JP5716153B2 - Reporter assay system for circadian rhythm control by DNA methylation of clock genes - Google Patents

Description

  The present invention relates to a reporter assay for monitoring a circadian rhythm control mechanism by DNA methylation of a clock gene and an established cell line used for the reporter assay.

In addition to hereditary sleep disorders, various sleep disorders associated with 24-hour society have become social problems. The onset of a series of sleep disorders called circadian rhythm sleep disorders is thought to be related to the biological clock composed of clock genes, but the detailed mechanism is unknown.
As a treatment method for sleep disorders, high-intensity phototherapy requiring a special device, administration of vitamin B12 (Non-patent Document 1), and melatonin preparation (Patent Document 1) are generally used.
In addition, benzodiazepine drugs (Non-patent Document 2) are generally used as sleeping drugs, and various half-life drugs have been developed from long-time types to short-time types, intermediate types, and long-term types. . However, treatment methods for sleep disorders using these sleeping drugs are coping therapy, and it is difficult to fundamentally treat sleep disorders. In addition, there are many side effects such as sleepwalking symptoms, and the usage thereof requires careful attention. Recently, a 4-oxoimidazolidine-2-spiropiperidine derivative having an action as an agonist of a nociceptin receptor is expected to have a circadian rhythm sleep disorder treatment effect (Patent Document 2), but is still under research and development. Side effects are also a concern.
Research and development of sleep-improving agents from natural ingredients and foods and drinks is also active, using tea leaves derived teaamine (Patent Document 4), adding whey such as fermented whey with endogenous melatonin secretion effect (patent In addition to literature 5), sleep improvement agents using ginseng extract (non-patent literature 3), phosphatidylcholine (non-patent literature 4) contained in salmon oil have been proposed. These are all derived from materials used for food and drink, so they are safe and can be taken on a daily basis, but their detailed sleep improvement effects and mechanisms of action are fully elucidated. However, there is a great individual difference in the effect.
As described above, most of the conventional treatment methods have an unknown mechanism of action, or are intended to normalize the biological rhythm by adjusting the phase of the biological clock, and the period of the biological clock is abnormal. It is not a fundamental cure for disorders caused by.

On the other hand, circadian rhythm is controlled by a circadian clock, and in recent years it has become clear that the rhythm of the circadian clock is engraved by a series of genes called clock genes. The biological clock has two functions: phase adjustment of biological rhythm and adjustment of cycle length. Regarding circadian rhythm phase regulation, the expression of clock genes is induced by many humoral factors, at least in cell culture systems, and it is assumed that there are many regulatory factors in living organisms. (Non-Patent Document 5).
From the results of recent knockout experiments using circadian rhythm-related genes, it has been almost established that the gene that is the center of biological clock regulation is the Bmal1 gene (Non-patent Document 6).
Traditionally, reporter assays at the cultured cell level using NIH3T3 cells into which a plasmid containing the RORE sequence in the Bmal1 gene promoter, a foreign SV40 promoter, and a luciferase gene have been introduced for transient expression have been performed in mammals. It has been known that circadian rhythm via Bmal1 gene in the body can be reproduced (Non-patent Document 7). However, in such a conventional reporter assay method using cultured cells, the state of the plasmid in the cell is unstable, and the cell culture is continued in a state where the test substance is added to the medium, and the influence is accurately determined. It was not possible to measure.

By the way, the expression of clock genes including Bmal1 gene is controlled by DNA methylation in the promoter region, but a correlation between abnormal DNA methylation in the human Bmal1 gene promoter region and the malignancy of blood tumors has been pointed out, It has been suggested that the circadian rhythm is disturbed by gene inactivation due to methylation of the promoter region of the human Bmal1 gene, thereby causing a hematological malignancy (Non-patent Document 8). Therefore, elucidating the mechanism of rhythm disorders caused by DNA methylation in the promoter region of the Bmal1 gene not only aims at improving sleep disorders due to circadian rhythm disorders, but also elucidates the mechanism of hematological malignancies, and consequently It is also very useful for prevention and treatment.
However, reporter gene knock-in mice and conventional reporter assay methods using established cells that stably hold a reporter gene are basically normal, so the rhythm disorder model system by DNA methylation is Don't be.
That is, the conventional screening method for circadian rhythm disorders is a search based on biological rhythm in normal clock gene expression using normal individuals or cells, and the clock gene is DNA such as cancer. It was not suitable for the development of treatments when inactivated by methylation.
Accordingly, there has been a strong demand for a reporter assay method that can monitor the expression control mechanism by DNA methylation of a clock gene and the establishment of a cell line therefor.

Japanese National Patent Publication No. 8-502259 WO2003 / 010168 WO2003 / 063907 WO2005 / 097101 WO2005 / 097101 JP 2009-183197 A

“Clinic of circadian rhythm sleep disorder” Shigeru Chiba, Kenichi Honma, Emerging Medical Publishers, 2003 “Sleep disorder response and treatment guidelines, Makoto Uchiyama, Jiho, 2002” Young Ho Rhee, et al., Psychopharmacology, 101, p. 486-488 (1990) Hidehiko Hibino, Food Style 21, Vol. 7, No. 3, p. 50-53 (2003) Curr Biol 2000,10: 1291-4 Cell, 2000,103,1009-1017 Nature, 2002,418,534-539 Caner Res., 2009,69,8447-8454 Mol.Cell.Biol., 2008,28,3477-3488 Neuron, 2004,43,527-537 Bioscience Rep., 2010, in press Methods in Enzymol., 2005, 393, 269-288

  An object of the present invention is to provide an established cell line suitable for screening a substance capable of regulating the suppression of expression by DNA methylation of a clock gene, and to provide a reporter assay method using the cell line. .

Transcriptional regulation with circadian rhythm in the Bmal1 gene (Non-patent Document 6), which is considered to be the center of biological clock regulation, can be detected at the level of cultured cells by a reporter assay using a luciferase gene ( Non-patent document 7), however, in such a conventional reporter assay method using cultured cells, the state of the plasmid in the cells is unstable, and the cell culture is continued with the test substance added to the medium. The effect could not be measured accurately.
Thus, unlike the reporter assay method, which was previously intended to search for a substance or gene that acts transiently on a specific region such as the promoter region of the conventional Bmal1 gene, the present inventors previously formed circadian rhythms. A reporter assay system for searching for a substance that acts on the cycle itself for a long period of time and accelerates or slows the cycle has been constructed and has been applied for a patent (Japanese Patent Application No. 2009-175576). Specifically, first, a reporter plasmid (FIG. 1) in which a luciferase gene serving as a reporter is linked to a region (positions −202 to +27) containing a “RORE” sequence and a transcription initiation site in a mouse Bmal1 gene promoter is prepared. It was introduced into the NIH3T3 cell line, and a cell line that produces luciferase with stable circadian rhythm was established. And, it is an assay system in which various substances are allowed to act on the cell line to monitor its luciferase activity. This reporter assay system is extremely effective for the discovery of substances that act on the transcriptional regulatory site of the Bmal1 promoter region and regulate the circadian rhythm cycle. However, since the cell line used is derived from normal cells, the clock gene DNA is not methylated and basically engraves a normal circadian rhythm. Therefore, rhythm disorders due to DNA methylation in the clock gene promoter region It cannot be used for the elucidation of substances that cause the circadian sensation or the search for substances for improving the circadian rhythm disorder.

By the way, in leukemia cells and the like, it is known that the expression of the Bmal1 gene is suppressed by DNA methylation of the promoter region, and there is a correlation between the degree of methylation of the region and the malignancy of the tumor. It has been reported (Non-Patent Document 8). Based on this finding, the present inventors examined the promoter region of the Bmal1 gene in the genomic sequence of human leukemia cell RPMI8402 in detail, and as a result, confirmed that the promoter region was highly DNA methylated. . Further, it was found that the RPMI 8402 cells were treated with 5-aza-2'-dCTP (Sigma), which is a well-known DNA demethylating agent, to restore the expression of Bmal1 gene and circadian rhythm. These results suggest that, in RPMI8402 cells, Bmal1 gene expression was suppressed by DNA methylation of the promoter region of Bmal1 gene, resulting in loss of circadian rhythm.
Therefore, similarly to the reporter plasmid, a minimal human Bmal1 gene promoter region (Non-patent Document 11 and Patent Document 6) necessary for monitoring intracellular circadian rhythm transcription was connected to the luciferase gene. A reporter plasmid (FIG. 1) was introduced into RPMI 8402 cells to establish a cell line in which the plasmid was stably maintained. In this established cell line, it was confirmed that the circadian rhythm was not recorded, and the influence of the demethylation treatment on the transcription rhythm in the established cell line was examined. When the established cell line was cultured in the presence of 5-aza-2'-dCTP, expression of the Bmal1 gene due to demethylation of the endogenous Bmal1 gene promoter region was observed, and circadian rhythm expression of the reporter gene was observed. (Figure 4).
Based on the above results, by monitoring the luciferase activity using this established cell line, it is possible to know the biological rhythm through transcription of the endogenous Bmal1 gene in RPMI8402 cells easily and accurately, and it is specific to human lymphocyte tissue. It was also possible to evaluate the effects on various circadian rhythms. Moreover, it became possible to evaluate the influence of various substances on circadian rhythm when the expression of Bmal1 gene was suppressed by DNA methylation.
By obtaining the above knowledge, the present invention was completed.

That is, the present invention includes the following inventions.
[1] A lymphoid cancer cell line transformed with a reporter plasmid having a reporter gene downstream of the Bmal1 promoter, which is an established cell line stably holding a reporter gene transcribed by the Bmal1 promoter.
[2] The lymphoid cancer cell line is human leukemia cell RPMI8402, and a reporter plasmid in which a luciferase gene is linked to a region of −201 to +24 containing “RORE” of the human Bmal1 promoter is used as a reporter plasmid, The established cell line according to 1].
[3] A method for screening for an agent for improving circadian rhythm disorders caused by DNA methylation of a clock gene, wherein a test substance is added to a medium containing the established cell line described in [1] or [2] And a method comprising measuring the luminescence or fluorescence intensity of the reporter protein over time and determining whether to circadian rhythm.

According to the present invention, an established cell line in which the clock gene promoter region including the Bmal1 gene is controlled to be suppressed by DNA methylation, and the reporter gene capable of evaluating the recovery of circadian rhythm by releasing the suppression mechanism It was possible to provide a lymphoid cancer-derived established cell line that stably holds
By using the established cell line of the present invention, it is possible to search for a fundamental treatment method for circadian rhythm disorders caused by abnormalities in the period of the circadian clock due to DNA methylation of the clock gene, which could not be observed until now. It was also possible to estimate the effect of drugs on lymphocyte tissue-specific rhythms.

Construction of reporter plasmid The positions of RORE (white) and SAF-A binding sequence (ash) in the genetic maps of the mouse and human Bmal1 promoter regions are shown. The region of −201 to +24 in the human Bmal1 promoter containing RORE was inserted upstream of the luciferase gene of the plasmid pGL3-dLuc. Methylation of Bmal1 promoter region in human leukemia cell RPMI8402 The methylation state of the Bmal1 promoter region in human leukemia cell RPMI8402 is shown by independent 4 clone analysis by Bisulfite sequence. CpG nucleotides that can be methylated (vertical line), methylated cytosine (black), unmethylated cytosine (white). Induction of Bmal1 gene expression by demethylation of DNA RPMI8402 cells are treated for 48 hours in the presence of 2 μM 5-aza-2'-dCTP (+). Actin and Bmal1 gene expression (RT-PCR) is shown. Regulation of circadian rhythm by 5-aza-2'-dCTP Chemiluminescence measurement in a medium containing 2μM 5-aza-2'-dCTP and the luminescent substrate luciferin of the established cell line (CPT-K stable clone 3) of the present invention Results are shown. Luminescence is measured for 1 minute every 10 minutes. The time course of chemiluminescence in the presence (Aza-dCTP) and absence (control) of 2 μM 5-aza-2'-dCTP is shown. In the figure, the gray curve shows the actual measured value, and the black curve shows the graph fitted by the least square method.

1. Regarding circadian rhythm and Bmal1 gene, there are four main factors involved in the regulation of the body clock, the Bmal1 and Clock genes that promote the ticking of the body clock, and the Cry and Period genes that act as suppressors.
Among these genes, mice that knocked out the Bmal1 gene no longer have any behavioral rhythm, and behave as if the biological rhythm was completely destroyed. In the single clock knockout mice such as other clock genes such as Period and Cry, only abnormalities are observed in the activity cycle, and the Bmal1 gene is the only gene that does not show any behavioral rhythm formation when the single gene is destroyed. Furthermore, in Bmal1 gene knockout mice, expression of clock genes Period1 and Period2 is almost completely suppressed in the suprachiasmatic nucleus (SCN), which is the clock center. From the above, it is considered that the Bmal1 gene is the center of biological clock regulation (Non-patent Document 6).
RORE in the Bmal1 gene promoter region is also called RORα and REV-ERBα recognition sequence, and it is known that RORα and REV-ERBα bind to each other to activate and repress Bmal1 gene promoter activity, respectively (non- Patent Document 10) recently discloses a nuclear matrix formed by binding of SAF-A protein to the “SAF-A binding region (positions −27 to +266)” in the downstream region of the mouse Bmal1 gene promoter. The regulatory mechanism of circadian rhythm that regulates the transcription of Bmal1 gene by structure was elucidated (Non-patent Document 11 and Patent Document 6).

2. Construction of a reporter plasmid and establishment of a lymphoid cancer cell-derived cell line carrying the reporter plasmid The reporter plasmid used in the present invention is a transcriptional regulator of all clock genes related to circadian rhythm that functions normally in the transcriptional control of the Bmal1 gene in the cell. It is for monitoring that the expression was normally performed. In Examples of the present invention, pGL3-dLuc (Non-patent Document 7), which is a plasmid used for searching for a substance acting on a specific region of the Bmal1 gene promoter in Non-Patent Document 7, is used upstream of the luciferase gene. The region of −201 to +24 containing “RORE” of the human Bmal1 promoter region was inserted (FIG. 1). The specific region of the human Bmal1 promoter used here is preferably the smallest unit that exhibits Bmal1 promoter activity, but includes the region that exhibits circadian rhythm promoter activity, for example, the human Bmal1 promoter excluding the SAF-A binding region. The full length may be used. Instead of the -201 to +24 region of the human Bmal1 promoter region, the -202 to +27 region of the mouse Bmal1 promoter region or the mouse Bmal1 promoter region containing it, or a homologous region derived from another mammal may be used. . In this case, the base sequence of the human Bmal1 promoter region can be obtained from public base sequence databases such as GenBank ID: NC — 000011.9, and the base sequence of the mouse Bmal1 promoter region can be obtained from the same ID: NC — 000073.5. Further, as a reporter gene, a fluorescent gene such as a GFP gene may be used instead of the luciferase gene.
As the lymphoid cancer cells, AKATA cells can be used in addition to the RAJI cells described in Non-Patent Document 8, but in this example, human acute lymphoma that does not have any circadian rhythm as lymphatic cancer cells. Sexual leukemia cells (T cell-derived) RPMI8402 strain was selected. In this example, the Bmal1 promoter region in the genomic gene of the RPMI 8402 strain is methylated at high density (FIG. 2), and a demethylating agent (that can desorb a methyl group from methylated DNA ( For example, it has been confirmed that when 5-aza-2'-dCTP) is allowed to act, the Bmal1 promoter activity is restored and the circadian rhythm begins.
Then, the reporter plasmid is introduced into the RPMI8402 strain to obtain transformed RPMI8402 cells (according to the technique of Non-Patent Document 7), and subculture is continued to produce luciferase stably without circadian rhythm. A transformed RPMI8402 cell line was established and deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center under the name of “CPT-K stable clone 3” (Accession Number: FERM AP-22006, Accepted Date: 2010) August 24th).
The biological species derived from lymphoid cancer cells and the biological species derived from the Bmal1 promoter region in the reporter plasmid may be different, but are preferably the same. For example, when using mouse-derived lymphoid cancer cells, it is preferable to use a reporter plasmid in which the -202 to +27 region of the mouse Bmal1 promoter region is inserted upstream of the reporter gene.

3. Reporter assay method using established lymphoid cancer cell-derived cell line The test substance is added to the culture medium of the established lymphoid cancer cell-derived cell line of the present invention, and the luminescence intensity of luciferase is monitored over time while culturing. As a result, the methyl group of the Bmal1 promoter region endogenous to the established cell is removed, the Bmal1 promoter activity is restored, and the Bmal1 gene is expressed. At that time, the circadian rhythm oscillation mechanism will function if expression of other clock genes is also expressed at the same time. That is, the expressed BMAL1 protein and CLOCK form a BMAL1-CLOCK complex, which activates transcription of Period and Cry. A series of circadian rhythm formation cycles in which the expression of Period and Cry genes is controlled in order to suppress the transcriptional activation function of Periodic and Cry genes of BMAL1-CLOCK complex by transferring the generated Period and CRY proteins into the nucleus (Negative feedback loop) starts to work. Transcription is regulated by the binding of RORα and REV-ERBα to the RORE sequence present in the human Bmal1 promoter in the reporter plasmid that stably expresses luciferase in the established cells. The gene expression of these RORα and REV-ERBα genes is also regulated by the circadian rhythm formation cycle (negative feedback loop) by the above BMAL1, CLOCK, PERIOD, and CRY. That is, when the circadian rhythm formation cycle functions, RORα and REV-ERBα become functional with respect to circadian rhythm, and the luciferase luminescence intensity transcribed by the Bmal1 promoter that is transcriptionally regulated by RORα and REV-ERBα Engrave the circadian rhythm.
Therefore, by monitoring the luminescence intensity of luciferase in the established cells over time, there is a substance that can cancel the circadian rhythm suppression mechanism by methylation in the Bmal1 promoter region of the test substance and the expression control DNA region of other clock genes. It became possible to search for the first time.

4). Screening method
The screening method for substances that improve rhythm disorders by DNA methylation is performed according to the following procedure.
(1) 10 ⁴ to 10 ⁶ cells, preferably 10 ⁵ to 10 ⁶ cells, of the established lymphoid cancer cell-derived established cell line of the present invention are seeded at a time, and cultured for 2 hours in a medium containing 50% serum for 2 hours. To reset.
(2) A test substance and a luminescent substrate (for example, luciferin) are added to the medium and incubated at 37 ° C. for 2 hours.
(3) While performing cell culture at room temperature (25 ° C.) for 5 to 10 days, the luminescence intensity or fluorescence intensity of the reporter protein is monitored over time. In the case of luciferase luminescence, measurement is typically performed every 10 minutes for 1 minute. The measured value is preferably fitted by the least square method (Non-patent Document 12).
(4) When the change in the luminescence intensity of the reporter protein starts to circadian rhythm after a certain time has elapsed, it is determined that the test substance is a substance that improves rhythm disorders caused by DNA methylation abnormality.

5. An agent for improving sleep disorders caused by abnormal DNA methylation in the clock gene promoter region
The expression of clock genes such as the Bmal1 gene is regulated by DNA methylation in the promoter region. However, in lymphoid tumor cells, the Bmal1 gene promoter region is abnormally DNA methylated and the degree of DNA methylation There is a correlation between malignancy and tumor malignancy (Non-patent Document 8). Since such abnormal DNA methylation is thought to cause sleep disorders due to circadian rhythm disorders such as leukemia patients, the clock genes screened by the reporter assay system of the present invention have a DNA methylation-degrading activity. The test substance can be used in a pharmaceutical composition for preventing or treating sleep disorders caused by abnormal DNA methylation.
The target animal causing such sleep disorder is typically a human, but primates such as humans, pets such as dogs and cats, domestic animals such as cows and horses, and laboratory animals such as mice and rats. Including mammals.

EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not particularly limited to these examples.
Technical terms used in the present invention have meanings commonly understood by those skilled in the art unless otherwise defined. Unless otherwise specified, specific procedures and conditions such as genetic recombination techniques, PCR methods, and other techniques used in the examples of the present invention are as follows: Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition. Harbor Laboratory Press, Plainview, NY (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); DMGlover et al.ed., "DNA Cloning", 2nd ed., Vol. 1 to 4, ( The Practical Approach Series), IRL Press, Oxford University Press (1995); Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995; "Lecture 1, Genetic Research Method II", Tokyo Chemical Doujin (1986); edited by Japanese Biochemical Society, "Shinsei Chemistry Experiment Course 2, Nucleic Acid III (Recombinant DNA Technology)", Tokyo Chemical Doujin (1992); R. Wu ed. , "Methods in Enzymology", Vol.68 (Recombinant DNA), Academic Press, New York (1980); R.Wu et al.ed., "Methods in Enzymology", Vol.100 (Recombinant DNA, PartB) & 101 (Recombinant DNA, Part C), Academi c Press, New York (1983); R. Wu et al.ed., "Methods in Enzymology", Vol. 153 (Recombinant DNA, Part D), 154 (Recombinant DNA, Part E) & 155 (Recombinant DNA, Part F), Academic Press, New York (1987) or the like, the method described in the literature cited therein, or a method substantially similar thereto.
Moreover, the description content of the prior art literature or the patent application specification cited in the present invention is incorporated as the description of this specification.

(Example 1) Establishment of human leukemia cell RPMI8402 strain stably holding the luciferase gene transcribed by the Bmal1 promoter (1-1) Construction of reporter plasmid To monitor circadian rhythm transcription in cells As a minimal promoter region necessary for rhythmic expression of the human Bmal1 gene, a region of −201 to +24 (Non-patent Document 9) including “RORE” is used as a luciferase of plasmid pGL3-dLuc (Non-patent Document 7). A Bmal1 reporter plasmid was prepared by inserting the gene upstream (FIG. 1).

(1-2) Confirmation of methylation of Bmal1 promoter region in human leukemia cell RPMI8402 Gene DNA was extracted from human leukemia cell RPMI8402, and the methylation state of Bmal1 promoter region was analyzed by bisulfite sequence. The sequence results of 4 independent clones are shown in FIG. In the figure, vertical lines represent CpG nucleotides that can be methylated, black circles represent methylated cytosine, and white circles represent unmethylated cytosine.
FIG. 2 suggests that the human leukemia cell RPMI8402 is DNA methylated in the Bmal1 gene promoter region.
(1-3) An established cell line stably holding a luciferase gene transcribed by the Bmal1 promoter pTracer having an antibiotic (Zeocin) resistance marker gene together with the Bmal1 reporter plasmid prepared in (1-1) above -CMV (manufactured by Invitrogen) was introduced into human leukemia cells RPMI8402 (by the method described in Non-Patent Document 7) to obtain recombinant RPMI8402 cells, and further cloning by limiting dilution was repeated twice, using the Bmal1 promoter. A cell line stably retaining the transcriptionally regulated luciferase gene was established. The established cell line was deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the name “CPT-K stable clone 3” (reception number: FERM AP-22006, date of receipt: August 24, 2010). Day).
In this established cell line, by monitoring the luciferase activity, it became possible to easily and accurately know the biological rhythm through transcription of the endogenous Bmal1 gene in RPMI8402 cells.

(Example 2) Induction of Bmal1 gene expression by demethylation of DNA
In order to demethylate Bmal1 promoter region DNA, RPMI8402 strain was incubated for 48 hours in the presence of 2 μM 5-aza-2′-dCTP. Using the established cell line (control) not treated with the cell line, the expression of both Bmal1 genes was observed by RT-PCR. Actin gene was used as a control (FIG. 3). From the results shown in FIG. 3, the transcriptional repression is caused by the DNA methylation of the Bmal1 gene promoter region of RPMI8402 cells, and the methylation of the Bmal1 promoter region endogenous to RPMI8402 cells by 5-aza-2'-dCTP treatment. It is considered that the expression of Bmal1 gene was induced. Further, when Example (1-2) and Example 2 were examined using the established cell line of the present invention, the same results as the parent RPMI8402 cells were obtained. Due to this mechanism, it is considered that methylation of the Bmal1 promoter region endogenous to RPMI8402 cells was dissociated by 5-aza-2′-dCTP treatment, and expression of the Bmal1 gene was induced.

(Example 3) Circadian rhythm regulation by 2 μM 5-aza-2'-dCTP After seeding about 5 × 10 ^{5 of} the established cell line established in Example 1 in a 35 mm culture dish, 50% serum The biological rhythm is reset by culturing for 2 hours in the contained medium. Thereafter, cell culture was performed in a medium containing 2 μM 5-aza-2′-dCTP and the luminescent substrate luciferin, and chemiluminescence by the reporter gene was measured in real time. Luminescence was measured for 1 minute every 10 minutes. FIG. 4 shows time-dependent changes in chemiluminescence amounts in the presence of 2 μM 5-aza-2′-dCTP (Aza-dCTP) and the control (control). An actual measured value (ash) and a graph (black) fitted by the least square method (Non-Patent Document 12) are shown.
As seen from the results in FIG. 4, the expression of the Bmal1 gene was induced by 5-aza-2′-dCTP treatment by demethylating the endogenous Bmal1 promoter region of the RPMI8402 cell line, and also in the RPMI8402 cell line. It was observed that circadian rhythm returned to normal.

Claims (3)

A lymphoid cancer cell line transformed with a reporter plasmid having a reporter gene downstream of the Bmal1 promoter,
The lymphoid cancer cell line stably holds a reporter gene transcribed by the Bmal1 promoter ,
An established cell line , wherein the reporter plasmid is a reporter plasmid in which a luciferase gene is linked to a region of −201 to +24 containing “RORE” of the human Bmal1 promoter . Lymphoid cancer cell lines are human leukemia cell RPMI8402, established cell lines according to claim 1.   A method for screening an agent for improving circadian rhythm disorders caused by DNA methylation of a clock gene, comprising adding a test substance to a medium containing the established cell line according to claim 1 or 2, and a reporter protein A method comprising a step of determining whether to circadian rhythm by measuring luminescence or fluorescence intensity over time.