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Negative regulation of peroxiredoxin 6 (Prdx 6) transcription by nuclear oncoprotein DEK during leukemia cell differentiation Negative regulation of peroxiredoxin 6 (Prdx 6) transcription by nuclear oncoprotein DEK during...
Kim, Kee-Beom; Chae, Yun-Cheol; Han, Arim; Kang, Joo-Young; Jung, Hyeonsoo; Park, Jin Woo; Hahm, Ja Young; Kim, Seryeon; Seo, Sang-Beom
2014-09-03 00:00:00
Animal Cells and Systems, 2014 Vol. 18, No. 5, 318–323, http://dx.doi.org/10.1080/19768354.2014.950605 Negative regulation of peroxiredoxin 6 (Prdx 6) transcription by nuclear oncoprotein DEK during leukemia cell differentiation Kee-Beom Kim, Yun-Cheol Chae, Arim Han, Joo-Young Kang, Hyeonsoo Jung, Jin Woo Park, Ja Young Hahm, Seryeon Kim and Sang-Beom Seo* Department of Life Science, College of Natural Sciences, Chung-Ang University, Seoul 156-756, Republic of Korea (Received 23 April 2014; received in revised form 23 June 2014; accepted 29 July 2014) The oncogene protein DEK is an abundant and ubiquitous nuclear protein with implications in acute myelogenous leukemia, as translocation which results in the formation of a DEK-CAN fusion protein. In a previous study, we have identified that DEK negatively regulated peroxiredoxin 6 (Prdx 6) transcription synergistically with the p65 subunit of NF-κB. In this study, we further investigated DEK-mediated transcriptional regulation of Prdx 6 during leukemia cell differentiation. Using Chromatin Immunoprecipitation analysis and Prdx 6 reporter assays, we found that DEK operated as a negative regulator of Prdx 6 transcription during leukemia cell differentiation. DEK was highly expressed and recruited to Prdx 6 promoter along with p65 and repressed transcription after leukemia cell differentiation. Keywords: DEK; Prdx 6; leukemia; differentiation Introduction AP-2α and enhance the binding of AP-2α to DNA. The interaction between DEK and AP-2α leads to transcrip- The DEK protein was first identified in a specific tional activation of target gene (Campillos et al. 2003). chromosomal translocation t(6;9)(p23;q34) in acute mye- NF-κB is a transcriptional repressor with various logenous leukemia (AML), which results in the formation biological functions including cell growth, differentiation, of fusion gene with the CAN nucleoporin protein NUP214 and the suppression of apoptosis. NF-κB contains five (von Lindern et al. 1992). The nuclear pore complex is subunits – RelA (p65), c-Rel, RelB, p50, and p52 which composed of more than 30 different proteins, and CAN is form various hetero- and homo-dimers (Hatada et al. one of the members in the complex. Translocated fusion 2000). DEK interacts with p65 subunit of NF-κB complex gene can make the fusion protein in which part of N- (Sammons et al. 2006). terminal (1–349 amino acids) of DEK is fused with part of Peroxiredoxins (Prdxs) are conserved from bacteria to (813–2,090 amino acids) of CAN. In addition to these mammals (Wood et al. 2003a). There are six subgroups of findings, recent report provides that DEK can be secreted Prdx enzymes in mammals (Wood et al. 2003b). These and acts as a chemoattractant it elicits, leading to enzymes use redox-active cysteins to reduce peroxides inflammation (Mor-Vaknin et al. 2006). Besides the and are divided into two categories of 1-Cys and 2-Cys. immunoreactivity it elicits, DEK has long been implicated Prdx 6 is the member of 1-Cys Prx and has nonselenium in aggressive human cancers such as hepatocellular glutathione peroxidase and phospholipase A2 activities carcinoma, glioblastoma, and melanoma (Kondoh et al. (Chen et al. 2000). Prdx 6 is expressed in multiple tissues 1999; Grottke et al. 2000; Larramendy et al. 2002; Casas related to inhibiting of cellular oxidative stresses (Mane- et al. 2003; Carro et al. 2006). vich et al. 2002). Moreover, Prdx 6-deficient mice are DEK has functional domains including DNA-binding susceptible to oxidative stress by hyperoxia or paraquat multimerization and acidic domain (Alexiadis et al. 2000; Waldmann et al. 2002). Most of all, DEK can regulate the treatment, leading to increase of mortality and lung injury (Wang et al. 2003; Wang et al. 2006). Previously, we have transcriptional activity in either activation or repression of reported that DEK negatively regulated Prdx 6 transcrip- downstream target genes through its acidic domains. For example, DEK can recruit the transcriptional corepressors tion and that the NF-κB subunit p65 had a synergistic such as hDaxx and HDAC2 to chromatin (Hollenbach effect on this DEK-mediated repression (Kim et al. 2010). In this study, we discuss the negative regulatory role of et al. 2002). The promoter region of NF-κB-regulated genes, both cIAP2 and interleukin-8 (IL-8), are occupied DEK in Prdx 6 transcription during leukemia cell differen- by DEK, leading to transcriptional repression as a tiation. We found that DEK expression was repressed repressor (Sammons et al. 2006). DEK can interact with during leukemia cell differentiation. Additionally, DEK *Corresponding author. Email: sangbs@cau.ac.kr © 2014 Korean Society for Integrative Biology MOLECULAR & CELLULAR BIOLOGY Animal Cells and Systems 319 was recruited to the Prdx 6 promoter along with p65 and β-actin. The ΔΔC value was calculated as the difference functioned as a corepressor of the target gene. between the control ΔC and values obtained for each sample. The n-fold change in gene expression relative to the ΔΔC untreated control was calculated as 2 . Materials and methods Plasmid constructs Western blot analysis For eukaryotic expression, the following pCMX plasmid Total proteins were isolated from cells using Radio- constructs were used: pCMX-DEK (Ko et al. 2006). The immunoprecipitation Assay buffer (50 mM Tris-HCl [pH proximal 5′-flanking region of the human Prdx 6 gene 8.0], 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycho- promoter was obtained from HeLa genomic DNA through late, 1% NP-40, 1X protease inhibitor cocktail, and 1 mM using XhoI-HindIII site-linked primer pair (forward, 5′- EDTA), fractionated by sodium dodecyl sulfate–polyacry- CTCGAGACATTTCTCTATCGATAGGTACC-3′; reverse lamide gel electrophoresis (SDS–PAGE), and transferred 5′-AAGCTTCACGTACCGGATGCCAGCTTAC-3′). The to nitrocellulose membranes. The membranes were probed purified products were enzyme digested with XhoI and overnight with the indicated antibodies at 4°C. The blots HindIII and cloned into the pGL3-basic luciferase reporter were incubated with horseradish peroxidase-conjugated vector (Promega). goat anti-rabbit or anti-mouse antibodies (Enzo Life Sciences) and detected using an enhanced chemilumines- cence (ECL) system. Cell culture HL-60 and K562 cells were grown in RPMI 1640, and 293T cells were grown in Dulbecco’s modified Eagle’s Reporter assay medium (DMEM) containing 10% heat-inactivated fetal K562 cells were seeded in 6-well dishes and transfected bovine serum and 0.05% penicillin-streptomycin at 37°C by lipofectamine 2000 with pGL3-Prdx 6 and shDEK. with 5% CO in humidified air. K562 and 293T cells were After 24 h transfection, Hemin (30 μM) or dimethyl transfected with each construct using lipofectamine 2000 sulfoxide (DMSO) is treated for indicated time point. (Invitrogen) or polyethylenimine (Sigma), respectively. After indicated time point, cells were harvested and assayed for luciferase activity using a luciferase assay Knockdown of DEK mRNA and transient transfection system (Promega). Each value was expressed as the mean of three replicates from a single assay, and experiments We used the vector-based RNAi method to knockdown were performed at least three times. endogenous DEK and reduce the DEK expression level. pSM2c-DEK expressing DEK-specific short hairpin RNAs (nucleotides 654–674 from NM_003472), referred Chromatin immunoprecipitation (ChIP) analysis to as shRNA, was purchased (Open Biosystems). ChIP analysis was performed as described in the protocols from Millipore. Briefly, HL-60 cells were differentiated RNA extraction and real-time polymerase chain by all-trans-retinoic acid (ATRA) or 12-O-tetradecanoyl- reaction (PCR) phorbol 13-acetate (TPA) and harvested 48 h later. Cells Total RNAs were prepared from cells for the reverse were cross-linked with 1% formaldehyde in the medium transcription PCR. Total RNA extraction was carried out at room temperature for 10 min, followed by the addition using TRIZOL reagent (TakaRa), according to the manu- 125 mM glycine at room temperature for 5 min, and facturer’s instructions. Extracted total RNAs were sub- then scraped into sodium dodecyl sulfate (SDS) lysis jected to reverse transcription PCR. The quantified cDNA buffer (50 mM Tris-HCl [pH 8.1], 1% SDS, and 10 mM was subjected to DEK, and Prdx 6 mRNA expression EDTA) with antibodies against DEK, p65, and IgG. analysis. The following PCR primers were used: 5′-ACG- The immunoprecipitates were recovered with protein GAACAGTTCTGGAATGG-3′ (forward) and 5′- A/G agarose beads (GenDEPOT). After reversing the TGGTGGCTCCTCTTCACTTT-3′ (reverse) (DEK), 5′- cross-links, chromatin was subjected to proteinase K diges- CAATACCACCGTCGGCCGCA-3′ (forward) and 5′- tion, and the DNA was purified for PCR amplifica- AAGGCTGGGGTGTGTAGCGG-3′ (reverse) (Prdx 6). tion (QIAGEN). To analyze the Prdx 6 promoter region, Disassociation curves were generated after each PCR run primer sets consisting of the promoter region (position to ensure that a single product of appropriate length was 911 to 672 from translation start site, forward: 5′- amplified. The mean threshold cycle (C ) and standard GTTGACCTGCACACAGTAGGTCTC-3′, reverse: 5′- error (SE) were calculated from individual C values CCTACAGTGGAGTGGAGTGACTGCT-3′); final intron obtained from triplicates per stage. The normalized mean C region (position 9,353 to 9,708, forward: 5′- GTCATGGCTGTAAAAGTACTGGTG-3′, reverse: 5′- was estimated as the ΔC by subtracting the mean C of T T 320 K.-B. Kim et al. Figure 1. DEK represses Prdx 6 during leukemia cell differentiation. (A) The expression levels of DEK and Prdx 6 in 293T cells with ΔΔC transiently overexpressed DEK were detected via RT-PCR. The error bars represent 2 ± the SD of three independent experiments. ***P < 0.001. Total proteins were extracts from DEK transfected 293T cells and then were subjected to Western blot analysis using specific antibodies. (B) HL-60 cells were treated with TPA or DMSO. After 48 h, RT-PCR was performed to compare the expression levels of the target genes. Results are shown as means ± SDs; n = 3. **P < 0.01; *P < 0.05. (C) HL-60 cells were treated with ATRA or TPA. After 48 h, differentiated HL-60 cells were lysed and immunoblotted with anti-DEK, anti-β-actin, and anti-Prdx 6 antibodies. CACTGGAATGGAAGTTCTATGAGGG-3′); final exon C was estimated as ΔC by subtracting the mean C of T T T (position 9,989 to 10,347, forward: 5′-GCTTGGAGAA- input from that of the individual region. GAAGCTGCAGAA-3′, reverse: 5′-CTATCCCCATCC- TATTGAAAGAC-3′) were used. The amplification Statistical analysis reaction was performed under the following conditions: 45 cycles of denaturation at 94°C, annealing at 58°C, and Data are expressed as mean ± standard deviation (SD) of extension at 72°C. Disassociation curves were generated three or more independent experiments. Statistically signi- after each PCR was run to ensure that a single product of ficant effects (P < 0.05) were evaluated with Microsoft appropriate length was amplified. The mean C ± SE was Excel. Differences between groups were evaluated by one- calculated from individual C values obtained from way analysis of variance (ANOVA), followed by Student’s triplicate determinations per stage. The normalized mean t-test or Bonferroni’s test, as appropriate. Animal Cells and Systems 321 Figure 2. DEK negatively regulates Prdx 6 transcription during leukemia cell differentiation. (A) K562 cells were transfected with Prdx 6-luc and hemin (30 µM) was treated for indicate time points. Cell extracts were assayed for luciferase activity. Luciferase activities were normalized to those of β-galactosidase. Results are shown as means ± SDs; n =3. **P < 0.01; ***P < 0.001. (B) K562 cells were transfected with Prdx 6-luc and shDEK. Hemin (30 µM) was treated for indicated time points. Cell extracts were assayed for luciferase activity. Luciferase activities were normalized to those of β-galactosidase. Results are shown as means ± SDs; n =3.**P <0.01; *P< 0.05. Results and discussion of DEK in 293T cells (Kim et al. 2010). Consistent with our previous study, real-time PCR (RT-PCR) and immu- Identification of the Prdx 6 target gene during leukemia noblot analysis showed significant down-regulation of cell differentiation Prdx 6 expression in DEK overexpression (Figure 1A). A nuclear phosphoprotein, DEK, has been associated with To evaluate thepossibleroleof DEK-mediated regulation of certain human diseases including leukemia and auto- Prdx 6 during leukemia cell differentiation, we first mon- immune disorders. In the previous study, we monitored itored the expression levels of DEK and Prdx 6 after TPA differentially expressed proteins in DEK knockdown cells treatment in promyelocytic HL-60 cells. First, RT-PCR using high resolution two-dimensional gel electrophoresis analysis showed that DEK transcriptions were significantly (2-DE) and Matrix-assisted laser desorption-ionization up-regulated after leukemia cell differentiation by TPA time-of-flight mass spectrometry (MALDI-TOF MS) to treatments (Figure 1B). Up-regulation of DEK protein levels investigate the physiological role mediated by DEK (Kim were also confirmed by immunoblots (Figure 1C). Consistent et al. 2009). Among differential proteomic profiles in with previous proteomic studies, the expression levels of DEK knockdown HeLa cells, we have identified that Prdx Prdx6wererepressedduringleukemia cell differentiation by 6 was up-regulated in the absence of DEK (Kim et al. immunoblot analysis (Figure 1C). These results suggest that 2009). To further investigate the possibility of Prdx 6 DEK may operate as a negative regulator of Prdx 6 transcriptional regulation by DEK and their related role, transcription during leukemia cell differentiation. we monitored expression of Prdx 6 during overexpression 322 K.-B. Kim et al. Figure 3. Occupancy of DEK and the p65 subunit in the Prdx 6 promoter region during leukemia cell differentiation. (A) Schematic diagram of the primer pairs used for ChIP analysis. Black boxes represent the Prdx 6 exons. (B) ChIP analyses of the Prdx 6 promoter in ATRA, TPA-treated HL-60 cells were conducted using anti-DEK, and anti-Prdx 6 and examined via RT-PCR. The results are representative of at least three independent experiments (± SD). ***P < 0.001. *P < 0.05. Transcriptional regulation of Prdx 6 promoter by DEK DEK is recruited to Prdx 6 promoter and represses transcription To further investigate that DEK repressed Prdx 6 expres- sion during leukemia cell differentiation, we next con- Having established that DEK negatively regulates Prdx 6, we subsequently attempted to determine whether DEK ducted a reporter assay using a Prdx 6-luc reporter system to examine DEK-mediated transcriptional regulation of and p65 are recruited to Prdx 6 promoter via ChIP analysis. In our previous study, we reported that NF-κB Prdx 6. Initially, we checked transcription of Prdx 6 during leukemia cell differentiation. When we differen- subunit p65 has a synergistic effect on DEK-mediated tiated K562 cells using hemin treatment, transcription of Prdx 6 repression (Kim et al. 2010). Both proteins were recruited to the Prdx 6 promoter region and regulate its Prdx 6 was significantly decreased after 24 h (Figure 2A). Interestingly, when DEK was knocked down by shRNA, transcription (Kim et al. 2010). We noted that there were basal levels of DEK and p65 recruitment to Prdx 6 transcription of Prdx 6 was up-regulated (Figure 2B). All together, these data were consistent with our previous promoter prior to TPA and ATRA treatment (Figure 3B). During leukemia cell differentiation by TPA and ATRA results which showing that transcription of Prdx 6 is negatively regulated by DEK during leukemia cell treatment, the recruitment of both DEK and p65 to the Prdx 6 promoter region was increased significantly and differentiation. Animal Cells and Systems 323 Hollenbach AD, McPherson CJ, Mientjes EJ, Iyengar R, repressed Prdx 6 transcription (Figure 3B). By contrast, Grosveld G. 2002. Daxx and histone deacetylase II associate the Prdx 6 final intron and final exon were not occupied with chromatin through an interaction with core his- by DEK and p65 (Figure 3B). These data indicate that tones and the chromatin-associated protein Dek. J Cell Sci. both DEK and p65 proteins are recruited to the Prdx 6 115:3319–3330. promoter and negatively regulate its transcription during Kim DW, Chae JI, Kim JY, Pak JH, Koo DB, Bahk YY, Seo SB. 2009. Proteomic analysis of apoptosis related proteins leukemia cell differentiation. regulated by proto-oncogene protein DEK. J Cell Biochem. Previous reports suggested that the levels of Prdx 6 106:1048–1059. protein increase when DEK is knocked down (Kim et al. Kim DW, Kim JY, Choi S, Rhee S, Hahn Y, Seo SB. 2010. 2009). These reports suggest that DEK regulates Prdx 6 Transcriptional regulation of 1-cys peroxiredoxin by the protein level with p65 during leukemia cell differentiation. proto-oncogene protein DEK. Mol Med Rep. 3:877–881. 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