KR101850596B1 - Method for screening therapeutic agents of hematological malignancies by interaction RE-IIBP with RNF20 - Google Patents
Method for screening therapeutic agents of hematological malignancies by interaction RE-IIBP with RNF20 Download PDFInfo
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- KR101850596B1 KR101850596B1 KR1020160008080A KR20160008080A KR101850596B1 KR 101850596 B1 KR101850596 B1 KR 101850596B1 KR 1020160008080 A KR1020160008080 A KR 1020160008080A KR 20160008080 A KR20160008080 A KR 20160008080A KR 101850596 B1 KR101850596 B1 KR 101850596B1
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The present invention relates to a method of screening a therapeutic agent for hematologic cancer using RE-IIBP and RNF20 binding, and more particularly, to a method for screening a therapeutic agent for hematologic cancer comprising the step of screening a candidate drug that promotes RE-IIBP and RNF20 binding. On the other hand, the present inventors confirmed that when the degree of binding of RE-IIBP and RNF20 increases, H3K79 HMTase activity of RE-IIBP is increased and RE-IIBP-mediated MEIS1 transcription is promoted to increase blood cancer cell death. And the degree of RNF20 binding increases, it can be usefully used as a screening method for hematologic cancer treatment which can treat hematologic cancer.
Description
The present invention relates to a screening method for hematologic cancer therapy using an interleukin-5 [IL-5] response element II binding protein (RE-IIBP) and a ring finger protein 20 (RNF20) ≪ / RTI >
Chromatin remodeling promoted by various post-translational modifications of histones is considered to be an important factor in the transcriptional regulation of genes. During histone modification, lysine methylation, which is regulated by histone methyltransferases (HMTases) and demethylases, plays an important role in transcriptional regulation. Yeast Dotl and its human counterpart, DOT1L, are known to
Multiple myeloma (MM) is a malignant tumor of mature plasmatic cells, in which 70% to 80% of patients have translocation of immunoglobulin heavy chain (IgH) located on
On the other hand, the PBX-related homeobox gene MEIS1 is upregulated by RE-IIBP. MEIS1 was first identified in leukemia cells in a murine self-directed murine leukemia virus insertion mutant. MEIS1 is involved in vascular development and hematopoiesis during embryogenesis and is present in the bone marrow after birth. To maintain a continuous proliferative and differentiation arrest phase, MEIS1 works downstream of the MLL fusion protein and plays an important role before the leukemogenesis. In order to induce caspase-dependent apoptosis, MEIS1 and PBX1 interacting motifs are required.
It is an object of the present invention to provide a method for screening a therapeutic agent for blood cancer comprising the step of screening candidate drugs that promote RE-IIBP and RNF20 binding.
In order to accomplish the above object, the present invention provides a method for treating cancer, comprising: treating a candidate cancer drug with a blood cancer cell; Measuring the degree of binding of RE-IIBP and RNF20 in a blood cancer cell treated with the candidate drug; And selecting a candidate drug having increased degree of binding of RE-IIBP and RNF20 in comparison with a control sample.
The present invention relates to a method for screening a therapeutic agent for hematologic cancer using RE-IIBP and RNF20 binding, and the present inventors confirmed that RE-IIBP has H3K79 HMTase activity. Proteomics analysis revealed that RE-IIBP interacts with RNF20, and RE-IIBP-mediated H3K79 methylation was further increased by RNF20. In addition, H3K79 methylation by RE-IIBP was dependent on the RNF20-mediated ubiquitination of H2BK120. RE-IIBP induces apoptosis through transcriptional activation by the H3K79 HMTase activity of the MEIS1 gene. It was confirmed that the interaction with RE-IIBP and RNF20 induces further upregulation of MEIS1-mediated cell death. That is, when the degree of binding of RE-IIBP and RNF20 is increased, blood cancer cell death is increased, and therefore, it can be usefully used as a screening method for hematologic cancer treatment which can treat hematologic cancer.
Figure 1 shows the histone H3K79 HMTase activity of RE-IIBP. Results were mean ± SDs; n = 3. ** p < 0.01; *** p < 0.001.
Figure 2 shows the transcriptional activation of MEIS1 by RE-IIBP-mediated H3K79 methylation. Results were mean ± SDs; n = 3. ** p < 0.01.
Figure 3 shows that RNF20 further enhances transcriptional activation of MEIS1 by RE-IIBP. Results were mean ± SDs; n = 3. * p < 0.05; ** p < 0.01.
Figure 4 shows that RE-IIBP induces MEIS1-mediated cell death by H2BK120 ubiquitination and H3K79 methylation. Results were mean ± SDs; n = 3. * p < 0.05.
The present inventors confirmed that RE-IIBP has H3K79 HMTase activity. Proteomics analysis revealed that RE-IIBP interacts with RNF20, and RE-IIBP-mediated H3K79 methylation was further increased by RNF20. In addition, H3K79 methylation by RE-IIBP was dependent on the RNF20-mediated ubiquitination of H2BK120. RE-IIBP induces apoptosis through transcriptional activation by the H3K79 HMTase activity of the MEIS1 gene. The interaction with RE-IIBP and RNF20 was further up-regulated by further inducing MEIS1-mediated cell death. These results are the new results for H3K79 methyltransferase except for DOT1L, and various other mechanisms depending on various biological effects are mediated by H3K79 methylation. Thus, the present invention has been completed.
The present invention relates to a method for treating cancer, comprising the steps of treating a candidate drug to blood cancer cells; Measuring the degree of binding of RE-IIBP and RNF20 in a blood cancer cell treated with the candidate drug; And selecting a candidate drug having increased degree of binding of RE-IIBP and RNF20 in comparison with a control sample.
In detail, the degree of binding of RE-IIBP and RNF20 can be measured by immunoprecipitation and immunoblotting, but the present invention is not limited thereto.
In detail, when the degree of binding of RE-IIBP and RNF20 is increased, H3K79 HMTase activity of RE-IIBP is increased and RE-IIBP-mediated MEIS1 transcription is promoted to increase blood cancer cell death.
In particular, the blood cancer may be, but is not limited to, multiple myeloma, acute leukemia, chronic leukemia or lymphoma.
As used herein, "RE-IIBP" refers to NCBI accession no. EU733655, "RNF20" is NCBI accession no. NM_001163263.1, but is not limited thereto.
The term "candidate drug" used in referring to the screening method of the present invention is used to determine whether it affects the expression level of a gene, affects protein expression or activity, or affects binding between proteins Quot; means an unknown candidate substance used in screening. Such samples include, but are not limited to, chemicals, nucleotides, antisense-RNA, siRNA (small interference RNA) and natural extracts.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
< Experimental Example >
The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.
1. Plasmid production
The plasmid pCDNA3.1.RE-IIBP-His, pCDNA6-RE-IIBP-HA, GAL4-RE-IIBP, GAL4-RE-IIBP R477A, GFP-RE-IIBP and GST- Cell Biol 28, 2023-2034, 2008). pcDNA3.1-RNF20-myc-His has been previously reported (
2. Antibody
H3K79me1 (ab2886), H3K79me2 (ab3594), H3K79me3 (Abcam; ab2621), H3K27me2 (07-452), caspase3 (04-439), H2BK120ub (17-650), H3K4me2 (Sc-1059), GFP (sc-577),? -Actin (sc-47778), c-myc (sc-9996), H3 (sc-8654), His (sc-53073) and HA (Santa Cruz Biotechnology; sc-805).
3. Cell culture
293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM). K562 and H1299 cells were cultured in RPMI 1640 containing 10% heat-inactivated fetal bovine serum and 0.05% penicillin-streptomycin at 37 ° C, 5 % CO 2 . Using polyethylenimine (PEI) or Lipofectamine 2000 (Invitrogen), 293T, K562 and H1299 cells were transfected with the indicated DNA constructs. 293T cells were induced with 1 nM SGC0946 and harvested 48 hours later.
4. Histone Tablets
Acid extraction of the histones was performed as previously reported. Briefly, approximately 5 × 10 6 cells were collected and washed once with PBS. The cell pellet was resuspended in 1 ml TEB lysis buffer (0.5% Triton X-100, 2 mM PMSF, and 1 × protease inhibitor cocktail) And reacted at 4 ° C for 30 minutes to promote hypotonic swelling of the cells. The cells were collected by centrifugation, resuspended in 400 쨉 l of 0.2 MH 2 SO 4 , and reacted at 4 째 C overnight in an agitator. After centrifugation, the supernatant was transferred and treated with 132
5. HMTase analysis
50 mM Tris-HCl [pH 8.5 ], 20 mM KCl, 10
6. Immunocytochemical analysis
293T and H1299 cells were inoculated on collagen-coated slides. The next day, cells were transfected with the indicated plasmid. The cells were then washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and permeabilized with 0.2% triton X-100. Cells were reacted with labeled antibody and then reacted with FITC-conjugated anti-mouse and Cy3-conjugated anti-rabbit antibodies (Jackson ImmunoResearch Laboratories) after removing unspecific binding of antibody and substrate with 1% BSA. Finally, the slides were mounted with GEL / MOUNT (Biomeda) and images were acquired via confocal laser scanning microscopy (Zeiss LSM 700).
7. Immunoprecipitation and mass spectrometry
293T cells were transfected with GFP-RE-IIBP. After 48 hours, the cell lysates were immunoprecipitated with anti-GFP antibodies. Protein A / G agarose beads were then added and stirred at 4 ° C for 2 hours. Binding proteins were separated on SDS-PAGE and visualized by silver staining. The extracted gel was applied to LC-MS / MS sequence analysis and data analysis at the Korea Basic Science Institute. For analysis of interaction with RNF20, isoformally expressed GFP-RE-IIBP and myc-RNF20 cell lysates were immunoprecipitated with anti-GFP or anti-myc antibodies. Protein A / G agarose beads (GenDEPOT) were then added and stirred at 4 ° C for 2 hours. Binding proteins were analyzed by immunoblotting with anti-GFP and anti-myc antibodies.
8. Luciferase analysis( Luciferase assays)
Luciferase assay was performed using the MEIS1 promoter reporter system. Using PEI, 293T cells were co-transfected with the MEIS1 promoter reporter construct and the indicated DNA construct. Forty-eight hours later, cells were harvested and analyzed for luciferase activity using the luciferase assay system (Promega). Each value was expressed as an average of 5 replicate measurements, and the experiment was performed more than 3 times.
9. Reverse transcription And real time PCR
Total RNA was isolated from transfected 293T cells using RNAiso Plus (TaKaRa). After cDNA synthesis, cDNA was quantified and applied to mRNA expression analysis. Disassociation curves were drawn after each PCR reaction to ensure that a single product of the appropriate length was amplified. The mean threshold cycle (C T ) and standard error were calculated from the respective C T values obtained from three repeated measurements per step. The normalized mean C T was estimated through the calculated ΔC T minus the mean C T of β-actin. The ΔΔC T values were calculated as the difference between the control ΔC T and the values obtained from each sample. For gene expression, the n-fold change for the untreated control was calculated as 2 -ΔΔCT .
10. Liquid Chromatography - Mass Spectrometry
For HMTase analysis using GST or GST-RE-IIBP, synthetic peptide (H3N5) (100 mM) was used as a substrate. The reaction was stopped by precipitation with 10% TCA at 4 DEG C for 10 minutes. The supernatant was recovered by centrifugation at 4000 rpm for 10 min. The methylated peptides were analyzed by liquid chromatography-mass spectrometry (LC-MS) at Korea Basic Science Institute. The extracted peptides were separated on a Luna column (
11. Chromatin Immunoprecipitation immunoprecipitation ) analysis
CHIP analysis was performed according to the previously reported method (Mol Cell Biol 28, 2023-2034, 2008). Briefly, 293T and DOT1L knockdown stable 293T cells were transfected with the indicated plasmids. All cells were harvested after 48 hours. After 1% formaldehyde was added to the medium and DNA-proteins inside the cells were cross-linked for 10 minutes at 37 ° C, 125 mM glycine was added at room temperature and reacted for 5 minutes . The cells were then lysed in SDS lysis buffer and the samples were sonicated and immunoprecipitated using the indicated antibodies. The immunoprecipitates were eluted, reversing the mutual binding. Thereafter, the DNA fragment was purified and PCR amplification was performed for quantification using each pair of primers. Disassociation curves were drawn after each PCR reaction to ensure that a single product of the appropriate length was amplified. The mean threshold cycle (C T ) and standard error were calculated from the respective C T values obtained from two repeated measurements per step. Through the ΔC T calculated by subtracting the average C T of MEIS1, it estimated the standardized average C T.
12. MTT (3- (4,5- dimethylthiazole -2- yl ) -2,5- 두henyltetrazolium bromide) analysis
293T cells were inoculated into 48-well plates (2.5 x 10 4 cells / well) and transiently transfected with RE-IIBP, RE-IIBP R477A, shRE-
13. FACS analysis
In order to determine the effect of RE-IIBP on apoptosis, K562 shRNA-IIBP-stabilized cells were treated with 5 μM etoposide (SIGMA). In addition, K562 cells were transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen) and harvested 48 hours later. Cells were trypsinized, washed and fixed in ice-cold 70% ethanol for 30 min. Immediately prior to flow cytometry, cells were treated with RNase A (20 mg / ml) and stained with propidium iodide (SIGMA) for 30 minutes. Subsequently, transfected cells were applied to FACS analysis using BD Accuri C6 cytometer (BD Biosciences) and the results were analyzed using BD Accuri C6 software (BD Biosciences). Cells treated with ethoposide were applied to FACS analysis using the FACSCalibur system (BD Biosciences).
<
Example
1>
Histone
H3 for
Previous reports indicate that the MMSET and RE-IIBP isomers, including the SET domain, have a variety of histone lysine specificities including methylation of H3K4, H3K27, H3K36 and H4K20. In view of the fact that the modulation of the MMSET protein and the alteration in response to the welfare modification are related to various human diseases, the present inventors sought to confirm lysine specificity for the yet unknown RE-IIBP HMTase activity.
To investigate additional lysine specificities for RE-IIBP, methylation levels of purified histones were confirmed after RE-IIBP transfection. In addition to the previously identified H3K27me2, H3K79 di-methylation (H3K79me2) was elevated (Figure 1A). Transfection with RE-IIBP R477A, a point mutation lacking HMTase activity, did not result in an increase in H3K79me2 levels (Figure 1A). In contrast to H3K79 methylation, RE-IIBP did not alter H3K9 and H3K4 methylation levels in this assay. Through a lentiviral infection with two independent shRNAs, a 293T cell line stably knocked down RE-IIBP was prepared and H3K79me2 levels were significantly down-regulated (FIG. 1B). In order to further confirm the H3K79 HMTase activity of RE-IIBP, in vitro HMTase analysis was performed using GST-RE-IIBP fusion protein and core histones or nucleosomes, and a consistent result was obtained (Fig. 1C). RE-IIBP showed similar HMTase activity to core histones and nucleosomes. Immunocytochemistry was also performed using 293T cells transiently overexpressing RE-IIBP. Again in this assay, RE-IIBP expression increased H3K79me2 levels, confirming the H3K79 HMTase activity of RE-IIBP (FIG. 1D). However, no change in H3K79me2 levels was detected in RE-IIBP R477A overexpressing cells (Fig. 1D). Both H3K79me2 images contain spots showing patterns similar to y-tubulin, presumed to be non-histone proteins that are detected nonspecifically in the centrosome. It was confirmed that RE-IIBP can induce H3K79 mono -, di - or tri - methylation, and RE - IIBP could induce H3K79me1, H3K79me2 and H3K79me3 1E). Next, more specific HMTase assays were performed with histone H3 peptides, each of which is a 6-amino acid peptide containing one lysine residue. As a negative control, only the peptide H3N5 reacted with the GST protein was analyzed. The reaction with the peptide H3N5 greatly enhanced the methylation activity, as did the other peptides tested, indicating that the histone H3K79 is the methylation target residue of the RE-IIBP (Fig. 1F). To further confirm the lysine specificity of RE-IIBP, histone peptides were analyzed by LC-MS after HMTase analysis. The predicted molecular weight of the H3N5 peptide is 866 Da, which increases by 14 to 15 Da, respectively, when the methyl group is added. Unmethylated H3N5 peptides show a major peak at 866.4 Da, while di- and trimethylated peptides have 23 Da mass of Na + in the preparation of peptide samples after HMTase analysis, representing 919.4 and 911.4 Da, respectively (Fig. 1G ). Spectroscopic analysis revealed that the histone peptide H3N5 is di- and trimethylated in H3K79. Without the addition of RE-IIBP, no peaks associated with the methylated form of the H3N5 peptide were observed.
In the absence of DOT1L, 293T cells were treated with SGC0946, a DOT1L specific inhibitor, to confirm the H3K79 methylation activity of RE-IIBP, followed by a significant decrease in H3K79me2 (Fig. 1H). As a result, when overexpressing RE-IIBP in SGC0946-treated cells, H3K79 methylation was successfully restored, demonstrating the H3K79 HMTase activity of RE-IIBP (Fig. 1H). Lenti-virus infections were used to generate DOT1L rust-down stabilized cell lines, and overexpression of RE-IIBP successfully up-regulated H3K79me2 levels. Taken together, these results strongly suggest that RE-IIBP has H3K79 HMTase activity.
< Example 2> H3K79 methylation-mediated transcriptional activity of MEIS1 by RE-IIBP
Various studies have been reported relating to the relationship between H3K79 methylation and transcriptional regulation. Therefore, the present inventors have searched for potential target genes of RE-IIBP. Selected genes were identified using real-time PCR. JMJD6, HSPA1A, WDR and the transcription level of MEIS1 is raised by the RE-IIBP overexpression - were controlled, USP36 levels did not change. The TALE family member, MEIS1, is a cofactor of HOX and increases HOX-DNA binding affinity. Increased HOX transcriptional activity by MEIS1 is known to contribute to accelerating the progression of leukemia. In addition, the binding of HOXA7 or HOXA9 to MEIS1 has been confirmed in several forms of human cancer. Similar to native cancer-gene c-Myc-mediated proliferation and apoptosis, overexpression of MEIS1 has been shown to induce apoptosis in various types of cells through interaction with the co-factor PBX1.
In addition, the present inventors previously reported that RE-IIBP is up-regulated in blood cells of several leukemia patients (Mol Cell Biol 28, 2023-2034, 2008). The present inventors have estimated that RE-IIBP may regulate leukemia-related gene expression. The present inventors searched the RE-IIBP target gene in a leukemia cell line and confirmed that MEIS1 was regulated by RE-IIBP. An intensive study has been carried out on the role of MEIS1 in normal hematopoiesis and leukemia. To further elucidate the molecular mechanism of MEIS1 regulation by RE-IIBP, we first examined the effect of RE-IIBP overexpression on MEIS1 levels in 293T cell lines using real-time PCR and Western blotting. As expected, RE-IIBP overexpression up-regulated MEIS1 expression (Figure 2A). In the RE-IIBP green-down stabilized 293T cell line, MEIS1 expression was significantly down-regulated (FIG. 2B). In order to accurately verify that the activation of transcription by MEIS1 RE-IIBP is due to H3K79 HMTase activity of the RE-IIBP, MEIS1 - operating luciferase; a reporter analysis was carried out in 293T cells by using the (luciferase luc) reporter system. When RE-IIBP was transfected in a dose-dependent manner, the MEIS1 promoter activity was increased (FIG. 2C). The RE-IIBP using R477A, MEIS1 promoter activity increased by RE-IIBP showed that depends on the HMTase activity of the H3K79 methylation (Fig. 2C). As expected, when RE-IIBP was knocked down with two different shRE-IIBP RNAs, a decrease in MEIS1 promoter activity was confirmed (FIG. 2C). Since the RE-IIBP antibody recognizes both RE-IIBP and MMSET, a K562-stabilized cell line was prepared which only recognized MMSET but did not recognize RE-IIBP through lenti- virus infection using shRNA against MMSET HMG domain . Interestingly, MMSET green-down had no effect on MEIS1 expression (Figure 2D).
To confirm the transcriptional regulation mechanism of MEIS1 by H3K79 HMTase activity of RE-IIBP, ChIP analysis was performed using real-time PCR. As expected, when RE-IIBP was overexpressed, the degree of RE-IIBP binding and the level of H3K79me2 in the MEIS1 promoter increased (Fig. 2E). Taken together, these results indicate that RE-IIBP up-regulates MEIS1 transcription by H3K79 methylation through migration to the MEIS1 promoter site.
< Example 3 > to RNF20 MEIS1 RE-IIBP synergistically activates transcription
In order to obtain more information on the proteins interacting with RE-IIBP, GFP-RE-IIBP was overexpressed in 293T cells and GFP affinity purification was performed. Immunoprecipitated proteins were analyzed by liquid chromatography-mass spectrometry (LC-MS). Among the proteins that appeared to interact with RE-IIBP, RNF20, known as E3 ubiquitin ligase, was identified (Figure 3A).
To confirm the interaction between RE-IIBP and RNF20, GST pull-down assays were first performed using purified GST-RE-IIBP and RNF20 overexpressing cell extracts (FIG. 3B). In addition, the present inventors conducted an inverse experiment to confirm the interaction, that is, the ectopically expressed lysate derived from RE-IIBP was reacted with GST-RNF20 (FIG. 3B). As a result of the above experiment, there was a remarkable interaction between RE-IIBP and RNF20. The interaction between the two proteins was confirmed by in-vivo co-immunoprecipitation (IP) analysis (Fig. 3C). RNF20 influenced transcriptional regulation of MEIS1 by RE-IIBP. Interestingly, the RE-IIBP-mediated up-regulation of MEIS1 expression was further activated, confirming the increased expression of MEIS1 in real-time PCR and Western blot analysis (FIG. 3D). To clarify the effect of RNF20 on RE-IIBP activity, reporter assays were performed using the MEIS1- luc promoter. As a result, it was confirmed that RNF20 further increased RE-IIBP-mediated transcriptional activation for MEIS1 , confirming that this result is consistent with the above results (FIG. 3E). , DOT1L rust to confirm the role of the RE-IIBP / RNF20 complex in MEIS1 promoter-down stable cells was performed ChIP analysis of MEIS1 promoter. When RNF20 was co-transfected, the degree to which RE-IIBP migrated to the MEIS1 promoter was further increased (Fig. 3F). In addition, H3K79 HMTase activity by RE-IIBP was upregulated in the presence of RNF20 (Fig. 3F). Taken together, these results indicate that RNF20 interacts with RE-IIBP and promotes RE-IIBP-mediated MEIS1 transcription by increasing the H3K79 HMTase activity of RE-IIBP.
<Example 4> MEIS1-mediated cell death induction by RE-IIBP dependent on H2BK120 ubiquitination
Since RE-IIBP interacts with RNF20, we have hypothesized that RNF20 may recruit RE-IIBP to increase H2BK120ub-mediated H3K79 methylation. To confirm this possibility, immunocytochemistry was performed using RE-IIBP and RNF20 co-transfected H1299 cells. Within the nucleus, RNF20 was co-located with RE-IIBP and the level of RE-IIBP-mediated H3K79 methylation was further increased (FIG. 4A). To exclude DOT1L-mediated H3K79 methylation, the 293T cell line was treated with SGC0946, which resulted in a decrease in H3K79 methylation levels (Fig. 4B). Upon overexpression of RE-IIBP in SGC0946 treated cells, H3K79 methylation levels were restored (FIG. 4B). Interestingly, RNF20 overexpression not only increased the level of H3K79 methylation, but also increased the level of H2bK120ub (Fig. 4B). It was also confirmed that the DOT1L green-down stabilized cell line was consistent with the above results. This strongly suggests that RNF20 increases RE-IIBP-mediated H3K79 methylation independent of H3K79 methylation by DOT1L. RNF20 is the target gene MEIS1 ChIP analysis was performed using real-time PCR to determine if the promoter could regulate the movement of RE-IIBP. ChIP analysis showed that upon overexpression of RNF20 , the migration of RE-IIBP and the level of H3K79me2 were increased in the MEIS1 promoter (Fig. 4C). Overexpression of the RING domain deficient mutant RNF20 DELTA RING did not change the level of H3K79me2. However, the migration of RE-IIBP was increased in both RNF20 and RNF20 DELTA RING overexpression (Fig. 4C). Indicating that the interaction of RNF20 and RE-IIBP is independent of the RING domain. Still, RE-IIBP-mediated H3K79 methylation was dependent on H2BK120ub by RNF20. Taken together, these results indicate that RNF20 interacts with RE-IIBP and induces RE-IIBP to the promoter of the target gene for improved H2BK120ub-mediated H3K79 methylation by RE-IIBP.
MEIS1 overexpression induces apoptosis in various cell types via a caspase-dependent process. The present inventors have assumed that RE-IIBP may activate MEIS1 expression and induce apoptosis. To determine if RE-IIBP induces apoptosis, the RE-IIBP dependent cell viability was measured at the indicated time points. Cell viability was decreased by RE-IIBP overexpression but increased in RE-IIBP rust-down. To determine the effect of RE-IIBP in survival, MTT assays for measuring cell survival were performed at different time points and results consistent with cell survival were obtained (FIG. 4D). Co-transfection of RNF20 and RE-IIBP further reduced cell viability (Figure 4D).
Since RNF20 further enhanced the expression of RE-IIBP-mediated MEIS1, we sought to determine the effect of RNF20 on RE-IIBP-mediated cell death. When RE-IIBP and RNF20 were overexpressed individually,
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (4)
Measuring the degree of binding of RE-IIBP and RNF20 in a blood cancer cell treated with the candidate drug;
Selecting a candidate drug having increased degree of binding of RE-IIBP and RNF20 compared to a control sample; And
Determining that increasing the degree of binding of RE-IIBP and RNF20 increases H3K79 HMTase activity of RE-IIBP and promotes RE-IIBP-mediated MEIS1 transcription to increase blood cancer cell death; .
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Non-Patent Citations (3)
| Title |
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| Animal Cells and Systems, 2012, Vol. 16, pp 289-294. |
| Biomolecules & Therapeutics, 2009, Vol. 17, pp 12-16. |
| 혈액종양에서의 히스톤 H3K27메칠화 효소 RE-IIBP의 DNA 손상반응에 의한 세포사멸유도, 중앙대학교(2014)* |
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