Function of high-mobility group A proteins in the DNA damage signaling for the induction of apoptosis

O6-Methylguanine produced in DNA can pair with thymine during DNA replication, thus leading to a G-to-A transition mutation. To prevent such outcomes, cells harboring O6-methylguanine-containing mispair undergo apoptosis that requires the function of mismatch repair (MMR) protein complex. To identify the genes involved in the induction of apoptosis, we performed gene-trap mutagenesis and isolated a clone of mouse cells exhibiting an increased resistance to the killing effect of an alkylating agent, N-methyl-N-nitrosourea (MNU). The mutant carries an insertion in the Hmga2 gene, which belongs to a gene family encoding the high-mobility group A non-histone chromatin proteins. To elucidate the function of HMGA proteins in the apoptosis pathway, we introduced siRNAs for HMGA1 and/or HMGA2 into human HeLa MR cells defective in O6-methylguanine-DNA methyltransferase. HMGA1- and HMGA2-single knockdown cells showed an increased resistance to MNU, and HMGA1/HMGA2-double knockdown cells exhibited further increased tolerance compared to the control. The phosphorylation of ATR and CHK1, the appearance of a sub-G1 population, and caspase-9 activation were suppressed in the knockdown cells, although the formation of mismatch recognition complex was unaffected. These results suggest that HMGA family proteins function at the step following the damage recognition in the process of apoptosis triggered by O6-methylguanine.

by MMR proteins, might induce apoptosis 8,14 . In this regard, it is important to know which proteins function directly, after the MMR complex formation, in the induction of apoptosis.
Gene-trap mutagenesis is a useful technique for identifying genes that function in various cellular processes. By applying this method to a mouse-derived cell line, we previously isolated the new genes Mapo1 and Mapo2, which are involved in the O 6 -meG-induced apoptosis 12,15 . The inhibition of the expression of these genes in human-derived cells suppressed the induction of several apoptosis-related activities, including the dimerization of Bak, mitochondrial membrane depolarization, and caspase-3 activation, although the activation of the DNA damage response was unaffected. Further investigations revealed that MAPO1 forms a complex with foliculin (a tumor suppressor protein encoded by FLCN) and AMP-activated protein kinase (AMPK; an energy sensor composed of AMPKα, β, and γ subunits), implying a role of MAPO1 in the signal transduction pathway in the cytoplasm 16,17 .
By extending the gene-trap mutagenesis screening, we found that high-mobility group A (HMGA) family proteins are involved in MNU-induced apoptosis. HMGA proteins are non-histone chromatin proteins encoded by two distinct genes: HMGA1 and HMGA2 18 . HMGA proteins per se do not possess intrinsic transcriptional regulatory activity; however, by interacting with transcription factors they have ability to alter chromatin structure and regulate gene transcription 19,20 . HMGA family proteins were found to be over-expressed in different types of malignant tumors. Furthermore, the overexpression of HMGA was shown to suppress the DNA repair ability of the cells, thereby sensitizing cells to DNA damage [21][22][23] . HMGA2 overexpression is also involved in doxorubicin-induced G 2 -M cell cycle delay and induces persistent phosphorylation of H2AX by modulating the activation of ATM 24 . These data suggest possible roles of HMGA proteins in the signaling pathway in response to DNA damage.
In this report, we present evidence supporting a novel function of the HMGA family proteins HMGA1 and HMGA2 at an early step of the apoptosis pathway triggered by O 6 -meG.

Results
Isolation of a mouse cell line defective in Hmga2. Retrovirus-mediated gene-trap mutagenesis was performed to isolate cells defective in genes functioning in the MNU-induced apoptosis pathway. YT102 cells, established from lung fibroblast of Mgmt-knockout mice, were infected with the gene-trap vector containing a promoterless hygromycin B resistance gene, and hygromycin-resistant (Hyg r ) cells were selected. From the collection of Hyg r -cells, MNU-resistant clones were isolated as candidates that have an insertional mutation of the vector sequence in genes involved in O 6 -meG-induced apoptosis. One of the isolated clones, termed KH102, exhibited significant resistance to MNU in comparison to the parental cell line YT102, although the level of resistance was lower than that of the YT103 cell line, which is defective in both Mgmt and Mlh1 (Fig. 1a). To identify the gene disrupted in KH102, an inverse polymerase chain reaction (PCR) was performed, which amplifies DNA fragments spanning the junctions between the genomic DNA and the integrated vector sequences, and the nucleotide sequences of the amplified fragments were determined. A database search revealed that the vector DNA was integrated into a sequence corresponding to the large third intron between exons 3 and 4 of the gene encoding high-mobility group AT-hook 2 (HMGA2), located on mouse chromosome 10. An immunoblotting analysis revealed that the expression level of Hmga2 in KH102 was less than half that in YT102, and a band for a protein with a smaller molecular mass was detected incidentally (Fig. 1b), suggesting the expression of a truncated form of Hmga2 from a gene-trapped allele of the gene.
To clarify the involvement of HMGA2 in the induction of apoptosis triggered by O 6 -meG, we measured the caspase-3 activity in three cell lines after administration of 0.4 mM MNU. As shown in Fig. 1c, the caspase-3 activity in YT102 cells increased to more than 2.5 times the level of untreated cells, whereas no increase was observed in Mlh1-defective YT103 cells. In KH102 cells, the activation of caspase-3 was hardly detected, even after the treatment with MNU. These results suggest that Hmga2-deficient KH102 cells acquired resistance to MNU, due to the inability to induce apoptosis.
Involvement of human HMGA proteins in O 6 -methylguanine-induced apoptosis. The mammalian HMGA family is composed of two members-HMGA1 and HMGA2-and they have some redundant molecular functions 25 . To see if either or both proteins function in the O 6 -meG-induced apoptosis, siRNAs specific for HMGA1 and/or HMGA2 were introduced into HeLa MR cells. The expression of HMGA1 was significantly reduced in siHMGA1/siHMGA2-double knockdown cells compared to that in cells treated with siCont ( Fig. 2a). A quantitative real-time PCR analysis revealed a 16% reduction in the HMGA2 mRNA level relative to that of the control in siHMGA2-transfected cells (Fig. 2b). It should be noted that, under these conditions, the expression of the MMR proteins MSH2, MSH6, MLH1, and PMS2 was unaffected (Fig. 2a). siRNA-treated cells were exposed to various doses of MNU, and the survival fractions were determined. As shown in Fig. 2c, HMGA1-or HMGA2-single knockdown cells showed considerable degrees of resistance to the treatment with MNU. Furthermore, HMGA1/HMGA2-double knockdown cells exhibited further increase in the resistance level to MNU. These results suggest that both HMGA proteins may be involved in the process of apoptosis triggered by O 6 -meG.
Formation of mismatch recognition complex in HMGA-knockdown cells. An MMR complex composed of MutSα (MSH2 and MSH6) and MutLα (MLH1 and PMS2) is formed on chromatin when human cells are exposed to MNU 7 . To see if such a complex formed in HMGA-knockdown cells, we performed immunoprecipitation experiments using chromatin extracts prepared from HeLa MR cells stably expressing Flag-tagged PMS2. As shown in Fig. 3, using an anti-Flag antibody, the stable association of MLH1 and MNU-induced interactions of MSH2 and MSH6 with Flag-PMS2 were detected in siCont-transfected cells. Even when both siHMGA1 and siHMGA2 were introduced, the same level of complex formation was achieved. It appears that HMGA proteins are dispensable for the formation of mismatch recognition complex. We therefore next examined if HMGA-knockdown cells have any defects in downstream events following mismatch recognition.

Suppression of DNA damage response in HMGA-knockdown cells. DNA damage responses, includ-
ing the phosphorylation of checkpoint kinases, such as ATR and CHK1, occur in the course of MNU-induced apoptosis 26 . The activation of those kinases is dependent on O 6 -meG among various types of alkylated bases, as the phosphorylation of CHK1 in the HeLa S3 cell line, which is proficient in MGMT, is not observed without adding O 6 -benzylguanine, a specific inhibitor of MGMT, even after treatment with MNU ( Supplementary  Fig. S1). To determine if defects in HMGA proteins affect these processes, the phosphorylation status of CHK1 in HMGA-knockdown cells was analyzed. HeLa MR cells, transfected with control siRNA (siCont) or HMGA-specific siRNAs, were treated with 0.4 mM MNU for 1 h, followed by cultivation for 24 and 48 h, and the whole-cell extracts were subjected to an immunoblotting analysis (Fig. 4a). In the siCont-transfected cells, the phosphorylation of the serine residue at position 317 of CHK1 was clearly detected after MNU treatment. When a mixture of siHMGA1 and siHMGA2 (siA1&A2) was introduced, the levels of the phosphorylated form of CHK1 were reduced at both 24 and 48 h after the drug administration. Almost the same levels of reduction were achieved in HMGA1 (siA1)-and HMGA2 (siA2)-single knockdown cells. It should be noted that the total amounts of CHK1 were unaffected, even after these treatments. Similar results were obtained in the case of ATR, which is a master kinase of ATR/CHK1-mediated DNA damage response. In comparison with the control (siCont), the phosphorylation of the threonine residue at position 1989 of ATR after treatment of MNU was suppressed when siRNAs targeting either or both HMGA genes were introduced (Fig. 4b). Furthermore, the phosphorylation of histone H2AX, a downstream event of the ATR/CHK1 axis, was attenuated in the HMGA-knockdown cells (Fig. 4c). In the mouse KH102 cells we isolated, where one of the alleles of Hmga2 has an insertional mutation, the Mlh1-dependent phosphorylation of H2AX was also found to be decreased compared with the parental cell line, YT102 (Supplementary Fig. S2). These results imply that the HMGA family proteins function prior to ATR/ CHK1 activation in the DNA damage signaling at an early step of O 6 -meG-induced apoptosis.   Effects of apoptosis-related events in HMGA-knockdown cells. We further examined the effects of HMGA-knockdown on the appearance of a sub-G 1 population and the activation of caspase-9, both of which are hallmarks for the induction of apoptosis. A flow cytometric analysis showed that the sub-G 1 population increased gradually after MNU treatment in cells transfected with either type of siRNA (Fig. 5a). However, the degree  of increase was significantly slighter in the HMGA-knockdown cells than in the control cells. In the HMGA1/ HMGA2-double knockdown cells, the degree of reduction (12.0%) at Day 3 after MNU treatment was almost half that of control (23.3%). Similar levels of reduction were obtained in cells introduced with siHMGA1 (16.7%) and siHMGA2 (10.7%).
We next analyzed the activation of caspase-9 in HMGA-knockdown cells. An immunoblotting analysis using an antibody that recognizes both pro-and cleaved-caspase-9 revealed that the cleavage of caspase-9 started at 48 h and became more evident at 72 and 96 h after MNU treatment in siCont-transfected cells (Fig. 5b). In contrast, in all types of HMGA-knockdown cells, the signals for cleaved-caspase-9 at 72 and 96 h were significantly weaker than in the controls. Furthermore, mitochondrial outer membrane permeabilization following MNU treatment was also suppressed in HMGA1/HMGA2-knockdown cells compared to the control (Supplementary Fig. S3). These results are consistent with the above-mentioned view that HMGA family proteins may function at an early step of apoptosis.

Discussion
In the present study, the gene-trap mutagenesis screening of mouse-derived cells revealed Hmga2 as a new gene functioning in the MNU-induced apoptotic pathway. An insertional mutation in the Hmga2 gene rendered cells significantly resistant to MNU, with concomitant reduction in the activation of caspase-3 even after MNU treatment. We further showed that HMGA1, another HMGA family protein, also functions in the induction of apoptosis in response to O 6 -meG. HeLa MR cells transfected with siRNA specific for either HMGA1 or HMGA2 acquired certain degrees of resistance to MNU, and double-knockdown cells treated with both siHMGA1 and siHMGA2 exhibited further resistance to the agent, implying that these two proteins cooperate in the process of apoptosis triggered by MNU.
To obtain further evidence supporting the notion that HMGA proteins are involved in the O 6 -meG-induced apoptosis, we examined several apoptosis-related events. Our results showed that not only the phosphorylation of ATR, CHK1, and H2AX but also the appearance of the sub-G 1 population and activation of caspase-9 occur in HMGA-dependent manners. In contrast, the formation of the MMR complex takes place normally in HMGA-suppressed cells. Taken together, these findings suggest that, in the pathway of apoptosis induction, HMGA proteins function at a step immediately after the MMR complex formation and prior to the phosphorylation of ATR/CHK1 as shown in a model for the transfer of an apoptosis induction signal (Fig. 6). The ATM/CHK2 axis is also activated at a later time point than that of ATR/CHK1 in the course of O 6 -meG-induced apoptosis 26 . It is not still clear whether HMGA proteins are involved in the activation of the ATM/CHK2 signaling pathway during apoptosis triggered by O 6 -meG.
HMGA proteins are abundant non-histone chromatin proteins that contain three DNA binding domains, named AT-hooks, and a highly acidic carboxy-terminal region. Although HMGA proteins do not possess intrinsic transcriptional regulatory activity, these proteins have been reported to interact with transcription factors and regulate gene transcription 19,20 . It has also been shown that HMGA proteins bound to chromatin recruit chromatin remodeling factors and histone chaperons, thereby enhancing the alteration of chromatin structure 27,28 . It is uncertain, at present, how these activities of HMGA proteins are related to the function of the proteins in the induction of apoptosis. Some other activities of HMGA proteins may also be involved in the activation of DNA damage signaling. HMGA proteins are implicated in predisposition to tumors 29 . Neoplastic phenotypes appeared in Hmga1 −/− and Hmga1 +/− mice, suggesting a possible role of HMGA proteins in tumor suppression 30 . The rearrangement of the HMGA2 sequence, which yields a truncated protein lacking the C-terminus tail, has been found in benign tumors 31 . It has also been reported that the overproduction of HMGA proteins correlates with the metastatic ability of malignant tumors [32][33][34] . Notably, the mouse KH102 cells isolated as an MNU-resistant clone in the present gene-trap screening had an insertion of vector DNA in the third intron of one of the Hmga2 alleles, thereby yielding a truncated form of the protein (see Fig. 1b). The truncated protein might compete with the full-length form of Hmga2 for DNA binding and inhibit activities of the full-length one. The dramatic suppression in the induction of apoptosis exhibited by KH102 cells might be related to the predisposition to the development of human benign tumors, in which a chimeric or a truncated form of HMGA2 protein is expressed.
Understanding the precise roles of HMGA family proteins in the process of apoptosis will require learning if any other components are recruited to damaged chromatin through the interaction with HMGA proteins. Studies along this line are in progress in the laboratory.

Methods
Cell lines and cell culture. YT102 and YT103 were established from mouse fibroblasts derived from the lung tissue of Mgmt −/− and Mgmt −/− Mlh1 −/− mice, respectively 35 . A human-derived HeLa MR cell line defective in the MGMT function was obtained from H. Hayakawa 36 . All of the cell lines were used from our laboratory stocks and cultivated at 37 °C in 5% CO 2 in Dulbecco's modified Eagle's medium (Wako) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Thermo Fisher Scientific).
Gene-trap mutagenesis. Gene-trap mutagenesis was performed as described previously 12 . Briefly, the retrovirus vector pLHΔU3L carrying a promoter-less hygromycin B-resistance gene was introduced into ΨMP34 retrovirus-packaging cells (Takara Bio Inc.), and retroviral particles were produced. YT102 cells defective in Mgmt were infected with the retrovirus and incubated for 15 h. The cells were selected in a medium containing 0.6 mg/ml of hygromycin B for 55 h and then treated with 0.4 mM MNU in serum-free medium for 1 h, followed by further incubation in drug-free medium. The colonies formed were subjected to a survival assay with various concentrations of MNU, and MNU-resistant clones were obtained.
Survival assay. The cells were treated with various concentrations of MNU in serum-free medium containing 0.02 M HEPES-NaOH (pH 6.0) for 1 h at 37 °C and then cultivated in a complete medium for 10 days at 37 °C. The number of colonies formed was counted, and the survival rates were calculated. siRNA transfection. Silencer siRNA for HMGA1 gene, 5′-CCUGGGAUCUGAGUACAUATT-3′, and Stealth RNAi for the HMGA2 gene, 5′-GGCCAAGAGGCAGACCUAGGAAAUG-3′, were purchased from Thermo Fisher Scientific. After culturing 8 × 10 4 cells per well in a 24-well plate for 1 day, the cells were transfected with 40 nM siRNA using the Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) in accordance with the manufacturer's protocol. For the control transfection, Silencer select negative control No. 1 siRNA (Thermo Fisher Scientific) was used.
Chromatin immunoprecipitation. Following treatment with control-or HMGA-siRNA, HeLa MR cells expressing Flag-tagged PMS2 were exposed to 0.2 mM MNU for 1 h and further incubated in complete medium. The cells were permeabilized on a dish with buffer A (20 mM HEPES-KOH [pH 7.5], 5 mM KCl, 1.5 mM MgCl 2 , and 0.1 mM dithiothreitol) containing 100 μg/ml of digitonin (Wako) and then treated with 1% formaldehyde (Wako) for 5 min at room temperature. After the addition of 1 M Tris-HCl (pH 8.0), the cells were harvested and collected by centrifugation at 3,000 g for 5 min at 4 °C. The cell pellet was suspended in buffer B (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40) containing a protease inhibitor cocktail (Roche Diagnostics) and then sonicated. The material was centrifuged at 20,000 g for 10 min at 4 °C, and the supernatant fraction was used as the chromatin extract. For immunoprecipitation, anti-FLAG M2-agarose (Sigma) was added to the chromatin extract and incubated for 12 h at 4 °C. After extensive washing of the beads with buffer B, the proteins bound to the beads were eluted in 40 μl of 2 × SDS sample buffer and subjected to immunoblotting. Analyses of apoptosis-related events. For the detection of the sub-G 1 population, the cells were treated with 0.4 mM MNU as described above and incubated in a complete medium for the indicated times. The cells were collected; suspended in PBS containing 0.1% Triton X-100, 0.025 mg/ml propidium iodide, and 0.1 mg/ml RNaseA; and then subjected to flow cytometry using a FACS Calibur (BD Biosciences). An assay for caspase-3 activity was performed in accordance with the instructions included with the EnzChek caspase-3 assay kit #2 (Thermo Fisher Scientific).