, AAH92517, AAI26228, NP_001370), Dnmt3a (NCBI accession numbers: AAH 18214 , AAH23612, Q9Y6K1 NP_715640, NP_783329, NP_783328, NP_072046, AAH51864, AAY14761, AAH43617, AAH32392, AAL57039, AAD33084) and Dnmt3b (NCBI accession numbers: CAB53071, CAB53069, CAB53070, Q9UBC3, NP_787046, NP_787045, NP_787044, NP_008823, ABC48951, AAL57040, AAF04015, AAD53063, AAD53062, AAD31434, AAD31433, AAD31432). These methyltransferases transfer a methyl group of coenzyme S-adenosyl-L-methionine ("AdoMet") to the nucleic acids to be methylated, in particular DNA ("target DNA"). In many diseases, a change in DNA methylation pattern of genomic DNA is observed over the usual methylation pattern of genomic DNA. Such aberrant DNA methylation is considered to be a trigger or promoter of the emergence and / or progression, especially of tumor diseases. Therefore, it has often been attempted to develop DNA methylation inhibitors in order to use them as drugs for preventing or treating aberrant DNA methylations. Known DNA methylation inhibitors such as sinefungin are based on the principle to bind to the coenzyme binding site of the respective DNA methyltransferase and thus to prevent them from binding to the coenzyme AdoMet. However, these DNA methylation inhibitors also bind to the coenzyme binding sites of other enzymes that require AdoMet as coenzyme. Due to this cross-reactivity, these DNA methylation inhibitors cause numerous side effects.

Further, nucleotide analogs such as zebularin and decitabine are used to reduce DNA methylation. Once incorporated into DNA, the nucleotide analogs covalently bind the DNA methyltransferases to the DNA. This reduces the cellular supply of available DNA methyltransferases. Disadvantageously, this also leads to considerable DNA damage and to a very high DNA repair activity.

There is therefore a need to find further nucleic acid methylation inhibitors which, if possible, should not have the disadvantages of conventional DNA methylation inhibitors described above. The object of the present invention was therefore to provide a method and, in particular, a method suitable for screening for determining the inhibiting effect of an inhibitor on a eukaryotic nucleic acid methylation reaction.

The object is achieved by a method for determining the inhibition effect of an inhibitor on a eukaryotic nucleic acid methylation reaction, comprising the steps:

1. Providing a Methylierungsansatzes containing

(a) the components required for carrying out the nucleic acid methylation reaction,

(b) a reporter nucleic acid of preselected methylation degree, wherein the reporter nucleic acid Optionally, a section for episomal replication of reporter nucleic acid and

a reporter gene expressed as a function of the degree of methylation of the reporter nucleic acid, and

(c) the inhibitor to be tested;

2. Performing the methylation reaction in the methylation batch;

3. Determining the expression of the reporter gene in the presence of the inhibitor to be investigated; and

4. comparing the expression of the reporter gene determined in step 3 with the expression of the reporter gene at a preselected level of inhibition to determine the inhibitory effect of the inhibitor to be tested on the nucleic acid methylation reaction.

The method according to the invention makes it possible in an advantageously simple manner to determine the inhibiting effect of a (suspected) inhibitor of a eukaryotic nucleic acid methylation reaction under standardized conditions. The method is particularly useful as part of a screening method for finding a nucleic acid methylation inhibitor. It is particularly expedient to carry out at least step 3 in a high-throughput approach. Due to its standardisability and simplicity, the method according to the invention is particularly well suited for use in high-throughput approaches, more details below.

Essential for the process according to the invention is the structure of the report nucleic acid used. This optionally includes a section for episomal replication of the report nucleic acid. The reporter nucleic acid can therefore be propagated in a eukaryotic and in particular a mammalian cell line without having to integrate into the genome. expediently example, the reporter nucleic acid is replicable independently of viral transcomplementing proteins in the cell line. The reporter nucleic acid is then replicable in a variety of cell lines without the cell lines having to be previously transformed with viral genes needed for replication for trans-complementing factors. In particular, the reporter nucleic acid according to the invention avoids a limitation of the method according to the invention to COS7 cells and the SV40 Large Transforming Antigen System. Accordingly, the method according to the invention can be carried out in a cell line which is unchanged from the reporter nucleic acid. The method according to the invention is therefore particularly suitable for circumventing artifacts which can be caused by the expression of trans-complementing proteins.

Also preferably, the reporter nucleic acid can be stably transfected or otherwise expressably introduced into a target cell or cell line, preferably a eukaryotic and especially a mammalian cell line. In this case, the reporter nucleic acid is expediently integrated into the genome, for example a chromosome, of the target cell or target cell line and replicated with the genome or chromosome. In this way, it is possible to produce cell lines (reporter cell lines) which, even in the absence of a selection pressure, are significantly more stable than in the case of episomally reproducible reporter nucleic acids expressing the reporter gene in a manner permitting investigation of nucleic acid methylation inhibition. This contributes to an improved reproducibility of the nucleic acid methylation analyzes and also facilitates the handling of the reporter cell lines.

Particularly preferred are such episomally replicable reporter nucleic acids comprising a nuclear scaffold / matrix attachment region (S / MAR) Such a region is adapted for binding to the chromating scaffold of a eukaryotic cell nucleus, and preferably one Such reporter nucleic acids, but also reporter nucleic acids stably integrated into the genome of a target cell, advantageously allow the study of nucleic acid methylation inhibition in Mammalian cells and preferably in human cells. A method according to the invention, which makes use of these reporter nucleic acids, is therefore set up particularly advantageously, suitable nucleic acid

Methylation inhibitors especially for the treatment of aberrant DNA methylation patterns in mammalian cells and especially in human cells. The method according to the invention therefore particularly supports the development of medicaments for the prevention or treatment of tumor diseases.

The reporter nucleic acid may further comprise one or more genes for antibiotic resistance or other phenotypic trait (control feature) effective in prokaryotic and / or eukaryotic cells. This makes it possible to check the presence of the reporter nucleic acid in a pro- or eukaryotic cell independently of the expression of the reporter gene.

The reporter nucleic acid used in a method according to the invention additionally comprises a reporter gene expressed as a function of the degree of methylation of the reporter nucleic acid, preferably a gene for a fluorescent or luminescent protein, in particular green fluorescent protein (GFP, EMBL accession no .: gi | 1277124 and gb | AAC53684.1 ), Enhanced GFP (Enhanced Green Fluorescent Protein, EEFP, EMBL Accession No .: gi | 1377914 and gb | U55763.1 |), Red Fluorescent Protein (RFP, EMBL Accession No .: gi | 21464837 and gb | AF506027. 1), luciferase (EMBL Accession No .: gi | 2582510, gb | AAB82573.1, gi | 1469268 and emb | CAA59282.1) The products of these reporter genes are either readily detectable by their fluorescence or they generate one In both cases, the signal associated with the product of the respective reporter gene (fluorescence or luminescence) is characteristic of the report so that false signals can be excluded by other cell components. The inventive method thus allows a largely error-free determination of the degree of methylation of the reporter nucleic acid. The reporter gene is preferably under the control of a methylation of the reporter nucleic acid dependent promoter which suppresses the expression of the reporter gene in the methylated state of reporter nucleic acid and allows in the demethylated state, preferably a cytomegalovirus immediate early promoter.

A reporter nucleic acid according to the invention preferably also contains an origin of replication effective in prokaryotes The reporter nucleic acid according to the invention can therefore also be advantageously propagated in prokaryotic cells and harvested by conventional methods and processed for the transfection of eukaryotic cells.

Preferably, the methylation reaction is carried out in the inventive method in a cell line. For this purpose, the cell line is usually transformed with the reporter nucleic acid and subsequently amplified in the cell line, preferably by episomal replication, or by proliferation of cells with stably integrated reporter nucleic acid. The reporter nucleic acid is optionally methylated under the action of methyltransferases of the cell line. It is particularly preferable to transform the cell line with a methylated reporter nucleic acid. In episomal replication of reporter nucleic acid, the replication products are also methylated; a reporter nucleic acid integrated into the genome is methylated during the replication of the genome. The effect of a nucleic acid methylation inhibitor can then be demonstrated by the fact that the replication products of the reporter nucleic acid are no longer or no longer completely methylated. In particular, when using preferred reporter nucleic acids whose reporter gene is under the control of a repressed in the methylated state promoter, the effect of a methylation inhibitor can be detected by the detection of the reporter gene product. This detection is possible in a very simple and reproducible manner, particularly in the case of fluorescent or luminescent reporter gene products, so that a corresponding method according to the invention can be carried out in a high-throughput assay. The methylation mixture, in particular the cell line transformed with the reporter nucleic acid, preferably further comprises, therefore optionally in addition to a gene for a control feature as described above, a control nucleic acid comprising a control reporter gene which can be expressed independently of the degree of methylation of the control nucleic acid. In such a methylation approach, in addition to checking the effect of a nucleic acid methylation inhibitor, the viability of each cell cell to be tested can be checked, thus preventing the lack of viability of the cell from occurring Conveniently, only those cells of the cell line are considered in step 4 of the method according to the invention which express the control reporter gene, in which case it makes sense to use the control reporter gene for a coded by the reporter gene different fluorescent or luminescent gene product.

In a high-throughput approach, the cells of the cell line transformed with the report nucleic acid and preferably also with the control nucleic acid are examined for the expression of the reporter gene and preferably also of the control reporter gene. It is expedient to submit the transformed cells of the cell line in individual microtiter vessels, to multiply therein and thereby to pressurize the inhibitor to be examined. The application may be effected in any desired manner, for example by adding the inhibitor or by inducing the expression of an inhibitor gene. The cells of the cell line are propagated after or with continued exposure to the inhibitor, wherein the reporter nucleic acid and, if present, the control nucleic acid are also increased. Depending on the effectiveness of the inhibitor, the report nucleic acid is methylated if the inhibitor has no or only a slight effect in the particular concentration present, or if it has no or less methylated if the inhibitor has a nucleic acid methylation-inhibiting activity in the particular concentration present. Furthermore, the expression of the reporter gene and optionally the control reporter gene is determined, preferably by determining the fluorescence intensity on the Wavelength of the gene product of the reporter gene and optionally of the control reporter gene. By comparing the particular expression of the reporter gene, optionally taking into account only those cells which express the control reporter gene, with the expression of the reporter gene at a preselected degree of inhibition, the inhibitory effect of the inhibitor to be examined on the nucleic acid methylation reaction can be simple and reproducible Be determined manner.

In preferred embodiments of the method according to the invention, in step 4 the expression of the reporter gene in the presence of the inhibitor to be tested is compared with an expression of the reporter gene in the presence of a preselected concentration of a standard methylation inhibitor selected from the group consisting of 5'-azacytidine and a peptide of up to 17 amino acids in length, containing an amino acid sequence according to Figure 7, wherein "x" is phosphorylated serine, and particularly preferably the amino acid sequence EKIY (I or M) SKI (V or L) (V or I ) E, where the letters are individual amino acids according to IUPAC

(http://www.chem.qmul.ac.uk/iupac/AminoAcid/). Here, "standard methylation inhibitor" means that this nucleic acid

Methylation inhibitor is used as a standard for several rounds of the inventive method or for several inhibitors to be examined. Particularly preferred is a standard methylation inhibitor consisting of a peptide of up to 17 amino acids comprising the amino acid sequence E K I Y I phosphorylated S K I V V E. This peptide has proven to be a particularly good nucleic acid methylation inhibitor.

Unless otherwise stated, any combination of features of embodiments of the method according to the invention with one another and with features of an embodiment of a reporter nucleic acid according to the invention and embodiments of a control nucleic acid described here is possible.

The invention will be described in more detail below with reference to the examples, without the examples intended to limit the scope of the claims. Example 1: Preparation of a reporter nucleic acid

The reporter nucleic acid should be episomally replicable in plasmid form in eukaryotic cells, especially in human cell lines. The reporter gene selected was a gene for green fluorescent protein (enhanced green fluorescent protein, eGFP, EMBL accession number: see above).

An approximately 2 kb backbone / matrix adhesion region (S / MAR range; Jenke et al., Proceedings of the National Academy of Sciences USA, 101 (2004), 11322-11327) was transformed into a peG FP-C 1 - Vector (Clontech) downstream of the eGFP gene. The vector also contains a gene for neomycin phosphotransferase as an antibiotic resistance gene active in prokaryotes and eukaryotes (kanamycin and neomycin resistance), as well as a pUC "origin of replication" as a bacterial replication initiation area.

FIG. 1 shows a map of the reporter nucleic acid.

Example 2: Methylation of reporter nucleic acid

The reporter nucleic acid was isolated with a methyltransferase from Spiroplasma sp. (M.Sssl, New England Biolabs). Methyltransferase methylates all cytosine residues in double-stranded cytosine guanine dinucleotides. Methylation was carried out in vitro according to the manufacturer's instructions.

Methylation was used: 10 μg reporter nucleic acid, 1 × buffer (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH 7.9 at 25 ° C.), 160 μM S-adenosylmethionine (Sigma), 20 U M.Sssl (NEB, 5 μl) in a total volume of 50 μl. The mixture was incubated for one hour at 37 ⁰ C. The methylation was stopped by 20 minutes heating at 65 ⁰ C. The methylated reporter nucleic acid was nigt aufgerei- by column purification. The completeness of methylation of reporter nucleic acid was checked by restriction digestion with Hpall and subsequent gel electrophoresis. This restriction nuclease cleaves only unmethylated CCGG nucleic acid segments. As a control, the methylated reporter nucleic acid was also digested with Mspl, a methylation insensitive isoschizomer of Hpall. The completeness of the methylation could be confirmed. Fig. 2 shows an electrophoresis gel of reporter nucleic acid digested with Hpall.

Example 3: Transfection of HEK293 cells with the methylated reporter nucleic acid

The methylated reporter nucleic acid and an unmodified, red fluorecent protein (EMBL accession number: see above) encoding plasmid (control nucleic acid) were transfected with the transfection reagent FuGene-6 (Roche) in HEK293 cells. For this purpose, the HEK 293 cells were first in Dulbecco's Modified Eagle medium (DMEM), supplemented with 5% fetal bovine serum (FBS), 100 U / ml penicillin / streptomycin and 2 mM glutamine in a humid incubator at 5% CO ₂ and 37 ⁰ C incubated. The cells were in the 25th-35th Passage. The day before transfection, cells were seeded in 6-well plates. DNA (reporter nucleic acid and control nucleic acid) in a total amount of 1 μg per well were mixed with 4 μl of the transfection reagent according to the manufacturer's recommendations and, after 20 minutes of incubation, added dropwise to the cells.

The transfected cells were examined for red and green fluorescence with a Carl Zeiss LSM501 meta-microscope. Green and red fluorescence were plotted against each other. The highest fluorescence value of non-transfected cells was used as limit for a detected fluorescence.

Example 4: Methylation of the reporter nucleic acid leads to significant transcriptional repression of the reporter gene Hek293 cells were transfected with methylated and non-methylated reporter nucleic acid and simultaneously with control nucleic acid as described in Example 3. Fig. 3 shows the transfection results.

When transfected with unmethylated reporter nucleic acid, cells which fluoresced both red and green were evenly distributed in the cell culture on the fifth day post-transfection (Figure 3A). The fluorescence distribution (Fig.

3C) shows that the fluorescence of the cell culture is distributed to cells with green

Fluorescence (region 1), red fluorescence (region 2) and simultaneously green and red fluorescence (region 3). The number (FIG. 3E) of the green or red fluorescing cells is approximately the same; this indicates an approximately equally strong

Expression of Reportemucleic Acid and Reporter Fluorescence Reporter Genes

Control nucleic acid out.

Upon transfection with methylated report nucleic acid, no green fluorescent cells were observed on the fifth day post transfection, only red fluorescent cells were detected (Fig. 3B). Accordingly, the fluorescence distribution (FIG. 3D) shows a clear accumulation in the area of the only red-fluorescing cells (area 2). The number of both green and red fluorescent cells (Figure 3F) is negligible compared to the number of red fluorescent cells alone.

The results show that the transfection was successful (as red fluorescence could be detected) and that methylation of the reporter nucleic acid results in an almost complete loss of the expression of the reporter gene eGFP (since no green fluorescence could be detected in this case).

Example 5: Inhibition of endogenous DNA methylation leads to the recovery of expression of the reporter gene of methylated reporter nucleic acids

Hek293 cells were transfected with methylated reporter nucleic acid and unmethylated control nucleic acid as described in Example 3. On the day of Transformation, the cells were treated with either the demethylating reagent 5'-azacytidine (Sigma) or buffer control. This time was taken as the "time 0 h" all expression studies. The reagent was added freshly for 7 days daily at a final concentration of 2 μM, while in the control experiment only buffer and solvent of the reagent were added. The experiments were repeated twice.

FIG. 4 shows the development of the expression of the reporter gene of reporter nucleic acid and demethylating and under non-demethylation cultivation conditions. Already on the second day, an increase in the green fluorescence and thus an increase in the expression of the reporter gene of the reporter nucleic acid could be detected (FIG. 4C). The increase in reporter gene expression was continuously observed until the 7th day of the experiment, then the experiments were discontinued. After culturing for 7 days under demethylating conditions, about 75% of the expression of the reporter gene was observed, as is achieved when using non-methylated reporter nucleic acid (maximum achievable expression). However, under non-demethylating culture conditions, expression of the reporter gene of the methylated reporter nucleic acid was only 5% of the maximum achievable expression.

FIG. 5 shows the expression of the reporter gene of the reporter nucleic acid and of the control nucleic acid. HEK293 cells cultured under non-demethylating conditions showed virtually no expression of the reporter gene eGFP (Fig. 5A) in accordance with the findings of Example 4. In contrast, HEK293 cells cultured under demethylating conditions showed strong expression of the reporter nucleic acid reporter gene (Figure 5B).

Example 6: High throughput approach to finding nucleic acid methylation inhibitors

HEK293 cells were transfected with methylated reporter nucleic acid and non-methylated control nucleic acid as described in Example 3, and after transfecting into 96-well microtiter plates. The cell cultures were measured for green and red fluorescence with a Safire XFLU0R4 (Tecan) fluorimeter. The cell cultures were treated as described in Example 5 5'-azacytidine (Sigma) for preparing demethylierender cultivation conditions. Figure 7 shows schematically the flow of the approach.

Under demethylating conditions, expression of the reporter gene eGFP, which was 120-fold stronger than that achieved under non-demethylating conditions, was determined five days after transfection. The expression of the reporter gene of the control nucleic acid was about 4 times more potent under demethylating culture conditions than under non-demethylating conditions. This indicates a low non-specific transcriptional activation by 5'-azacytidine. However, the increase in the expression of the reporter gene of the reporter nucleic acid can not be explained by this, but is the result of the demethylation of the reporter nucleic acid.

The mixture was repeated using, instead of the demethylating reagent 5'-azacytidine, a peptide having the amino acid sequence EKIYISKIWE with phosphorylated serine residue. Here, too, a significantly stronger eGFP expression after 5 days under demethylating cultivation conditions could be detected in comparison to non-demethylating cultivation conditions.

Claims

Expectations

1. Method for determining the inhibitory effect of an inhibitor on a eukaryotic nucleic acid methylation reaction, comprising the steps:

1. Providing a methylation approach containing

(a) the components required to carry out the nucleic acid methylation reaction,

(b) a report nucleic acid with a preselected degree of methylation, where the report nucleic acid

- optionally a section for episomal replication of the report muleic acid, and

a reporter gene expressed depending on the degree of methylation of the reporter nucleic acid, and

(c) the inhibitor to be examined;

2. Carrying out the methylation reaction in the methylation approach;

3. Determine the expression of the reporter gene in the presence of the inhibitor to be examined; and

4. Comparing the expression of the reporter gene determined in step 3 with the expression of the reporter gene at a preselected degree of inhibition to determine the inhibitory effect of the inhibitor to be examined on the nucleic acid methylation reaction.

2. The method according to claim 1, characterized in that the expression of the reporter gene is controlled by a regulatory element which suppresses the expression of the reporter gene in the methylated state of the reporter nucleic acid and enables it in the demethylated state.

3. The method according to claim 1 or 2, characterized in that the methylation reaction is carried out in a cell line.

4. The method according to claim 3, characterized in that the reporter nucleic acid can be replicated in the cell line independently of viral trans-complementing proteins.

5. The method according to claim 3 or 4, characterized in that the reporter nucleic acid is transfected into the cell line in the methylated state.

6. The method according to any one of the preceding claims, characterized in that the methylation approach further comprises a control nucleic acid which comprises a control reporter gene which can be expressed independently of the degree of methylation of the control nucleic acid.

7. Method according to one of the preceding claims, characterized in that the reporter gene and/or the control reporter gene is a gene for a fluorescent protein.

8. Method according to one of the preceding claims, characterized in that step 3 is carried out in a high-throughput approach.

9. The method according to any one of the preceding claims, characterized in that in step 4 the expression of the reporter gene in the presence of the inhibitor to be examined is compared with an expression of the reporter gene in the presence of a preselected concentration of a standard methylation inhibitor which is selected from the group consisting from 5'- azacytidine and a peptide up to 17 amino acids long containing the amino acid sequence EKIY (I or M) SKI (V or L) (V or I) E.

10. Reporter plasmid, comprising

1. a bacterial replication start area (origin of replication),

2. a reporter gene for green fluorescent protein (GFP) or enhanced GFP (eGFP) under the control of a promoter active in eukaryotic cells,

3. a core framework/matrix attachment region (S/MAR) downstream of the promoter, and optionally

4. one that is effective in prokaryotic and/or eukaryotic cells

Antibiotic resistance gene

11. Use of a reporter plasmid according to claim 10 as reporter nucleic acid for carrying out a method according to one of claims 1 to 9.

12. A method of preparing a medicament for treating aberrant DNA methylation, comprising

a) carrying out a method according to one of claims 1 to 9 to provide one or more DNA methylation inhibitors,

b) selecting a DNA methylation inhibitor provided in step a), and

c) mixing the DNA methylation inhibitor selected in step b) in a pharmaceutically effective amount.