Use for regulating DNA damage repair by set7-mediated TIP60 methylation

Abstract

The present invention relates to the use of regulating DNA damage repair through SET7-mediated TIP60 methylation. Specifically, the present invention confirmed that DNA damage repair is regulated through SET7-mediated TIP60 methylation, and confirmed that SET7 regulates HR-mediated DSB repair by methylating TIP60, and that SET7 targets TIP60 for the methylation of K137 to methylate TIP60 and promote the HR-mediated DSB repair process. Accordingly, the present invention has the advantage of being provided as a therapeutic agent screening method.

Description

Technology Field

[0001] The present invention relates to the use of regulating DNA damage repair through SET7-mediated TIP60 (Tat interactive protein 60kDa) methylation. Background Technology

[0002] DNA damage repair plays a crucial role in maintaining genomic stability. Failure of DNA damage repair leads to a combination of mutagenic effects, increasing the risk of numerous pathological processes, including tumorigenesis. Among DNA lesions, double-strand breaks (DSBs) are generated by endogenous and exogenous DNA damaging factors, such as ionizing radiation, ultraviolet (UV) radiation, and certain highly toxic chemicals. Non-homologous end joining (NHEJ) and homologous recombination (HR) are the two major DSB repair pathways. Error-prone NHEJ is a DSB repair mechanism that is a DSB end ligation response activated throughout the cell cycle. Furthermore, NHEJ is not dependent on a template. In contrast, error-free HR requires intact template DNA. Therefore, HR occurs only during the late S / G2 phase. HR repair is effectively induced by DSB end ablation using the Mre11-Rad50-NBS1 (MRN) protein complex, ExoI, and CtIP. In particular, post-translational modifications (PTMs) of non-histone proteins are essential for supporting DNA damage repair. Many proteins involved in the DNA repair system are regulated by the control of PTMs for a rapid DNA damage response (DDR). This is because failure to repair DNA leads to genomic instability and the development of cancer. For example, RPA1 acetylation by PCAF is required for nucleotide excision repair, and PRMT5-dependent methylation of RUVBL1, a TIP60 co-activator, is essential for HR. Regulation by the dephosphorylation of PP4 plays a crucial role in DNA repair and cell survival.

[0003] This describes the function of the acetyltransferase TIP60, which is ubiquitously expressed in various signaling pathways such as transcriptional regulation, histone acetylation, and DNA repair. TIP60 acetylates key histones H2A, H3, H4, and various non-histone proteins such as p53 and Twist. Various PTMs control the acetyltransferase activation of TIP60. For example, S86 phosphorylation plays a crucial role in regulating autophagy and apoptosis mediated by TIP60 under various stress conditions. Furthermore, TIP60 plays an important role in DSB repair by maintaining genomic stability and regulating DNA repair through histone acetyltransferase (HAT) activity.

[0004] SET7, a histone H3K4-specific monomethyltransferase, is a major methyltransferase for non-histone proteins. To date, more than 30 non-histone SET7 targets involved in various cellular processes, such as transcriptional regulation, differentiation, and responses to DNA damage, have been identified. In particular, PARP1 methylated by SET7 has been reported to increase enzymatic activity and be required to activate DDR proteins. Therefore, SET7 is known to play an important role in DDR by activating or regulating the enzymatic activity of DDR proteins.

[0005] The inventors confirmed that SET7-mediated TIP60 is methylated at K137 in response to DNA damage, confirmed that DNA damage caused by hydroxyurea (HU), a potent DNA damage agent, induces SET7-mediated TIP60 methylation and promotes HR repair, confirmed that LSD1 induces demethylation of TIP60 and regulates the DNA damage repair process, and confirmed that the methylation of TIP60, which is essential for HR-mediated DSB repair, promotes cell proliferation of HCT116 colon cancer cells, thereby completing the present invention. Prior art literature

[0006] Korean Patent Publication No. 10-2023-0048234 (April 11, 2023) The problem to be solved

[0007] The objective of the present invention is to provide a cancer treatment screening method comprising the step of selecting a test substance in which the degree of methylation of TIP60 by SET7 is reduced.

[0008] Another objective of the present invention is to provide a cancer treatment screening method comprising the step of selecting a test substance in which the degree of demethylation of TIP60 by LSD1 is increased.

[0009] Another objective of the present invention is to provide a reagent composition for inducing TIP60 methylation in cancer cells in vitro, comprising SET7 as an active ingredient.

[0010] Another objective of the present invention is to provide a reagent composition for inducing TIP60 demethylation in cancer cells in vitro, comprising LSD1 as an active ingredient.

[0011] Another objective of the present invention is to provide a method for inducing DNA damage repair comprising the step of inducing TIP60 methylation by SET7 of cancer cells in vitro.

[0012] Another objective of the present invention is to provide a method for inhibiting DNA damage repair comprising the step of inducing TIP60 demethylation by LSD1 in cancer cells in vitro. means of solving the problem

[0013] The present invention provides a cancer treatment screening method comprising: (1) a step of contacting a test substance with cancer cells; (2) a step of measuring the degree of methylation of TIP60 by SET7 in the cancer cells contacted with the test substance; and (3) a step of selecting a test substance in which the degree of methylation of TIP60 by SET7 is reduced compared with a control sample.

[0014] In addition, the present invention provides a cancer treatment screening method comprising: (1) a step of contacting a test substance with cancer cells; (2) a step of measuring the degree of demethylation of TIP60 by LSD1 in the cancer cells contacted with the test substance; and (3) a step of selecting a test substance in which the degree of demethylation of TIP60 by LSD1 increases compared with a control sample.

[0015] In addition, the present invention provides a reagent composition for inducing TIP60 methylation of cancer cells in vitro, comprising SET7 as an active ingredient.

[0016] In addition, the present invention provides a reagent composition for inducing TIP60 demethylation of cancer cells in vitro, comprising LSD1 as an active ingredient.

[0017] In addition, the present invention provides a method for inducing DNA damage repair comprising the step of inducing TIP60 methylation by SET7 of cancer cells in vitro.

[0018] In addition, the present invention provides a method for inhibiting DNA damage repair comprising the step of inducing TIP60 demethylation by LSD1 in cancer cells in vitro. Effects of the invention

[0019] According to the present invention, it was confirmed that DNA damage repair is regulated through SET7-mediated TIP60 methylation, and that SET7 regulates HR-mediated DSB repair by methylating TIP60, and that SET7 targets TIP60 for the methylation of K137 to methylate TIP60 and promote the HR-mediated DSB repair process. Accordingly, the present invention has the advantage of being provided as a therapeutic agent screening method. Brief explanation of the drawing

[0020] Figure 1 illustrates that SET7 / 9 methylates TIP60 in vitro and in vivo. (A) Purified GST-TIP60 was incubated overnight at 30°C while increasing the amount of GST-SET7. The reaction mixture was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie or exposed to radiographic film. (B) SET7 was overexpressed in HCT116 cells. IP using an anti-methyllysin antibody was performed. Methyllysin levels were normalized to the amount of TIP60 administered. (C) HCT116 cells were treated with 1 uM SET7 inhibitor (R)-PFI-2 for 24 hours. IP using an anti-methyllysin antibody was performed. Methyllysin levels were normalized to the amount of TIP60 administered. (D) Purified GST-TIP60 deletion mutants were incubated with GST-SET7 overnight at 30°C. The reaction mixture was separated by SDS-PAGE and analyzed by radiographic observation. (E) Mass spectrometry (LC-MS / MS) was performed to determine the methylation status of K137 residues. Purified GST-TIP60△2 or GST-TIP60△2 point mutants were incubated with GST-SET7 overnight at 30°C. The reaction mixture was separated by SDS-PAGE and analyzed by radiographic observation. (F) HEK293T cells were transfected with the labeled plasmids, and IP was performed using an anti-methyllysine antibody. Methyllysine levels were normalized to the amount of TIP60 added. (G) pcDNA3.1-SET7 and Flag-TIP60 were overexpressed in HCT116 cells. Cell lysates were immunoprecipitated with an anti-SET7 antibody. The related proteins were eluted, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotted using labeled antibodies. Figure 2 shows that SET7 methylates TIP60 at Lys 137, and that this region is conserved across various species. (A) HCT116 cells were transfected with the indicated plasmid and immunoprecipitated with an antimethyllysin antibody. The associated proteins were eluted, analyzed by SDS-PAGE, and then immunoblotted with the designated antibody. Methyllysin levels were normalized to the amount of TIP60 added. (B) Conserved region across various species. The arrow indicates K137 of TIP60. Figure 3 indicates that SET7 does not affect TIP60-mediated acetylation and intracellular localization. (A, B) 293T cells and HCT116 cells transfected with pcDNA3.1-SET7, pcDNA3.1-SET7 H297A were lysed and immunoblotted using the labeled antibodies. (C) HCT116 cells transfected with pcDNA3.1-SET7, pcDNA3.1-SET7 H297A, or an empty vector were separated into nuclear and cytoplasmic fractions. H3 and β-tubulin were used as loading controls. Figure 4 shows that SET7 interacts with TIP60 in vitro and in vivo. (A) Extracts from HCT116 cells transfected with SET7 were cultured with purified GST or GST-TIP60. The relevant proteins were eluted, analyzed by SDS-PAGE, and immunoblotted (top). The amount of TIP60 in the cell extracts was determined by Coomassie staining (bottom). (B) Flag-EV or Flag-TIP60 and pcDNA3.1-SET7 were overexpressed in HCT116 cells. Cell lysates were immunoprecipitated with an anti-SET7 antibody. The relevant proteins were eluted, analyzed by SDS-PAGE, and immunoblotted using the labeled antibody. Figure 5 shows that hydroxyurea (HU)-mediated DNA damage induces SET7-dependent methylation of TIP60 by SET and induces homologous recombination (HR). (A) HCT116 cells were treated with 5 mM hydroxyurea (HU) for 4 and 8 hours. Apoptotic cells were measured via Fluorescence Activated Cell Sorting (FACS). (B) HCT116 shNC and shSET7 cells treated with 5 mM HU for 4 hours were immunoprecipitated using an antimethyllysine antibody. The immunoprecipitate was eluted, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotted using the labeled antibody. Methyllysine levels were normalized to the amount of TIP60 added. (C) HCT116 cells were transfected with the labeled plasmid and treated with 5 mM HU for 4 hours. Cell extracts from control cells and damaged cells were immunoprecipitated using anti-SET7 antibodies, and the associated proteins were pulled out using A / G agarose beads. The beads were extensively washed, and the bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted using the labeled antibodies. (D) HR analysis was performed on PcDNA3.1-SET7, PcDNA3.1-SET7 H297A, or Flag-TIP60. Results are expressed as mean ± SEM. n = 3, **P < 0.01, NS: No significant difference. (E) HCT116 cells with TIP60 knockdown were transfected with TIP60 WT or TIP60 K137R. Rad51 centers were examined after 5 mM HU treatment for 4 hours. Figure 6 illustrates that hydroxyurea (HU)-mediated DNA damage induces SET7-dependent methylation of TIP60 and leads to homologous recombination (HR). (A) HCT116 cells were treated with 5 mM HU for 4 hours. IP using an anti-methyllysine antibody was performed. Methyllysine levels were normalized by the amount of TIP60 administered. (B) HCT116 cells were arrested at the G1 / S checkpoint by double thymidine block / release, and then the cells were treated with 5 mM HU for 4 hours. IP using an anti-methyllysine antibody was performed. Methyllysine levels were normalized by the amount of TIP60 administered. Results are expressed as mean ± SEM. n = 3, ***P < 0.001, **P < 0.01. (C) Diagram of the HR reporter. HR analysis was performed on PcDNA3.1-SET7, Flag-TIP60 WT, or Flag-TIP60 K137R. Results are expressed as mean ± SEM. n = 5, *P < 0.05, NS: No significant difference. (D) HCT116 cells with TIP60 knockdown were transfected with TIP60 WT or TIP60 K137R. RPA centers were examined for 4 hours after treatment with 5 mM HU. (E) HCT116 shTIP60 cells overexpressing SET7 were transfected with the indicated plasmids. Subsequently, cells were treated with HU and neutral comet analysis was performed. Tail moments were determined using CaspLab software, and 90 individual comets were counted for each sample (bottom panel). Results are expressed as mean ± SEM. n = 90, ***P < 0.001. Figure 7 shows that LSD1 demethylates TIP60 methylation. (A) Cell extracts from HCT116 cells were immunoprecipitated with an anti-TIP60 antibody. The immunoprecipitate was eluted, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotted with the labeled antibody. (B) HCT116 cells were treated with 500 nM GSK-LSD1 for 24 hours. IP using an anti-methyllysin antibody was performed. Methyllysin levels were normalized to the amount of TIP60 administered. (C) HCT116 cells were transfected with Flag-EV of Flag-LSD1 and treated with 5 mM HU for 4 hours. IP using an anti-methyllysin antibody was performed. Methyllysin levels were normalized to the amount of TIP60 administered. (D) Homologous recombination (HR) analysis was performed on Flag-LSD1, Flag-TIP60 WT, or Flag-TIP60 K137R. Results are expressed as mean ± SEM. n = 5, ***P < 0.001, NS: No significant difference. Figure 8 shows that LSD1 demethylates TIP60 methylation. HCT116 cells were transfected with Flag-EV or Flag-LSD1 in the control group, or LSD1 stably knocked out the cells. Lysates were immunoprecipitated using an anti-methyllysine antibody. Methyllysine levels were normalized to the amount of TIP60 administered. Figure 9 shows that HU induces apoptosis in TIP60 K137R compared to TIP60 WT. (A) HCT116 cells were transfected with the labeled plasmid and treated with 5 mM hydroxyurea (HU) for 4 hours. Apoptotic cells were measured via Fluorescence Activated Cell Sorting (FACS). (B) HCT116 cells were transfected with the labeled plasmid and treated with 5 mM HU for 4 hours. Apoptotic cells were measured via Fluorescence Activated Cell Sorting (FACS). Results are expressed as mean ± SEM. n = 3, *P < 0.05, NS: no significant difference. (C) Cell viability was determined using the MTT assay. HCT116 cells were transfected with the Flag-empty vector (EV) or Flag-TIP60. Cells were treated with 500 nM GSK-LSD1 for 24 hours and cultured in fresh medium for 0–72 hours. Results are expressed as mean ± SEM. n = 3, **P < 0.01, *P < 0.05, NS: No significant difference. Figure 10 shows that TIP60 methylation by SET7 promotes cell proliferation. (A) HCT116 cells were transfected with the labeled plasmids and treated with 5 mM HU for 4 hours. Apoptotic cells were measured by fluorescence-activated cell sorting (FACS), and protein expression was confirmed by Western blot analysis. Results are expressed as mean ± SEM. n = 3, **P < 0.01, NS: No significant difference. (B) Representative colony formation analysis was performed using HCT116 cells with TIP60 knockdown transfected with TIP60 WT or TIP60 K137R. Cells were treated with 5 mM HU for 4 hours and cultured in fresh medium for 7 days. Results are expressed as mean ± SEM. n = 3, *P < 0.05, NS: No significant difference. (C) Cell viability was determined using the MTT assay. HCT116 cells with TIP60 knockdown were transfected with TIP60 WT or TIP60 K137R. Cells were treated with 5 mM HU for 4 hours and cultured in fresh medium for 0–72 hours. Results are expressed as mean ± SEM. n = 3, **P < 0.01, NS: no significant difference. (D) Schematic diagram showing how SET7-mediated TIP60 methylation promotes the homologous recombination (HR)-mediated DNA repair pathway. Specific details for implementing the invention

[0021] The inventors confirmed that methyltransferase SET7 regulates HR-mediated DSB repair by methylating histone acetyltransferase and the tumor suppressor TIP60, which are involved in gene expression and protein stability, and that SET7 targets TIP60 for the methylation of K137, thereby methylating TIP60 and promoting the HR-mediated DSB repair process. Furthermore, they confirmed that TIP60 methylation, which increases significantly during the S phase, effectively promotes HR, and that TIP60 is methylated by SET7, and that SET7-dependent methylation of TIP60 induced by DNA damage is essential for the HR repair pathway. Accordingly, the inventors completed the present invention by identifying the regulatory mechanism based on the TIP60 methylation status by SET7 and LSD1 in the double-strand break (DSB) repair pathway.

[0023] The present invention will be described in more detail below.

[0025] The present invention provides a cancer treatment screening method comprising: (1) a step of contacting a test substance with cancer cells; (2) a step of measuring the degree of methylation of TIP60 by SET7 in the cancer cells contacted with the test substance; and (3) a step of selecting a test substance in which the degree of methylation of TIP60 by SET7 is reduced compared with a control sample.

[0026] In the present invention, the methylation of TIP60 may be methylated at TIP60 K137, but is not limited thereto.

[0027] In the present invention, if the degree of methylation of TIP60 by SET7 is reduced, DNA damage repair can be inhibited, thereby increasing cancer cell death.

[0028] In addition, the present invention provides a cancer treatment screening method comprising: (1) a step of contacting a test substance with cancer cells; (2) a step of measuring the degree of demethylation of TIP60 by LSD1 in the cancer cells contacted with the test substance; and (3) a step of selecting a test substance in which the degree of demethylation of TIP60 by LSD1 increases compared with a control sample.

[0029] In the present invention, the demethylation of TIP60 may be performed on TIP60 K137, but is not limited thereto.

[0030] In the present invention, if the degree of demethylation of TIP60 by the LSD1 increases, DNA damage repair can be inhibited, thereby increasing cancer cell death.

[0031] In the present invention, the cancer may be selected from the group consisting of colon cancer, rectal cancer, breast cancer, liver cancer, lung cancer, brain cancer, stomach cancer, head and neck cancer, esophageal cancer, biliary tract cancer, bladder cancer, small intestine cancer, multiple myeloma, sarcoma, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, bladder cancer, cervical cancer, ovarian cancer, kidney cancer, lymphoma, leukemia, and skin cancer, but is not limited thereto.

[0032] In the present invention, "test substance" refers to an unknown candidate substance used in screening to test whether it affects the expression level of a gene, affects the expression or activity of a protein, or affects the binding between proteins. The sample includes, but is not limited to, chemical substances, nucleotides, antisense RNA, siRNA (small interference RNA), and natural product extracts.

[0033] In addition, the present invention provides a reagent composition for inducing TIP60 methylation of cancer cells in vitro, containing SET7 as an active ingredient.

[0034] In addition, the present invention provides a reagent composition for inducing TIP60 demethylation of cancer cells in vitro, containing LSD1 as an active ingredient.

[0035] In addition, the present invention provides a method for inducing DNA damage repair comprising the step of inducing TIP60 methylation by SET7 of cancer cells in vitro.

[0036] In addition, the present invention provides a method for inhibiting DNA damage repair comprising the step of inducing TIP60 demethylation by LSD1 in cancer cells in vitro.

[0037] In the present invention, "SET7" may be NCBI accession no. NP_085151.1.

[0038] In the present invention, "LSD1" may be NCBI accession no. NP_001009999.1.

[0039] In the present invention, "TIP60" may be NCBI accession no. NP_874369.1.

[0040] Meanwhile, since the corresponding features can be substituted in the aforementioned section, their description is omitted.

[0042] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0044] Example 1. Materials and Method

[0046] 1.1. Plasmid Construction

[0048] TIP60 (residues 1-461), TIP60 K137R, and partial constructs of human TIP60 were subcloned into the pGFP-C1 vector (Clontech) and the bacterial expression vector pGEX-4T2 (Invitrogen). SET7 (residues 1-366) of human SET7 (#24084; Addgene) was amplified by PCR and then subcloned into the bacterial expression vector pGEX-4T2. Double-stranded oligonucleotides for shRNA plasmid construction were produced using primers extending from the 5' to the 3' ends. The shTIP60 and shSET7 RNA oligonucleotide sequences are shown in Table 1 below:

[0050] Name Sequence (5'- to -3') shTIP60 Top strand: 5'-CCGGTCGAATTGTTTGGGCACTGATCTCGAGATCAGTGCCCAAACAATTCGATTTTTG-3'(Sequence No. 1) Bottom strand: 5'-AATTCAAAAATCGAATTGTTTGGGCACTGATCTCGAGATCAGTGCCC AAACAATTCGA-3'(Sequence No. 2) shSET7 Top strand: 5'-CCGGGCCAGGGTATTATTATAGAATCTCGAGATTCTATAATAATACCCTGGCTTTTT-3'(Sequence No. 3) Bottom strand: 5'-AATTCAAAAAGCCAGGGTATTATTATAGAATCTCGAGATTCTATAATAATACCCTGG-3'(Sequence No. 4) Top strand: 5'-CCGGAGGAAGGCTCTTCTAGCAATACTCGAGTATTGCTAGAAGAGCCTTCCTTTTTTG-3'(Sequence No. 5) Bottom strand: 5'-AATTCAAAAAAGGAA GGCTCTTCTAGCAATACTCGAGTATTGCTAGAAGAGCCTTCCT-3'(Sequence No. 6)

[0052] These oligonucleotides were inserted into the Age I / EcoR I region of the pLKO.1 TRC vector. The siRNA sequences of the negative control (siNC) are as shown in Table 2 below:

[0054] Name Sequence (5'- to -3') siNC Negative control: 5'-CCUCGUGCCGUUCCAUCAGGUAGUU-3'(Sequence No. 7)

[0056] 1.2. Antibodies

[0058] Antibodies against SET7 (sc-390823), p-histone H2A.X (sc-517348), RPA (sc-56770), GFP (sc-9996), β-actin (sc-47778), TIP60 (sc-166323), and LSD1 (sc-271720) were purchased from Santa Cruz Biotechnology. Methyl-lysine (Me-K; ab174719, ab23366) (Abcam), Flag (F3165) (Sigma-Aldrich), anti-caspase 3 (AB1899) (millipore), and Rad51 (GTX70230) (Genetex) were used.

[0060] 1.3. Cell Culture and Transfection

[0062] HEK293T and U2OS cells were cultured in Dulbecco Modified Eagle Medium (DMEM) (Gibco), and HCT116 cells were cultured in RPMI 1640 medium (Gibco) containing 10% heat-inactivated fetal bovine serum (Gibco) and 0.05% penicillin-streptomycin (Welgene) at 37°C in a 5% CO2 atmosphere. HCT116 WT and U2OS cells were transfected with labeled DNA constructs using polyethyleneimine (PEI) (Polyscience) or Lipofectamine 2000 (Invitrogen).

[0064] 1.4. In vitro methylation analysis

[0066] Methylation was performed at 30°C for 3 hours in a 30 μL volume containing 2 μg of 50 mM Tris-HCl [pH 8.5], 20 mM KCl, 10 mM MgCl2, 10 mM β-mercaptoethanol, 1.25 M sucrose, 100 nCi of [14C]-SAM (Perkin Elmer), GST-TIP60, GST-TIP60 △1 (residues 1–104), GST-TIP60 △2 (residues 105–220), GST-TIP60 △3 (residues 221–461), and GST-SET7. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by radiography. TIP60 K137R was synthesized based on the N-terminal amino acid sequence of H3 histone (Cosmo Genetech), filtered using p81 filter paper (Upstate), and washed three times with cold 10% trichloroacetic acid (TCA) and 95% ethanol at room temperature for 5 minutes. After air-drying the filter, 2 mL of Ultima Gold (Perkin Elmer) was added, and [14C]-SAM was quantified using a scintillation counter.

[0068] 1.5. Immunoprecipitation (IP) Analysis

[0070] Cells were lysed in lysis buffer (50 mM Tris-HCl [pH 7.5], 200 mM NaCl, 0.5% NP-40, 1x protease inhibitor cocktail) and incubated overnight at 4°C with the indicated antibodies. Subsequently, protein A / G agarose beads (GenDEPOT) were added, and the mixture was gently spun at 4°C for 3 hours. The bound proteins were analyzed by immunoblotting using the indicated antibodies.

[0072] 1.6. LTQ-Orbitrap Mass Spectrometry

[0074] Samples were separated by SDS-PAGE and gel extraction. After overnight trypsin digestion at 37°C, eluted peptides were separated using a C18 column at a flow rate of 300 nl / min with a linear gradient (A: 100% H2O, 0.1% formic acid, and B: 100% ACN). Typically, 2 μL of sample was injected. Mass spectrometry was performed using a dual mass spectrometer (LTQ Orbitrap Velos, Thermo Scientific) coupled to a nano-LC system (EASY nLC, Thermo Scientific). This method consisted of cycles combining a single full MS scan (mass range: 150–2000 m / z). Proteins were identified from the MS / MS spectra using SEQUEST.

[0076] 1.7. DNA Repair Analysis (HR Reporter Analysis)

[0078] An integrated DNA repair reporter system was used to determine HR efficiency. U2OS cells integrated with the HR reporter were transfected with the labeled construct and the I-SceI plasmid, which induces DSBs. Cells were harvested 48 hours after transfection, and the proportion of GFP-positive cells was determined by Fluorescence-Activated Cell Sorting (FACS) analysis using a BD Accuri C6 cell counter (BD Biosciences). Data were analyzed using BD Accuri C6 software (BD Biosciences). Repair frequency represents the average of at least three independent experiments.

[0080] 1.8. Immunofluorescence staining

[0082] HCT116 TIP60 knockdown cells were cultured on coverslips and transfected with Flag-TIP60 WT or methylation-deficient TIP60 (Flag-TIP60 K137R) using Lipofectamine 2000 Reagent (Thermo Fisher Scientific). After treatment with 5 mM HU for 4 hours, cells were fixed in 4% paraformaldehyde for 1 hour and permeated in 0.2% Triton X-100 solution for 10 minutes at room temperature. Cells were blocked with 1% bovine serum albumin in PBS and incubated with the primary antibody for 2 hours. Cells were washed with PBS and incubated with the secondary antibody for 1 hour. Coverslips were mounted on glass slides and visualized using an ECLIPSE 80i fluorescence microscope (Nikon, Tokyo, Japan).

[0084] 1.9. Comet Analysis

[0086] After treating cells with HU for 4 hours, the medium was replaced with fresh medium, and the cells were cultured at 37°C. A neutral comet assay was performed the following day after harvesting. Cells were mixed with 1% UltraKem LE Agarose (Young Science; 1:10 [v / v]), and once the agarose coagulated, the cells were lysed overnight at 37°C in lysis buffer (2% sarkosyl, 0.5 M EDTA-Na2, and 0.5 mg / mL proteinase K, pH 8.0). Subsequently, the cells were washed three times for 20 minutes each with electrophoresis buffer (90 mM Tris buffer, 90 mM boric acid, and 2 mM EDTA-Na2, pH 8.5). Proteins from the washed gel were electrophoresed at 20V for 25 minutes. Slides were stained with propidium iodide (Sigma-Aldrich) and fluorescence images were captured using an Olympus BX53 fluorescence microscope (Olympus). Tail moments were quantified using CASP version 1.2.3 beta2 (CaspLab).

[0088] 1.10. MTT Analysis

[0090] HCT116 cells (shTIP60) and control cells were transfected with FLAG-TIP60 WT or FLAG-TIP60 K137R and 5 x 10 cells per well 3 Cells were seeded into 48-well plates at a certain density. After 24 hours, the cells were treated with 5 mM HU for 4 hours, and the medium was replaced with fresh medium. At 24, 48, and 72 hours after HU treatment, MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) was added to the cells (final concentration 0.5 mg / mL), and the cells were cultured for an additional 2 hours at 37°C. Afterward, the medium was removed and dimethyl sulfoxide (DMSO) was added (200 μL / well). OD was measured using a microplate spectrophotometer (BioTek) at a wavelength of 575 nm.

[0092] 1.11. Analysis of Colony Formation

[0094] 5 x 10 cells per well transfected with FLAG-TIP60 WT or FLAG-TIP60 K137R 3 Canine cells were inoculated into a 35 mm culture dish at a cell density. After 24 hours, the cells were treated with 5 mM HU for 4 hours, and the medium was replaced with fresh medium. After culturing for 6 days, the surviving colonies were stained with 0.005% crystal violet.

[0096] 1.12. Statistical Analysis

[0098] Data are expressed as the mean ± standard error (SEM) of three independent experiments. Data were analyzed using GraphPad Prism (version 9; GraphPad Software, USA). Differences between groups were evaluated using Student's t-test. P<0.05 was considered statistically significant.

[0100] Example 2. Experimental Results

[0102] 2.1. SET7 methylating TIP60 of Lys 137 in vitro and in vivo

[0104] TIP60 is an acetyltransferase that plays a role in DNA repair and apoptosis by acetylating histones, and it is reported to play a crucial role in DDR signaling induction. To further investigate post-translational modifications of TIP60 during the DNA repair process, TIP60 methylation by SET7 was determined. SET7 was selected among methyltransferases because it is known as a major methyltransferase for various non-histone proteins. First, recombinant GST-TIP60 and GST-SET7 were cultured to perform an in vitro methylation assay. TIP60 was dose-dependently methylated by SET7. However, the catalytic null mutant SET7 H297A did not methylate TIP60 (Fig. 1A).

[0105] To determine whether SET7 methylated TIP60 in vivo, the methylation levels of TIP60 were tested via IP analysis using antimethyllysine antibodies. An increase in TIP60 methylation levels was observed in HCT116 cells overexpressing SET7 (Fig. 1B). In SET7 knockdown cells, ectopic overexpression of SET7 resulted in increased TIP60 methylation levels. In contrast, SET7 H297A failed to rescue TIP60 methylation levels (Fig. 2A). The methyltransferase activity of SET7 is known to be inhibited by (R)-PFI-2. When cells were treated with (R)-PFI-2, TIP60 methylation was significantly inhibited in HCT116 cells (Fig. 1C).

[0106] To accurately investigate the methylation sites of TIP60, in vitro methylation analysis was performed using the TIP60 deletion constructs TIP60 △1, △2, and △3. Among the deletion constructs tested, only TIP60 △2 was methylated by SET7 (Fig. 1D). To accurately identify the major methylation sites of TIP60, LC-MS / MS was performed on a high-resolution orbital device following in vitro methylation analysis using recombinant SET7 and TIP60. Mass spectrometry results showed that the methylated lysine residue of TIP60 was K137 (Fig. 1E, top panel). To confirm the LC-MS / MS analysis results, in vitro methylation analysis was performed using SET7, in which K137 was replaced by arginine, and the TIP60 △2 mutant (TIP60 △2 K137R). Compared to TIP60 △2, no methylation of TIP60 was detected at TIP60 △2 K137R (bottom panel of Fig. 1E). TIP60 K137 is an evolutionarily conserved residue across various species, indicating its functional importance (Fig. 2B). This suggests that K137 is the major methylation site of TIP60. When comparing methylation levels between TIP60 WT and TIP60 K137R in vivo, the methylation level of TIP60 K137R was lower than that of TIP60 WT (Fig. 1F). Overall, the data show that SET7 methylates K137 of TIP60 both in vitro and in vivo.

[0107] Next, the interaction between TIP60 and SET7 was investigated. First, it was confirmed that TIP60 methylation by SET7 does not affect TIP60 expression levels or TIP60-mediated acetylation (Figs. 3A, B). TIP60 is primarily localized to the nucleus. Therefore, cell fractions were analyzed to confirm that SET7 does not affect TIP60 localization (Fig. 3C). Co-IP analysis was performed on pcDNA3.1-SET7 or Flag-TIP60 overexpressing cells, and the results indicate the interaction between SET7 and TIP60 in vivo (Fig. 1G). To confirm the interaction between TIP60 and SET7 in vitro, a GST pull-down assay was performed using the TIP60 construct (Fig. 4A). Subsequently, to demonstrate the interaction between SET7 and TIP60 in cells, co-IP analysis was performed on Flag-empty Vector (EV) or cells overexpressing Flag-TIP60 and pcDNA3.1-SET7, and the results indicated that TIP60 interacts with SET7 in vivo (Fig. 4B). This data suggests that SET7 interacts with TIP60 both in vitro and in vivo.

[0109] 2.2. Induction of TIP60 Methylation by HU-Mediated DNA Damage and Promotion of HR for DSB Repair

[0111] Hydroxyurea (HU) is a replication inhibitor that depletes the nucleotide pool and induces DSBs by causing replication fork arrest. Since it was confirmed that apoptosis was sufficiently induced under these conditions, experimental conditions for HU treatment were established at a concentration of 5 mM HU for 4 hours (Fig. 5A). To investigate whether TIP60 methylation is important for DNA damage signaling, TIP60 methylation levels were checked after treating HCT116 cells with HU. TIP60 methylation levels increased significantly due to HU-induced DNA damage (Fig. 6A). However, HU did not induce TIP60 methylation when SET7 was depleted (Fig. 5B). To investigate whether the interaction between TIP60 and SET7 is affected by HU-induced DNA damage, IP analysis was performed with and without HU. The interaction between TIP60 and SET7 increased significantly in response to DNA damage caused by HU (Fig. 5C). It is known that dynamic HU, which occurs mainly during the S phase of the cell cycle, inhibits replication to induce HR in mammalian cells, that HR and SET7 are associated with DDR by catalyzing the methylation of the DDR protein ARTD1, and that HR occurs during the S and G2 phases of the cell cycle. Therefore, we focused on the effect of TIP60 methylation by SET7 on the HR repair mechanism.

[0112] TIP60 methylation levels were assessed at each stage of the cell cycle to determine the relationship between TIP60 methylation and its role in HR. The results showing increased TIP60 methylation levels during the S phase in Figure 6B suggest that TIP60 methylation may play a significant role in HR. These results demonstrated that TIP60 methylation is induced by SET7 in response to DNA damage. Therefore, an integrated reporter assay was performed to determine whether TIP60 methylation affects HR efficiency (Figure 6C, top panel). Since TIP60 methylation by SET7 induces the HR process, we tested whether methylated TIP60 K137R could promote HR. The HR reporter assay showed that TIP60 WT promoted HR in SET7-overexpressing cells but not in TIP60 K137R-expressing cells (Figure 6C, bottom panel). In addition, SET7 WT and SET7 H297A (catalytic mutant) were overexpressed in HCT116 cells and HR efficiency was measured, and it was found that TIP60 methylation by SET7 promoted HR (Fig. 5D).

[0113] Immunofluorescence staining was performed on HU-treated shTIP60 cells to confirm the effects of TIP60 on the formation of RPA and Rad51 foci at DNA damage sites. In contrast to the effects of TIP60 WT, RPA foci were blocked in cells transfected with TIP60 K137R (Fig. 6D). Additionally, Rad51 foci were identified in TIP60 WT (Fig. 5E).

[0114] Next, a neutral comet assay was performed using shTIP60 cells to determine whether TIP60 methylation induced by HU promotes DNA repair. The length of the comet tail moment indicates the degree of DNA breakage. In TIP60 K137R cells, the comet tail moment increased significantly after HU treatment, whereas in TIP60 WT cells, the tail moment was shorter. Additionally, compared to the control group, the comet tail moment of TIP60 K137R cells increased more than that of TIP60 WT cells after HU treatment (Fig. 6E). The above data indicate that the degree of DNA breakage in TIP60 WT cells is significantly lower than that in TIP60 K137R cells.

[0115] In summary, the data suggest that HU increases SET7-mediated TIP60 methylation and promotes HR in DNA-damaged cells.

[0117] 2.3. Mediation of TIP60 Demethylation by LSD1

[0119] SET7 can methylate histone H3K4, and LSD1 is a major H3K4 dimethylase. Before investigating whether LSD1 can catalyze the demethylation of TIP60, an IP assay was performed to confirm the interaction between LSD1 and TIP60. The results indicate that endogenous TIP60 interacts with LSD1 in HCT116 cells (Fig. 7A). Subsequently, it was confirmed that TIP60 methylation levels increased in cells treated with the LSD1 inhibitor GSK-LSD1 (Fig. 7B), suggesting that LSD1 is involved in the demethylation of TIP60. To further test this, an IP assay was performed using an anti-methyllysine antibody in LSD1 knockdown HCT116 cells overexpressed with either an empty vector or Flag-LSD1. TIP60 methylation levels were significantly increased in LSD1 knockdown cells, whereas methylation levels were decreased in cells structured by LSD1 overexpression (Fig. 8).

[0120] Since the present invention confirmed that TIP60 methylation is damage-dependent, IP analysis was performed to determine whether the demethylation of TIP60 induced by LSD1 is damage-dependent. It was confirmed that the increased TIP60 methylation levels in HU-treated cells decreased when LSD1 was overexpressed (Fig. 7C). Furthermore, the effect of LSD1-induced TIP60 demethylation on HR was demonstrated by confirming HR efficiency using HR reporter analysis. Although LSD1 reduced HR efficiency with TIP60 WT, LSD1 did not affect HR efficiency in cells transfected with TIP60 K137R (Fig. 7D).

[0121] Overall, these results indicate that TIP60 methylation is regulated by SET7 and LSD1 during the DNA repair process.

[0123] 2.4. Promotion of Colon Cancer Cell Proliferation by SET7-Mediated TIP60 Methylation

[0125] Substances such as HU, thymidine, and camptothecin induce replication fork disruption and potently induce HR in mammalian cells. Since HR is required for cell survival, we investigated whether SET7-mediated TIP60 methylation regulates cell proliferation and apoptosis upon HU treatment using flow cytometry. TIP60 K137R was found to increase apoptosis in a HU-dependent manner compared to TIP60 WT (Fig. 9A, top panel of Fig. 10A).

[0126] Apoptosis was investigated through the expression level of cleaved caspase-3, a marker of apoptosis (bottom panel of Fig. 10A). Additionally, apoptosis in shLSD1 cells was measured by treating them with HU. In shLSD1 cells overexpressing TIP60, there was no change in apoptosis before and after HU treatment, while in control cells, the number of apoptotic cells increased compared to shLSD1 when treated with HU (Fig. 9B).

[0127] Next, the viability of cells overexpressing the methylation-deficient mutant treated with TIP60 WT and 5 mM HU for 4 hours was observed. Colony formation analysis was performed. The number of colonies increased in TIP60 WT cells. However, a decrease in the number of colonies was observed in cells treated with the methylation-deficient mutant (TIP60 K137R) (Fig. 10B). To further investigate these observations, an MTT assay was performed. TIP60 knockdown cells transfected with TIP60 WT exhibited higher cell proliferation compared to TIP60 knockdown cells. In contrast, cells transfected with the methylation-deficient mutant (TIP60 K137R) did not show a significant increase in cell proliferation compared to TIP60 knockdown cells (Fig. 10C). TIP60 knockdown cells showed increased proliferation compared to control cells, indicating that the overexpression of TIP60 reduces HCT116 proliferation.

[0128] In addition, an MTT assay was performed using the LSD1 inhibitor GSK-LSD1. Control cells showed increased cell proliferation when treated with GSK-LSD1, and similar results were obtained in TIP60 overexpressing cells (Fig. 9C).

[0129] Overall, these results suggest that SET7-dependent methylation of TIP60 promotes cancer cell proliferation.

[0131] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 (1) a step of contacting a test substance with cancer cells isolated from the body; (2) a step of measuring the degree of methylation of TIP60 by SET7 in the cancer cells contacted with the test substance; and (3) a step of selecting a test substance in which the degree of methylation of TIP60 by SET7 is reduced compared with a control sample. Claim 2 A cancer treatment screening method according to claim 1, wherein the methylation of TIP60 is methylated at TIP60 K137. Claim 3 A cancer treatment screening method according to claim 1, wherein a decrease in the degree of methylation of TIP60 by the SET7 inhibits DNA damage repair and increases cancer cell death. Claim 4 (1) a step of contacting a test substance with cancer cells isolated from the body; (2) a step of measuring the degree of demethylation of TIP60 by LSD1 in the cancer cells contacted with the test substance; and (3) a step of selecting a test substance in which the degree of demethylation of TIP60 by LSD1 increases compared with a control sample. Claim 5 A cancer treatment screening method according to claim 4, wherein the demethylation of TIP60 is demethylated at TIP60 K137. Claim 6 A cancer treatment screening method according to claim 4, wherein increasing the degree of demethylation of TIP60 by the above LSD1 inhibits DNA damage repair and increases cancer cell death. Claim 7 A cancer treatment screening method according to any one of claims 1 to 6, wherein the cancer is selected from the group consisting of colon cancer, rectal cancer, breast cancer, liver cancer, lung cancer, brain cancer, stomach cancer, head and neck cancer, esophageal cancer, biliary tract cancer, bladder cancer, small intestine cancer, multiple myeloma, sarcoma, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, bladder cancer, cervical cancer, ovarian cancer, kidney cancer, lymphoma, leukemia, and skin cancer. Claim 8 A reagent composition for inducing TIP60 methylation in cancer cells in vitro, comprising SET7 as an active ingredient. Claim 9 A reagent composition for inducing TIP60 demethylation in cancer cells in vitro, comprising LSD1 as an active ingredient. Claim 10 A method for inducing DNA damage repair comprising the step of inducing TIP60 methylation by SET7 in cancer cells in vitro. Claim 11 A method for inhibiting DNA damage repair comprising the step of inducing TIP60 demethylation by LSD1 in cancer cells in vitro.

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