KR102348602B1 - Use for regulating DNA damage repair by methylated UHRF1 and PARP1 interaction - Google Patents

Abstract

The present invention provides an anticancer drug screening method and a DNA damage repair control method of cancer cells capable of controlling a period of the cancer cells and perishing the cancer cells, by regulating DNA damage repair by methylated UHRF1 and PARP1 interaction. In the present invention, it has been confirmed that PARP1 introduces methylated UHRF1 in a DNA damaged area and accelerates UHRF1 medium homologous recombination (HR) by interaction with methylated UHRF1 to activate a DNA double strand damage (DSB). Also, it has been confirmed that the methylated UHRF1 and PARP1 interaction controls the DNA damage repair mechanism of cancer cells to regulate a period of cancer cells and cell proliferation, and thus a method of regulating methylated UHRF1 and PARP1 interaction may be provided as an anticancer drug screening method of controlling DNA damage repair of cancer cells to perish cells.

Description

{Use for regulating DNA damage repair by methylated UHRF1 and PARP1 interaction}

The present invention relates to the use of regulating DNA damage repair by regulating the interaction between methylated UHRF1 and PARP1.

DNA damage is known to be the cause of many diseases, including cancer and accelerated aging. To overcome this, there is a system that recognizes DNA damage and stops the cell cycle and repairs it. However, it has been observed that this system does not function normally in many diseased cells. Therefore, it is very important to prevent and treat DNA mutation-related diseases so that the DNA damage response is normally operated after DNA damage so that the damage can be repaired without mutation.

The DNA damage repair process plays an important role in maintaining gene stability. Certain epigenetic markers of chromatin-associated proteins activate the DNA double strand break (DSB) repair pathway.

UHRF1 (Ubiquitin-like with PHD and RING finger domain 1) plays a role in regulating DNA methylation by mobilizing DNMT1 (DNA methyltransferase 1) to replication forks, which is intrinsic from cell to cell during cell division. It is important to convey information. In addition, UHRF1 is important in cancer progression and is overexpressed in several types of tumors, such as bladder, prostate, or ovarian cancer. In a previous study, UV-induced DNA damage induced UHRF1 ubiquitination and proteasome degradation, and its phosphorylation was confirmed to be related to genomic destabilization following UHRF1 destabilization. It has been reported that UHRF1 methylation by SET7, a methyltransferase, enhances homologous recombination (HR) signaling through PCNA polyubiquitination.

PARP1 (Poly [ADP-ribose] polymerase 1) is recruited to DNA lesions and is involved in DNA repair pathways and genomic stability. Its poly(ADP)ribosylation (PARylation) activity is required for DNA repair and unaltered replication fork progression. In particular, PARP1 regulates homologous recombination (HR) by mobilizing various proteins during DNA repair. As described above, the roles of PARP1 and UHRF1 for DNA repair have been confirmed, but the effect of the interaction between PARP1 and UHRF1 on the DNA repair process has not been confirmed at all.

Republic of Korea Patent Publication No. 10-2010-0085315 (published on July 29, 2010)

The present invention provides a method for screening an anticancer drug capable of inducing cell cycle regulation and cancer cell death of cancer cells by regulating DNA damage repair through methylated UHRF1 and PARP1 interaction regulation, and a method for inhibiting DNA damage repair in cancer cells. .

The present invention comprises the steps of (1) treating cancer cells with a test substance; (2) confirming the degree of binding between UHRF1 and PARP1 in cancer cells treated with the test substance; and (3) selecting a test substance having a reduced degree of binding between UHRF1 and PARP1 compared to a control sample.

The present invention provides a method comprising: inducing inhibition of binding of methylated UHRF1 to PARP1 in cells in vitro; And it provides a method of inhibiting cellular DNA damage repair comprising the step of inhibiting the binding of the methylated UHRF1 to PARP1 inhibits DNA damage repair in the cell.

In addition, the present invention provides a reagent composition for promoting methylated UHRF1-mediated DNA damage repair in cells in vitro, containing PARP1 as an active ingredient.

According to the present invention, PARP1 introduces methylated UHRF1 into the DNA damage site and promotes UHRF1-mediated homologous recombination (HR) through interaction with methylated UHRF1 to activate the DNA double-stranded break (DSB) repair pathway. As it was confirmed that the methylated UHRF1 and PARP1 interaction regulates the DNA damage repair mechanism of cancer cells to regulate the cell cycle progression and cell proliferation of cancer cells, the method for regulating the methylated UHRF1 and PARP1 interaction is the It can be provided as a screening method for a cancer disease therapeutic agent that induces cell death by inhibiting DNA damage repair.

1 is a result confirming PARP1 interaction according to the methylation status of UHRF1, FIG. 1A is an immunoprecipitation analysis result using anti-UHRF1 me0 or anti-UHRF1 me1 antibody, immunoprecipitation (IP) purified from HEK 293T nuclear extract The complex was visualized by silver staining, and the protein was confirmed by mass spectrometry, and FIG. 1B shows that GFP-UHRF1, Flag-SET7 WT or catalyst-deficient Flag-SET7 H297A was transfected into HCT116 cells and treated with anti-GFP antibody. Results of immunoprecipitation (IP) analysis, Figure 1C is a result of immunoprecipitation (IP) analysis using an anti-PARP1 antibody after treating HCT116 cells with 500 nM GSK-LSD1 for 24 hours, Figure 1D is blank These are the results of transfection of vector or Flag-LSD1 into control cells or stably knocked down LSD1 cells, and immunoprecipitation (IP) analysis of cell lysates using anti-PARP1 antibody.
Figure 2 is the result confirming that for the methylation of the UHRF1 induced damage to enhance the interaction of UHRF1 and PARP1, Figure 2A is a control cell and SET7 handle 1 mM H ₂ O ₂ in the knockdown stable cell for 30 minutes and anti- It is the result of immunoprecipitation analysis using PARP1 antibody, Figure 1B is a UHRF1 knockdown stabilization cells overexpressing GFP-UHRF1 WT or K385R 1 mM H ₂ O ₂ After 30 minutes treatment with anti-PARP1 antibody to the cell lysate This is the result of immunoprecipitation analysis using
3 is a result of confirming the effect of PARP1 on UHRF1 recruitment and DNA damage repair process at the DNA damage site. were transfected with I-SceI for 24 hours into U2OS-DRGFP cells temporarily knocked down with shNC or shPARP1, and cross-linked for ChIP analysis 48 hours later. qPCR analysis was performed, and % Input is a result showing the relative UHRF1-binding chromatin fraction to the input material (total chromatin), and Figure 3C is DMSO or olaparib 24 in I-SceI transfected cells. After time treatment, cross-linking was performed for 48 hours for ChIP analysis, and ChIP-qPCR analysis was performed using an anti-UHRF1 antibody at the I-SceI DSB site. Flag-tagged UHRF1 WT or K385R was transfected with the addition of I-SceI, treated with olaparib for 24 hours, cross-linked for 48 hours for ChIP analysis, and anti-UHRF1 antibody at the I-SceI DSB site The results of ChIP-qPCR analysis using is the mean ± SEM, n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, and FIG. 3F shows that cells were temporarily knocked down with shUHRF1 and increased by 10 μM after transfection with UHRF1 WT or K385R to confirm HR efficiency. As a result of performing HR reporter analysis after treatment with parip for 24 hours, the result value is mean ± SEM, n = 3; **P < 0.01, NS means no difference.
4 is a result confirming that PARP1 and UHRF1 interact for cell cycle progression and cell proliferation in response to DNA damage, FIG. 4A shows DMSO or olaparib 24 After treatment for an hour, 1 mM H ₂ O ₂ was treated for 30 minutes, cells were cultured in fresh medium for 24 hours, fixed, and stained with propidium iodide (PI) for DNA content by FACS (fluorescence-activated cell sorting). 4B is a result of confirming apoptotic cells by treating cells transfected with UHRF1 WT or K385R with 10 μM olaparib for 24 hours and performing FACS analysis using Annexin-V and PI staining. % (Annexin-V positive + Annexin-V and PI double positive), Figure 4C is a result of confirming cell viability by performing MTT analysis, UHRF1 WT or K385R was transfected into cells and After treatment with olaparib for 24 hours, 1 mM H ₂ O ₂ was treated for 30 minutes and cultured for 4 days in fresh medium containing olaparib, the result values were mean ± SEM, n = 3; *P < 0.05, **P < 0.01, NS means no difference. Figure 4D shows the results of colony formation analysis using UHRF1 WT or K385R-transfected UHRF1 knockdown HCT116 cells. After treatment with olaparib for 24 hours, cells were treated with 100 μM H ₂ O ₂ for 4 days. is the result

Hereinafter, the present invention will be described in more detail.

According to a recent study, UHRF1 (ubiquitin-like with PHD and RING finger domain 1) methylation is regulated by SET7 and LSD1 (lysine-specific histone demethylase 1A) and is essential for homologous recombination (HR). In the above study, it was confirmed that SET7-mediated UHRF1 methylation promotes HR-induced polyubiquitination of proliferating cell nuclear antigen (PCNA). However, to elucidate the mechanism of UHRF1 methylation-induced HR, studies on the mediators that recruit UHRF1 to damaged lesions and interact with UHRF1 are needed. Accordingly, the inventors of the present invention confirmed that PARP1 (poly [ADP-ribose] polymerase 1) specifically interacts with UHRF1 induced by methylation by damage and induces HR progression by mediating UHRF1. completed.

The present invention comprises the steps of (1) treating cancer cells with a test substance; (2) confirming the degree of binding between UHRF1 and PARP1 in cancer cells treated with the test substance; and (3) selecting a test substance having a reduced degree of binding between UHRF1 and PARP1 compared to a control sample.

The UHRF1 may be methylated by SET7 or damaged DNA, and more specifically, UHRF1 K385 may be methylated by SET7, but is not limited thereto.

The PARP1 may induce DNA damage repair in cancer cells by introducing methylated UHRF1 into the DNA damage site and binding to the methylated UHRF1.

When the binding degree of UHRF1 and PARP1 is decreased, DNA damage repair by UHRF1 may be inhibited, thereby increasing apoptosis of cancer cells.

The binding degree of the UHRF1 and PARP1 is reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA), immunoprecipitation (immunoprecipitation), immunohistochemistry, Western blotting (Western Blotting) And it may be measured by any one selected from the group consisting of flow cytometry (FACS), but is not limited thereto.

The test substance may inhibit DNA damage repair induced by UHRF1 by inhibiting the binding or interaction of PARP1 with methylated UHRF1.

The cancer disease may be selected from the group consisting of colon cancer, kidney cancer, breast cancer, liver cancer, lung cancer, prostate cancer, brain cancer, uterine cancer, cervical cancer and leukemia.

The term "test substance" used while referring to the screening method of the present invention affects the expression level of a gene, affects the expression or activity of a protein, or affects the binding between proteins. It refers to an unknown candidate substance used for screening. The sample includes, but is not limited to, chemicals, nucleotides, antisense-RNA, small interference RNA (siRNA), and natural product extracts.

The present invention provides a method comprising: inducing inhibition of binding of methylated UHRF1 to PARP1 in cells in vitro; And it may provide a method of inhibiting cell DNA damage repair comprising the step of inhibiting the binding of the methylated UHRF1 to PARP1 inhibits DNA damage repair in the cell.

In addition, the present invention can provide a reagent composition for accelerating repair of methylated UHRF1-mediated DNA damage in cells in vitro containing PARP1 as an active ingredient.

"UHRF1" used in the present invention is NCBI accession no. It may be NP_001041666.1.

"PARP1" used in the present invention is NCBI accession no. It may be NP_001609.2.

"SET7" used in the present invention is NCBI accession no. It may be NP_085151.1.

"LSD1" used in the present invention is NCBI accession no. It may be NP_001009999.1.

Hereinafter, examples will be described in detail to help the understanding of the present invention. 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 embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Cell culture and reagents

293T and U2OS cells were cultured in DMEM medium (Gibco), and HCT116 cells were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (Gibco) and 0.05% penicillin/streptomycin (Welgene) at 37°C, Incubated in 5% CO _{2 .}

2. Immunoprecipitation assay

Cells were lysed with lysis buffer (50 mM Tris-HCl [pH 7.5], 200 mM NaCl, 0.5% NP-40, 1× protease inhibitor cocktail) and incubated overnight with the antibody at 4°C. Protein A/G agarose beads (GenDEPOT) were added and mixed by stirring at 4°C for 3 hours. The bound protein was confirmed by immunoblot.

3. Chromatin Immunoprecipitation

U2OS-DRGFP cells with integrated chromatin I-SceI sites were used.

Briefly, the DSB-derived I-SceI plasmid was transfected into cells and cross-linked with 1% formaldehyde 48 h later. Then 125 mM glycine was added for 5 minutes.

The collected cells were resuspended in SDS lysis buffer [1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1]. Cells were sonicated and the lysate was immunoprecipitated. The immunoprecipitate was isolated, reverse cross-linked, and the DNA fragment was purified. UHRF1-related DNA was analyzed by real-time PCR using a primer for the I-SceI site, and the primers were as follows: forward, 5'-AACCATGTTCATGCCTTCTT-3'; Reverse, 5'-CCTCGTGGGTCTT-CTACTTT-3'. All experiments were repeated three times to confirm similar results.

4. LTQ-orbitrap mass spectrometry

The IP complex was digested on SDS-PAGE and stained with Coomassie brilliant blue. Then, it was trypsinized overnight at 37° C., and the eluted peptide was separated using a C18 column with a linear gradient (A: 100% H ₂ O, 0.1% formic acid, and B: 100% ACN) and a flow rate of 300 nl/min.

2 μl of the sample was injected, and a dual-mass spectrometer (LTQ Orbitrap Velos; Thermo Scientific) mass spectrometer connected to a nano-LC system (EASY nLC; Thermo Scientific) was used.

The method consisted of a cycle including one full MS scan (mass range: 150-2000 m/z), and the protein was identified by searching the MS/MS spectrum using SEQUEST. Mass spectrometry and proteomic analysis were performed with the Ion Mobility Tandem Mass Spectrometer of the Korea Basic Science Institute.

5. DNA repair analysis

HR efficiency was confirmed using an integrated DNA repair reporter system.

Briefly, U2OS cells were transfected with DSB-induced I-SceI plasmid 24 h after shUHRF1 or shPARP1 virus infection. After 48 hours of transfection, cells were collected and the percentage of GFP-positive cells was confirmed by FACS (fluorescence-activated cell sorting) using a BD Accuri C6 cytometer (BD Biosciences), and data using BD Accuri C6 software (BD Biosciences) was analyzed. The repair frequency was expressed as the average value of at least 3 independent experiments.

<Example 1> Confirmation of UHRF1 and PARP1 interaction according to a methylation-dependent method

As it was reported that UHRF1 methylation of K385 by the methyltransferase SET7 is a prerequisite for the DNA double-strand break (DSB) repair mechanism, to confirm the regulatory mechanism of UHRF1-methylation-dependent HR progression or more, mass spectrometry proteomics analysis (mass spectrometry proteomic analysis) was performed to confirm the UHRF1 interacting protein.

Two different antibodies to unmethylated or methylated UHRF1 were used to precipitate UHRF1 methylation-dependent interacting proteins.

As a result, as shown in FIG. 1A, methylated UHRF1 exhibited a strong interaction with PARP1. In addition, it was confirmed that SET7 is responsible for UHRF1 methylation, and complete UHRF1 methylation does not appear in the catalyst-deficient mutant SET7 H297A. In addition, as shown in FIG. 1B, it was confirmed that UHRF1 methylation by SET7 is important for binding to PARP1.

As it was confirmed in the previous study that LSD1 demethylates UHRF1, it is assumed that LSD1 inhibits UHRF1-PARP1 interaction while LSD1 inhibition increases the interaction, as shown in FIG. 1C, methylation status and UHRF1-PARP1 binding In order to confirm the correlation between the proteins, the proximity of the proteins after LSD1 deficiency was confirmed.

As a result, it was confirmed that LSD1 knockdown induces a very strong interaction between UHRF1 and PARP1, as shown in FIG. 1D.

From the above results, it was confirmed that UHRF1 methylation status was regulated by SET7 and UHRF1-PARP1 interaction was determined by LSD1.

<Example 2> H ₂ O ₂ Confirmation of increased interaction of UHRF1 and PARP1 by mediated UHRF1 methylation

DSB promotes UHRF1 methylation. By confirming the interaction between UHRF1 and PARP1 under methylation-dependent conditions, it was confirmed whether DNA damage could increase the binding of UHRF1 and PARP1.

As a result, as shown in FIG. 2A , the interaction between the two proteins was improved after exposure to _{H 2} O ₂ , whereas the interaction between UHRF1-PARP1 by _{H 2} O _{2 was not increased in SET7 deficiency.} In addition, as shown in FIG. 2B , it was _{confirmed that DNA damage by H 2} O ₂ induced a higher UHRF1-PARP1 interaction in wild-type UHRF1 than in methylation-deficient UHRF1 K385R.

From the above results, it was confirmed that UHRF1 methylation induced by _{H 2} O _{2 mediates binding to PARP1.}

<Example 3> Confirmation of role of PARP1 in DNA damage repair process

UHRF1 entry into the injured site is regulated by methylation status. 1 and 2 , it was confirmed that PARP1 preferentially interacts with methylated UHRF1.

Since PARP1 is involved in various DNA repair pathways and functions as a modulator of damage repair proteins, it was confirmed whether PARP1 mobilized UHRF1 for DSB damage.

To confirm UHRF1 influx around DSB, ChIP analysis was performed in U2OS-DRGFP cells as shown in FIG. 3A.

As a result, it was confirmed that I-SceI ectopic expression produced DSB and introduced UHRF1 as shown in FIG. 3B. However, referring to FIG. 3B , it was confirmed that UHRF1 did not accumulate in DSB after PARP1 deficiency.

In addition, the effect of PARP1 on the influx of UHRF1 to the injured site was confirmed using olaparib, a PARP1 inhibitor.

As a result, consistent with the study result that PARP1 was delayed in lesions damaged by olaparib, it was confirmed that UHRF1 was also stagnated around the DSB as shown in FIG. 3C. Interestingly, however, referring to FIG. 3D , as UHRF1 K385R did not enter the injured lesion, support of the methylation-dependent UHRF1-PARP1 interaction was confirmed.

As the main role of PARP1 in homologous recombination (HR) was confirmed from the above results, it was confirmed whether the accumulation of UHRF1 methylated by PARP1 could promote HR.

As shown in Figure 3E, PARP1 knockdown in wild-type UHRF1 cells reduced HR efficiency. Interestingly, when K385 of UHRF1 was inhibited, PARP1 deficiency did not affect HR efficiency. In addition, as shown in FIG. 3F, incomplete HR was confirmed in the cells in which methylation-deficient UHRF1 was overexpressed both in the presence or absence of olaparib.

From the above results, it was confirmed that the role of PARP1 is to recruit UHRF1 to damaged lesions in HR progression.

<Example 4> Confirmation of the effect of UHRF1-PARP1 interaction on DNA damage

As the regulation of HR activity by PARP1-UHRF1 interaction was confirmed in the previous experiment, the effect of UHRF1 methylation and PARP1 cooperation on cell cycle progression was confirmed.

Consistent with previous studies (15, 16), _{DNA damage induced by H 2} O ₂ in wild-type UHRF1 cells increased the G2/M cycle ratio, but significant G2/M phase arrest was confirmed in UHRF1 K385R cells. Results indicate a decrease in DNA repair efficiency (34.6% WT vs 41.4% K385R).

In addition, as it was confirmed that olaparib treatment was similarly involved in cell cycle disorders after UHRF1 WT or K385R recovery as shown in FIG. 4A, PARP1 may be suggested to determine the DSB repair function of UHRF1.

Consistent with the cell cycle analysis results according to PI staining, as a result of the BrdU binding analysis, it was confirmed that methylation-deficient UHRF1 could not efficiently repair damaged DNA, and that PARP1 played a role in encapsulating chromatin after olaparib treatment.

Interestingly, as a result of immunocytochemical analysis, it was confirmed that ectopic expression of UHRF1 reduced γ-H2AX lesions through successful DSB repair, whereas remaining γ-H2AX lesions were confirmed in adjacent non-transfected cells. However, overexpression of UHRF1 K385R did not show an effect on the change in γ-H2AX level, which could be expected that cell cycle progression inhibition was not overcome by the absence of sub-DNA repair process.

Meanwhile, as shown in FIG. 4B , it was confirmed that methylation-deficient UHRF1 induces higher apoptotic cell death than wild-type UHRF1. However, despite overexpression of wild-type or methylation-deficient UHRF1 after olaparib treatment, the apoptotic signaling pathway was not inhibited.

Finally, cell viability according _{to H 2} O ₂ treatment in the presence or absence of olaparib was confirmed.

As a result, as shown in Figure 4C, olaparib significantly reduced cell viability in wild-type UHRF1 cells. However, there was no decrease in cell viability in UHRF1 K385R cells regardless of olaparib treatment. From the above results, high sensitivity to DNA damage was confirmed.

Meanwhile, to confirm the effect of UHRF1-PARP1 on damage tolerance, a colony formation assay was performed.

Referring to Figure 4D, consistent with the results of Figure 4C, when olaparib was treated, there was little effect on the cell survival of methylation-deficient UHRF1 cells. Accordingly, it was confirmed that the UHRF1-PARP1 interaction plays an important role in cell proliferation.

From the above results, it was confirmed that PARP1 regulates the influx of UHRF1 according to the methylation status, and the interaction between UHRF1 and PARP1 mediates HR progression and regulates cell cycle progression and cell proliferation in response to DNA damage.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

(1) treating cancer cells with a test substance;
(2) confirming the degree of binding between methylated UHRF1 and PARP1 in cancer cells treated with the test substance; and
(3) A cancer disease therapeutic screening method comprising the step of selecting a test substance with a reduced degree of binding between the methylated UHRF1 and PARP1 compared to a control sample. The method according to claim 1, wherein the methylated UHRF1 is methylated by SET7 or damaged DNA. The method according to claim 1, wherein the PARP1 introduces methylated UHRF1 into a DNA damage site and binds to the methylated UHRF1 to induce DNA damage repair in cancer cells. The method according to claim 1, wherein when the degree of binding is reduced, DNA damage repair by UHRF1 is inhibited, and apoptosis of cancer cells is increased. The method according to claim 1, wherein the degree of binding is reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA), immunoprecipitation (immunoprecipitation), immunohistochemistry, Western blotting (Western Blotting) ) and a cancer disease therapeutic screening method, characterized in that the measurement is performed by any one selected from the group consisting of flow cytometry (FACS). The method according to claim 1, wherein the test substance inhibits the binding or interaction of PARP1 and methylated UHRF1 to inhibit DNA damage repair induced by UHRF1. The method according to claim 1, wherein the cancer disease is selected from the group consisting of colon cancer, kidney cancer, breast cancer, liver cancer, lung cancer, prostate cancer, brain cancer, uterine cancer, cervical cancer and leukemia. inducing inhibition of binding of methylated UHRF1 to PARP1 in cells in vitro; and
Cellular DNA damage repair inhibition method comprising the step of inhibiting the binding of the methylated UHRF1 to PARP1 inhibits DNA damage repair in the cell. A reagent composition for accelerating repair of methylated UHRF1-mediated DNA damage in cells containing PARP1 as an active ingredient.

Images