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ZNF509S1 downregulates PUMA by inhibiting p53K382 acetylation and p53-DNA binding
ZNF509S1 downregulates PUMA by inhibiting p53K382 acetylation and p53-DNA binding Bu-Nam Jeon, Jae-Hyeon Yoon, Dohyun Han, Min-Kyeong Kim, Youngsoo Kim, Seo-Hyun Choi, Jiyang Song, Kyung-Sup Kim, Kunhong Kim, ManWook Hur PII: DOI: Reference:
S1874-9399(17)30096-2 doi:10.1016/j.bbagrm.2017.07.008 BBAGRM 1170
To appear in:
BBA - Gene Regulatory Mechanisms
Received date: Revised date: Accepted date:
15 March 2017 20 June 2017 26 July 2017
Please cite this article as: Bu-Nam Jeon, Jae-Hyeon Yoon, Dohyun Han, MinKyeong Kim, Youngsoo Kim, Seo-Hyun Choi, Jiyang Song, Kyung-Sup Kim, Kunhong Kim, Man-Wook Hur, ZNF509S1 downregulates PUMA by inhibiting p53K382 acetylation and p53-DNA binding, BBA - Gene Regulatory Mechanisms (2017), doi:10.1016/j.bbagrm.2017.07.008
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ZNF509S1 downregulates PUMA by inhibiting p53K382
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acetylation and p53-DNA binding
Bu-Nam Jeona, Jae-Hyeon Yoona, Dohyun Hanb, Min-Kyeong Kima, Youngsoo Kimb, Seo-
a
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Hyun Choia, Jiyang Songa, Kyung-Sup Kima, Kunhong Kima, and Man-Wook Hura,* Brain Korea 21 Plus Project for Medical Science, Severance Biomedical Research Institute,
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Department of Biochemistry and Molecular Biology, Yonsei University School of Medicine, b
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50-1, Yonsei-Ro, SeoDaeMoon-Ku, Seoul 03722, Republic of Korea Department of Biomedical Sciences and Biomedical Engineering, Seoul National
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University College of Medicine, Seoul 110-799, Republic of Korea
Running title: ZNF509S1 represses PUMA gene transcription
*Corresponding author Man-Wook Hur, Ph.D. Department of Biochemistry and Molecular Biology Yonsei University School of Medicine 50-1, Yonsei-Ro, SeoDaeMoon-Ku, Seoul 03722, Republic of Korea Tel: 82-2-2228-1678 Fax: 82-2-312-5041 E-mail: [email protected] 1
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Abstract
Expression of the POK family protein ZNF509L, and -its S1 isoform, is induced by p53
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upon exposure to genotoxic stress. Due to alternative splicing of the ZNF509 primary transcript, ZNF509S1 lacks the 6 zinc-fingers and C-terminus of ZNF509L, resulting in
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only one zinc-finger. ZNF509L and -S1 inhibit cell proliferation by activating p21/CDKN1A and RB transcription, respectively. When cells are exposed to severe DNA
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damage, p53 activates PUMA (p53-upregulated modulator of apoptosis) transcription. Interestingly, apoptosis due to transcriptional activation of PUMA by p53 is attenuated by
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ZNF509S1. Thus we investigated the molecular mechanism(s) underlying the transcriptional attenuation and anti-apoptotic effects of ZNF509S1. We show that ZNF509S1 modulation of p53 activity is important in PUMA gene transcription by
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modulating post-translational modification of p53 by p300. ZNF509S1 directly interacts
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with p53 and inhibits p300-mediated acetylation of p53 lysine K382, with deacetylation of p53 K382 leading to decreased DNA binding at the p53 response element 1 of the PUMA
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promoter. ZNF509S1 may play a role not only in cell cycle arrest, by activating RB expression, but also in rescuing cells from apoptotic death by repressing PUMA expression in cells exposed to severe DNA damage.
Highlights
• ZNF509S1 decreases apoptosis induced by severe DNA damage only in the presence of p53. • ZNF509S1 represses PUMA gene transcription induced by etoposide. • ZNF509S1 inhibits binding of p53 to the PUMA promoter via direct interaction with p53. • ZNF509S1 modulates acetylation of p53 K382 by p300.
Keywords: ZNF509S1 (ZBTB49), p53, PUMA, Post-translational modification, Apoptosis
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1. Introduction
The tumor suppressor p53 coordinates a regulatory network that supervises and
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responds to a variety of stress signals, including DNA damage, oncogenic activation, telomere erosion, ribosomal stress, loss of cell-cell or cell-matrix adhesion, and hypoxia [1,
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2]. Such coordination occurs by p53 regulation of genes affecting many important cellular processes, including cell cycle arrest, DNA repair, apoptosis, autophagy, senescence,
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metabolism, and oncogenesis [3-6]. p53 exerts irreplaceable anti-tumorigenic function at homeostasis and has been referred to as ‘the guardian of the genome’ [7]. Regardless of the
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type of stress, the final outcome of p53 activation is either cell survival, through cell cycle arrest and DNA repair, or cell death, but the mechanism leading to the choice between these outcomes has not been fully elucidated.
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Normally, p53 levels are kept low by HDM2, an E3 ubiquitin ligase that is regulated by
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p53 at the transcriptional level [8]. In response to a variety of cellular stresses, p53 expression is induced and activated and/or stabilized by post-translational modifications
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(PTMs) [5]. These PTMs have been shown to control p53 transcriptional activity, selection of growth inhibitory, DNA repair, or apoptotic gene targets, and other biological functions, in response to diverse cellular stresses [2]. PTM of p53 is highly complex. Its N-terminus is heavily phosphorylated, whereas its C-terminal domain (CTD) can be methylated, phosphorylated, acetylated, neddylated, ubiquitinated, and/or sumoylated [9, 10, and references therein).
In particular, acetylation is critical for p53 downstream gene transactivation. This PTM increases p53 protein stability, binding to specific promoter sequences, association with other proteins, and is required for specific checkpoint responses to DNA damage and activated oncogenes [5, 10]. Histone acetyltransferase (HAT) proteins such as p300/CBP, PCAF, and Tip60 have all been identified to acetylate p53 lysine residues, predominantly within its CTD. p300/CBP acetylates p53 at K373 and K382, while PCAF acetylates K320, and Tip60 acetylates K120 [11-14]. These modifications by HATs increase p53 stability and sequence-specific DNA-binding activity, both in vitro and in vivo, possibly due to 3
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conformational changes [15, 16]. Acetylated p53 can bind promoter sequences and recruit
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specific coactivators, mediate the transcriptional “activating” acetylation of histone H3 and H4 [17, 18]. Post-translational modification of p53 is a dynamic process that relays
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signaling to target gene expression.
PUMA, a primary p53 target gene, is critical to apoptosis. PUMA interacts with anti-
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apoptotic Bcl-2 family members such as Bcl-xL, Bcl-2, Mcl-1, Bcl-w, and A1, by inhibiting their interaction with the pro-apoptotic molecules Bax and Bak [19-21]. When
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the inhibitory binding is removed, Bax is translocated to, and elicits, mitochondrial dysfunction, resulting in release of the mitochondrial apoptotic proteins cytochrome C,
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SMAC, and apoptosis-inducing factor (AIF), eventually leading to caspase proteinase activation and cell death [19, 22-24]. PUMA is normally expressed at a very low level [25], but is rapidly induced in response to a wide range of stresses. p53 is a major transcription
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factor activating PUMA gene expression in cells exposed to DNA damage [25, 26]. p53
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acetylation at K382, by p300/CBP, increases PUMA gene transcription [27]. Within hours of DNA damage, acetylated p53 is recruited to two p53-responsive elements in the PUMA
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promoter, causing further protein acetylation, of histones H3 and H4, and PUMA transcriptional activation [25-27]. p53 and other transcription regulators, including p73, FoxO3a, E2F1, c-Myc, C/EBP, CREB, c-Jun, and Sp1 can also induce PUMA gene expression. Conversely, proteins such as CTCF, Scratch2, MYSM1, MYC, and Slug repress PUMA [28-30]. However, the molecular mechanism of how activated PUMA gene expression is ‘turned off’ in heavily damaged cells remains elusive [31]. Recently, we characterized four novel members of the POK family transcription factors, ZNF509 isoforms (ZNF509L, -S1, -S2, and -S3), generated by alternative splicing of the ZNF509 primary transcript [32, Fig. 1A], showing their induction by p53 following DNA damage. Moreover, these isoforms regulate two critical genes controlling cell proliferation. ZNF509L binds to the p21/CDKN1A promoter either alone or by interacting with MIZ-1 to recruit the co-activator p300 and activate p21/CDKN1A transcription. ZNF509S1, however, does not affect p21/CDKN1A transcription, but it does bind to the distal RB promoter to interact and interfere with the MIZF repressor, resulting in derepression of RB. 4
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Immunohistochemical analysis revealed that ZNF509 is highly expressed in normal
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epithelial cells, but was completely repressed in tumor tissues of the colon, lung, and skin, indicating a possible role as a tumor suppressor. We investigated this possible novel
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function of ZNF509S1 in cell cycle regulation and/or apoptosis, and found that ZNF509S1 induced not only cell cycle arrest, but also inhibited apoptosis, enabling cells to survive by
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repressing PUMA gene transcription, despite severe DNA damage, such as that caused by
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the topoisomerase inhibitor etoposide.
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2. Materials and methods
2.1. Cell culture
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HEK293, HCT116 p53+/+, and HCT116 p53-/- cells were cultured in media recommended
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by ATCC (Manassas, VA, USA).
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2.2. Plasmids, antibodies, and reagents
pcDNA3.1-p53, pcDNA3.1-p53K382R, and pcDNA3.0-FLAG-ZNF509S1 constructs were prepared by cloning cDNA fragments into pcDNA3.0 or pcDNA3.1 (Invitrogen, Carlsbad, CA, USA). To prepare recombinant GST-POZ ZNF509 protein, a cDNA fragment encoding the ZNF509 POZ (a.a. 25~121) domain was cloned into pGEX4T3 (Amersham Biosciences, NJ, USA). All plasmid constructs were verified by sequencing. The following antibodies were used: GAPDH (FL-335, sc-25778), p53 (DO-1, sc-126), p21 (C-19, sc-397), BAX (N20, sc-493), (Ac)-Lysine (AKL5C1, sc-32268), all from Santa Cruz Biotechnology (Santa Cruz, CA, USA), PUMA (ab33906), from Abcam (Cambridge, MA, USA), (Ac)-p53K382 (#2525), from Cell Signaling Technology (Danvers, MA, USA), and FLAG-tag (F3165), from Sigma (St. Louis, MO, USA). To obtain a rabbit polyclonal antibody against ZNF509, a white rabbit was immunized by subcutaneous injection with a recombinant GST-POZ polypeptide (a.a., 25~121) eight times, at 2-week intervals. Blood was then collected, 5
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incubated at 37°C for 90 min, and centrifuged. The supernatant was incubated with Affi-
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Gel 10 beads cross-linked to a recombinant ZNF509 POZ domain (Bio-Rad, Hercules, CA, USA.). The precipitated beads were washed with PBS, and the antibody was eluted (1.0 M
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2.3. Transient transfection and transcription assay
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Tris pH 7.6). Most of the chemical reagents were purchased from Sigma.
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pG13-Luc, pcDNA3.0-p53, pcDNA3.0-p53K382R, pcDNA3.0-ZNF509S1 and pCMVLacZ expression vectors in various combinations were transiently cotransfected into
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HCT116 p53-/- cells. After 24 h of incubation, cells were harvested and analyzed for luciferase activity. Reporter activity was normalized with cotransfected β-galactosidase activity for transfection efficiency. Independent assays were repeated three times in
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2.4. MTT assay
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triplicate.
To investigate the effect of ZNF509S1 on cell growth following etoposide treatment, cells were grown in 24-well dishes to 30~50% confluency, and then incubated for 4 days. The cell growth of each sample was determined by measuring the conversion of the tetrazolium salt MTT to formazan. After 0, 1, 2, 3 and 4 days of cell culture at 37°C, 20 l of MTT in PBS (2 mg/ml) was added to each well. After incubating for 4 hrs at 37°C, the supernatant was discarded and the precipitate was dissolved wit 0.5 ml of DMSO. Plates were then read on a microplate reader at 540 nm (Molecular Devices Corp., Sunnyvale, CA, USA). Independent assays were repeated twice in triplicate.
2.5. Flow cytometry cell cycle analysis
Cells were washed with PBS containing 1% horse serum, and stained with a FITC-annexin V apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA) at RT for 30 min in 6
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the dark. DNA content, cell cycle profiles, and forward scatter profiles were determined using a Becton Dickinson LSRII flow cytometer (San Jose, CA, USA), as analyzed by
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FlowJoTM software (Tree Star, Inc., Ashland, OR, USA). The assay was repeated three
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2.6. Quantitative real-time RT-PCR (qRT-PCR)
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times, and a representative result is shown.
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Total RNA was isolated from cells using TRIzol reagent (Invitrogen). cDNA was prepared using total RNA, random hexamers, and Superscript reverse transcriptase II (Promega,
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Madison, WI, USA). qRT-PCR was conducted in an ABI PRISM 7300 RT-PCR System using a SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA), with gene-specific primers (see Supplementary Table). GAPDH mRNA was used as a control
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2.7. Western blot analysis
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for normalization. Independent assays were repeated twice in triplicate.
Cells were washed, pelleted, and resuspended in RIPA buffer supplemented with protease inhibitors. Cell extracts were separated by 12% SDS-PAGE gel electrophoresis, transferred to Immun-Blot™ PVDF (polyvinylidene difluoride) membranes (Bio-Rad) and blocked with 5% skim milk (BD Biosciences) or BSA (bovine serum albumin). Blotted membranes were then incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies conjugated to HRP (horseradish peroxidase) (Thermo Scientific, Rockford, IL, USA), at RT for 2 h. Protein bands were visualized by ECL (enhanced chemiluminescence) solution (Thermo Scientific). GAPDH protein was used as a normalization control. Intensities of western blot bands were analyzed by MultiGauge image analysis program 3.0 (Fuji Film). The assay was repeated twice.
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2.8. GST-fusion protein purification and GST pull-down assays: in vitro transcription and
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translation (TNT) of p53 polypeptides
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Recombinant GST and GST-ZNF509S1 fusion proteins were prepared from E. coli BL21 (DE3) cells by glutathione-agarose 4-bead affinity chromatography (Peptron, Daejeon,
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Korea). [35S]-methionin-labeled p53 polypeptides were prepared using an in vitro TNT kit (Promega). GST and GST-fusion ZNF509S1 protein-agarose beads were incubated with
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[35S]-labeled p53 polypeptides in HEMG buffer, centrifuged, and the pellets then washed and separated by 12% SDS-PAGE. The gels were then exposed to X-ray film (Kodak,
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Rochester, NY, USA), to detect the GST-fused protein.
2.9. Immunoprecipitation (IP)
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Cell lysates were precleared, and supernatants incubated overnight with antibodies at 4°C,
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followed by incubation with protein A/G agarose beads. Beads were collected, washed, and resuspended in equal volumes of 5x SDS loading buffer. Immunoprecipitated proteins were
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separated by 12% SDS-PAGE and analyzed by western blotting, as described above. IgG was used as a negative control for IP.
2.10. Oligonucleotide pull-down assay
Oligonucleotide probes (see Supplementary Table) were annealed by heating at 95°C for 5 min, and cooled slowly to room temperature. Cells were lysed in HKMG buffer and the extracts incubated with 1 g of biotinylated double-stranded oligonucleotide for 16 h. The mixtures were further incubated with NeutrAvidin-agarose beads and precipitated by centrifugation. The precipitate was analyzed by western blot using antibodies against FLAG or p53. The ZNF509S1 3’ UTR region was used as a negative control. The assay was repeated twice.
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2.11. Chromatin immunoprecipitation (ChIP) assay
DNA-protein interactions with ZNF509S1, p53, or p53K382R, at the endogenous PUMA
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gene promoter, were analyzed by antibody-to-chromatin pull-down and standard ChIP assay protocols, as reported elsewhere [32]. Oligonucleotide primers sets were designed to
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amplify the promoter regions of interest (see Supplementary Table). The assay was
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repeated three times.
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2.12. In-gel trypsin digestion and LC-MS/MS analysis of p53
Excised gel pieces were destained, dehydrated, and rehydrated. Dried gel pieces were reduced with 10 mM DTT and then alkylated with 50 mM iodoacetamide (IAA). The gel
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pieces were then digested overnight with sequencing-grade modified trypsin (Promega) at
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an enzyme-to-protein ratio of 1:100 w/w (%). After overnight digestion, the peptides were sequentially extracted from the gel pieces and desalted using a C18 stage Tip. Desalted
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peptides were subjected to liquid chromatography tandem mass spectrometry analysis using a 75 m I.D. 15 cm C18 column on an EasyLC (Proxeon, Odense, Denmark) interfaced to a high-throughput tandem mass spectrometer (LTQ Velos ion trap MS, Thermo Scientific, Waltham, MA, USA). Data was acquired using a data-dependent Top10 method. For each cycle, one full scan MS survey spectra (m/z 300-2000) was followed by up to 10 MS/MS in the LTQ Velos for the most intense ions. Each sample was analyzed in triplicate. The raw data acquired was processed on a SEQUEST Sorcerer 2 platform (Sage-N Research, Milpitas, CA, USA). All MS/MS data were searched with a target-decoy database searching strategy against a composite database containing the International Protein Index (IPI) Human v3.74 database. The search included cysteine carbamidomethylation as a fixed modification and acetylation of lysine and oxidation of methionine as variable modifications. Identified acetyl-peptides were filtered and validated using Scaffold 3 proteomics analysis software (Proteome Software Inc., Portland, OR, USA). Details are available upon request and reported elsewhere [33, 34]. 9
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2. 13. Statistical analysis
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Student’s t-test was used for all statistical comparisons.
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3. Results
3.1 ZNF509S1 decreases apoptosis induced by severe DNA damage only in the presence of
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p53
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Recently, we showed that ZNF509S1 activates RB gene transcription by interacting with the MIZF (HINFP) repressor, resulting in RB derepression and inhibition of cell proliferation [32]. Similarly, p53-target proteins are induced by genotoxic stresses, cell
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cycle arrest, and can also induce apoptosis or senescence. Therefore, we investigated
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whether ZNF509S1 can affect proliferation of HCT16 p53+/+ and HCT116 p53-/- cells treated with etoposide dosage (50 M), reported to cause severe DNA damage. MTT assays
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showed that cells transfected with a ZNF509S1 expression vector were growth-inhibited one day prior to etoposide treatment, as previously reported [32]. After etoposide treatment, cells gradually arrested growth and began to show apoptosis. Interestingly, after 2 days of cell culture, cells with ectopic ZNF509S1 expression showed a relatively weak decrease in cell numbers, compared to controls. In contrast, cells transfected with siZNF509 RNA grew faster than control cells, from day 2 and beyond, of etoposide exposure. Interestingly, HCT116 p53-/- cells showed proliferation patterns similar to those of HCT116 p53+/+ cells, with regard to ZNF509S1 expression, over a 24-hr period. However, ZNF509S1 expression did not elicit similar effects, and cell numbers decreased similarly over a 2-4 day period of etoposide treatment (Fig. 1B). Microscopic examination of the HCT116 p53+/+ cells transfected with ZNF509S1 expression vector or siZNF509 RNA also showed that ZNF509S1 increased cell survival at day 4 after etoposide treatment, while knockdown of ZNF509S1 decreased cell survival. In contrast, HCT116 p53-/- cells transfected with ZNF509S1 expression vector or siZNF509 10
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RNA showed similar cell survival pattern at day 4 of etoposide treatment, suggesting
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ZNF509S1’s effect on cell survival requires p53 (Fig. 1C). Previously, we reported that ZNF509L could activate p21/CDKN1A transcription [32].
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Therefore, we examined whether ZNF509L could also affect cell survival. MTT assays showed ZNF509L to inhibit cell proliferation 24-hr prior to etoposide treatment. However,
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ZNF509L had no effect on cell viability of HCT116 p53+/+ or p53-/- cells exposed to 50 M etoposide (Supplementary Figs. S1A and B).
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We next tested, by flow cytometry, whether ZNF509S1 affects apoptosis of cells treated with high dosage etoposide. In HCT116 p53+/+ cells, flow cytometry analysis showed that
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ZNF509S1 decreased apoptotic cell numbers (annexin V-stained), compared to the control, after day 4 of etoposide treatment (from 53.3% to 24.9%). Analogously, knockdown of ZNF509S1 increased apoptosis (from 53.3% to 74.8%). In contrast, HCT116 p53-/- cells
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transfected with ZNF509S1 expression or siZNF509 RNA vectors showed apoptotic cell
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numbers similar to those seen following high-dose etoposide (Fig. 1D; Supplementary Fig. S2). These results suggest that ZNF509S1 increases cell survival by decreasing apoptotic
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death of cells treated with high-dose etoposide.
3.2. ZNF509S1 represses PUMA gene transcription induced by etoposide
DNA damage by etoposide treatment similarly increased expression of p53 target genes controlling cell cycle and apoptosis, including CDKN1A, BAX, and PUMA [35, 36]. We first tested whether ZNF509S1 affected transcriptional activity of p53. p53 and/or ZNF509S1 expression vectors, and pG13-Luc p53 reporter plasmids were transiently cotransfected into HCT116 p53-/- cells. ZNF509S1 effectively decreased transcription activation of reporter gene by p53 (Fig. 2A). ZNF509S1 potently repressed endogenous PUMA gene transcription induction, and knockdown of ZNF509S1 significantly increased PUMA gene transcription, by etoposide treatment, with little effect on CDKN1A and BAX expression (Fig. 2B and C). These data suggest that ZNF509S1 decreases PUMA gene transcription activation by p53, which may inhibit apoptotic cell death in cells exposed to 11
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high-dose etoposide.
3.3. ZNF509S1 inhibits binding of p53 to the PUMA promoter via direct interaction with
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p53
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Because ZNF509S1 decreased transcriptional activation of PUMA by p53, ZNF509S1 protein-protein interaction might negatively affect p53 activities important for downstream
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gene transcriptional activation. Co-immunoprecipitation and in vitro GST-fusion protein pull-down assays showed that ZNF509S1 and the DNA-binding domain of p53 interact
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directly (Fig. 3A and B). ChIP assays of HCT116 p53+/+ cells transfected with ZNF509S1 expression vector showed endogenous p53 binding to the PUMA promoter was low, but remained similar in the presence or absence of ZNF509S1. However, robust p53 binding to
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the PUMA promoter upon etoposide treatment, was decreased by ectopic ZNF509S1
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expression. Interestingly, ZNF509S1 alone did not bind to the p53-binding element within the promoter of the PUMA gene (Fig. 3C). Oligonucleotide pull-down assays also showed
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that ZNF509S1 alone did not bind to the two p53REs, but did strongly decrease p53 binding to p53RE1, and weakly to p53RE2 (Fig. 3D).
3.4. ZNF509S1 modulates acetylation of p53 K382 by p300
As described above, ZNF509S1 interacts with p53 to inhibit p53 binding to the p53RE1 of the PUMA promoter. Because ZNF509S1 did not affect TP53 transcription or protein expression, ZNF509S1 might affect p53’s functional activity important to DNA binding. We thus investigated whether ZNF509S1 regulates acetylation of p53 C-terminus lysine residues, which are important for p53 DNA-binding activity upon DNA damage. In HCT116 p53+/+ cells transfected with ZNF509S1 and then treated with etoposide, ZNF509S1 inhibited acetylation of the p53 protein (Fig. 4A). Furthermore, p53 acetylation of in vitro p53 acetylation reaction mixtures (p53 only; p53 + p300; p53 + p300 + ZNF509S1) was analyzed by LC-MS/MS. p53 K382 acetylation 12
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levels were quantified by comparing extracted ion chromatograms (XICs) of the acetylated
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peptides in the three different in vitro reactions, showing that ZNF509S1 inhibited acetylation of p53K382 by p300 (Fig. 4B; Supplementary Figs. S3 and S4). Western blot
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analysis of HCT116 p53+/+ cell extracts with ectopic ZNF509S1 expression decreased p53K382 acetylation induced by etoposide, while knockdown of ZNF509S1 increased
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p53K382 acetylation. These data support in vitro p53 acetylation data revealed by LC-
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MS/MS (Fig. 4C).
3.5. ZNF509S1 represses transcription of PUMA by inhibition of p53K382 acetylation and
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p53 binding
Having revealed that ZNF509S1 inhibits acetylation of p53K382 by p300, we next
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investigated the functional significance of acetylation inhibition. We prepared p53K382R, a
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mimic of deacetylated p53K382, and tested whether it could regulate transcription of PUMA and/or a pG13-Luc transcriptional reporter. In HCT116 p53-/- cells, transcriptional
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activation of pG13-Luc or PUMA by wild type p53 was repressed by ZNF509S1, possibly by deacetylation of p53K382. However, while transcriptional activation of pG13-Luc or PUMA was activated by p53K382R, the activation was relatively weak, compared to p53WT. Similarly, coexpression of ZNF509S1 did not further affect the reporter or PUMA gene transcription activation by p53K382R, suggesting inhibition of acetylation at p53K382 by ZNF509S1 may be sufficient to repress PUMA gene activation by p53 (Fig. 5A-C). By flow cytometry, we tested whether ZNF509S1 affects apoptosis of the cells transfected wild-type p53 or p53K382R expression vector. In HCT116 p53-/- cells, apoptotic cell death by ectopic p53 was attenuated by co-expressed ZNF509S1 (from 28.1% to 10.2%). In contrast, ZNF509S1 only weakly affected apoptosis by p53K382R (Fig. 5D; Supplementary Fig. S5). ChIP and oligonucleotide pull-down assays of HCT116 p53-/- cells transfected with various combinations of p53, p53K382R, and/or ZNF509S1 expression vectors, were performed to investigate whether inhibition of acetylation of p53 K382 affected PUMA 13
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promoter binding, both in vivo and in vitro. That assay showed that DNA binding activity of
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wild type p53, on p53RE1, was considerably decreased by ZNF509S1. As expected, compared to wild type p53, p53K382R showed relatively weak promoter DNA binding
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activity. Moreover, p53K382R-to-DNA binding to the PUMA p53RE1 was not further affected by coexpressed ZNF509S1 (Fig. 6A). Oligonucleotide pull-down assays showed
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differences in p53-binding events at p53RE1 and p53RE2, which could not be revealed by ChIP, due to proximity of the two sites. ZNF509S1 inhibited binding of wild type p53 to
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p53RE1, but the deacetyl-mimic p53K382R showed only weak binding that was not affected by ZNF509S1. However, p53 or p53K382R binding to p53RE2 was not inhibited
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significantly by ZNF509S1 (Fig. 6B). These results suggest that ZNF509S1 affects p53 binding to the PUMA promoter p53RE1 by inhibiting acetylation of p53K382 by p300,
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4. Discussion
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severe cellular DNA damage.
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resulting in PUMA downregulation and attenuation of apoptotic cell death, caused by
Previously, we identified ZNF509S1 to be induced by p53, inhibiting cell proliferation by upregulating the tumor suppressor RB. While ZNF509S1 did not bind DNA directly, it interacted with MIZF to displace a corepressor and activate the RB promoter [32]. ZNF509S1 is a p53 downstream target gene that reciprocally, interacts with p53 (Fig. 7A). Moreover, ZNF509S1 inhibited p53-binding activity through blocking p53K382 acetylation, following severe DNA damage. p53 activity is regulated by many post-translational modifications, including ubiquitylation, phosphorylation, acetylation, sumoylation, methylation, and neddylation [9, 10, and references therein]. These distinct PTMs then dictate p53 response to diverse cellular signals. While the regulation and effects of p53 ubiquitination and phosphorylation have been extensively studied, less is known about other p53 PTMs, including acetylation. Acetylation of one or more lysines can have profound functional effects by altering protein conformation and/or interactions with other proteins [5, 10]. Several C-terminal lysines of 14
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p53 (K370, K372, K373, K381, and K382) were hypothesized to be acetylated by various
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histone acetyltransferases, including PCAF, p300/CBP, Tip60, and GCN5. It has been postulated that p53 acetylation increases DNA binding activity and consequently, transcriptional activation of p53 target genes [11-14]. Acetylation of one or more lysine
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residues of p53 (the so-called “acetylation code”) confers DNA binding specificity, and
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appears to be critical for cell fate decisions, by triggering expression of a set of p53 target genes regulating cell cycle arrest, apoptosis, senescence, DNA repair, autophagy, etc [3].
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For example, p300-mediated p53 acetylation of K382, in cancer cells exposed to DNA damage, resulted in transcriptional activation of both apoptotic and cell cycle arrest genes
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like PUMA and CDKN1A [11, 31]. Our investigation into ZNF509S1 revealed an interesting mechanism that could attenuate apoptotic cell death determined by p300mediated p53K382 acetylation. While ZNF509S1 lacked direct or indirect binding to p53-
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response elements within the PUMA promoter, it specifically inhibited p53 binding to the
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PUMA promoter by interacting with p53 and inhibiting its acetylation at K382, an event important for apoptotic p53 target gene expression.
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DNA damage induces cell cycle arrest, DNA repair, and irreversible events such as senescence and apoptosis. p53, in damaged cells, decides which fate to take, depending on the severity of DNA damage [5, 35, 36]. Incomplete repair of damaged DNA prior to replication can result in the accumulation of genetic mutations. Consequently, cells in cell cycle arrest must block the function of DNA damage-induced, apoptosis-related proteins, until DNA repair is complete [39, 40]. Several proteins were shown to arrest the cell cycle and inhibit apoptosis. Bcl-2 not only inhibits cell cycle progression, but also restrains apoptosis [41]. Galectin-3 also modulates BCL2 anti-apoptotic activity, and cell cycle arrest, by downregulation of cyclin A and upregulation of cyclin D1, p21, and p27, in response to a loss of cell-substrate interactions via BCL2 phosphorylation [42]. p21 (CDKN1A) functions as a negative regulator of cell cycle progression, and also induces cellular senescence and repression of pro-apoptotic genes [43]. In addition, the transcriptional regulator TCF3/E2A is required for full p21 induction upon p53 activation, and also acts as a repressor of PUMA expression [44]. Although these reports suggested that the above15
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mentioned proteins could separate cell cycle arrest from scenescence or apoptotic cell death,
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detailed mechanisms need yet to be demonstrated. Our current study reveals a unique mechanism regarding how ZNF509S1 arrests cell proliferation by activating RB
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transcription, and rescuing cells from apoptotic death by repressing PUMA transcription, in cells exposed to severe DNA damage (Fig. 7B). These features of ZNF509S1 may be
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important in tumor suppressor function. Our finding may also provide an example of how molecular programs can influence a regulatory protein that induces cell cycle arrest, while
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also inhibiting apoptotic cell death, thereby driving cell fate to cell cycle arrest (and thus,
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survival).
Author contributions
B.-N.J. and M.-W.H. designed and performed experiments, analyzed data and wrote the
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Funding
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and K.K. analyzed data.
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manuscript; J.-H.Y., D.H., M.-K.K., Y.K., S.-H.C., and J.S. performed experiments. K.-S.K.,
This work was supported by JeonRack-HooSok Grant 2016R1E1A1A02921938 (to M.-W. H), Do-Yak Research Grant 2011-0028817 (to M.-W.H.), and MRC Research Grant 20110030086 (to M.-W.H.) from the National Research Foundation of Korea (NRF) of the Korean Government (MSIP).
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References
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[1] H. F. Horn, K. H. Vousden, Coping with stress: multiple ways to activate p53, Oncogene 26 (2007) 1306-1316.
RI
[2] B. Gu, W. G. Zhu, Surf the post-translational modification metwork of p53 regulation, Int. J. Biol. Sci. 8 (2012) 672-684.
SC
[3] K. H. Vousden, C. Prives, Blinded by the light: The growing complexity of p53, Cell 137 (2009) 413-431.
NU
[4] A. J. Levine, M. Oren, The first 30 years of p53: Growing ever more complex, Nat. Rev. Cancer 9 (2009) 749-758.
MA
[5] S. M. Reed, D. E. Quelle, p53 acetylation: regulation and consequences, Cancers 7 (2014) 30-69.
[6] C. F. Cheok, C. S. Verma, J. Baselga, D. P. Lane, Translating p53 into the clinic, Nat.
D
Rev. Clin. Oncol. 8 (2011) 25-37.
TE
[7] D. P. Lane, p53, Guardian of the genome, Nature 358 (1992) 15-16. [8] M. H. Kubbutat, S. N. Jones, K. H. Vousden, Regulation of p53 stability by Mdm2,
AC CE P
Nature 387 (1997) 299-303.
[9] A. M. Bode, Z. Dong, Post-translational modification of p53 in tumorigenesis, Nat. Rev. Cancer 4 (2004) 793-805.
[10] L. Feng, T. Lin, H. Uranishi, W. Gu, Y. Xu, Functional analysis of the roles of posttranslational modifications at the p53 C terminus in regulating p53 stability and activity, Mol. Cell. Biol. 25 (2005) 5389-5395. [11] W. Gu, X. L. Shi, R. G. Roeder, Synergistic activation of transcription by CBP and p53, Nature 387 (1997) 819-823. [12] N. A. Barlev, L. Liu, N. H. Chehab, K. Mansfield, K. G. Harris, T. D. Halazonetis, S. L. Berger,
Acetylation
of
p53
activates
transcription
through
recruitment
of
coactivators/histone acetyltransferases, Mol. Cell 8 (2001) 1243-1254. [13] L. Liu, D. M. Scolnick, R. C. Trievel, H. B. Zhang, R. Marmorstein, T. D. Halazonetis, S. L. Berger, p53 sites acetylated in vitro by PCAF and p300 are acetylated in vivo in response to DNA damage, Mol. Cell. Biol. 19 (1999) 1202-1209. 17
ACCEPTED MANUSCRIPT
[14] Y. Tang, J. Luo, W. Zhang, W. Gu, Tip60-dependent acetylation of p53 modulates the
PT
decision between cell-cycle arrest and apoptosis, Mol. Cell 24 (2006) 827-839. [15] W. Gu, R. G. Roeder, Activation of p53 sequence-specific DNA binding by acetylation
RI
of the p53 C-terminal domain, Cell 90 (1997) 595-606.
[16] J. Luo, M. Li, Y. Tang, M. Laszkowska, R. G. Roeder, W. Gu, Acetylation of p53
SC
augments its site-specific DNA binding both in vitro and in vivo, Proc. Natl. Acad. Sci. USA 101 (2004) 2259-2264.
NU
[17] R. A. Henry, Y. M. Kuo, A. J. Andrews, Differences in specificity and selectivity between CBP and p300 acetylation of histone H3 and H3/H4, Biochemistry 52 (2013)
MA
5746-5759.
[18] L. Xu, Y. Chen, Q. Song, D. Xu, Y. Wang, D. Ma, PDCD5 interacts with Tip60 and functions as a cooperator in acetyltransferase activity and DNA damage-induced
D
apoptosis, Neoplasia 11 (2009) 345-354.
TE
[19] J. Yu, Z. Wang, K. W. Kinzler, B. Vogelstein, L. Zhang, PUMA mediates the apoptotic
1936.
AC CE P
response to p53 in colorectal cancer cells, Proc. Natl. Acad. Sci. USA 100 (2003) 1931-
[20] J. E. Chipuk, T. Moldoveanu, F. Llambi, M. J. Parsons, D. R. Green, The BCL-2 family reunion, Mol. Cell 37 (2010) 299-310. [21] J. R. Jeffers, E. Parganas, Y. Lee, C. Yang, J. Wang, J. Brennan, K. H. MacLean, J. Han, T. Chittenden, J. N. Ihle, P. J. McKinnon, J. L. Cleveland, G. P. Zambetti, Puma is an essential mediator of p53-dependent and -independent apoptotic pathways, Cancer Cell 4 (2003) 321-328. [22] K. S. Yee, K. H. Vousden, Contribution of membrane localization to the apoptotic activity of PUMA, Apoptosis 13 (2008) 87-95. [23] L. Ming, P. Wang, A. Bank, J. Yu, L. Zhang, PUMA dissociated Bax and Bcl-XL to induce apoptosis in colon cancer cells, J. Biol. Chem. 28 (2006) 16034-16042. [24] J. E. Chipuk, L. Bouchier-Hayes, T. Kuwana, D. D. Newmayer, D. R. Green, PUMA couples the nucler and cytoplasmic proapoptotic function of p53, Science 309 (2005) 1732-1735. 18
ACCEPTED MANUSCRIPT
[25] J. Yu, L. Zhang, P. M. Hwang, K. W. Kinzler, B. Vogelstein, PUMA induces the rapid
PT
apoptosis of colorectal cancer cells, Mol. Cell 7 (2001) 673-682. [26] P. Wang, J. Yu, L. Zhang, The nuclear function of p53 is required for PUMA-mediated
RI
apoptosis induced by DNA damage, Proc. Natl. Acad. Sci. USA 104 (2007) 4054-4059. [27] N. G. Iyer, S. F. Chin, H. Ozdag, Y. Daigo, D. E. Hu, M. Cariati, K. Brindle, S.
SC
Aparicio, C. Caldas, p300 regulates p53-dependent apoptosis after DNA damage in colorectal cancer cells by modulation of PUMA and p21 levels, Proc. Natl. Acad. Sci.
NU
USA 101 (2004) 7386-7391.
[28] E. Rodríguez-Aznar, M. A. Nieto, Repression of Puma by scratch2 is required for
MA
neuronal survival during embryonic development, Cell Death Differ. 18 (2011) 11961207.
[29] J. I. Belle, J. C. Petrov, D. Langlais, F. Robert, R. Cencic, S. Shen, J. Pelletier, P. Gros,
D
A. Nijnik, Repression of p53-target gene Bbc3/PUMA by MYSM1 is essential for the
TE
survival of hematopoietic multipotent progenitors and contributes to stem cell maintenance, Cell Death Differ. 23 (2016) 759-775.
AC CE P
[30] S. Amente, J. Zhang, M. L. Lavadera, L. Lania, E. V. Avvedimento, B. Majello, Myc and PI3K/AKT signaling cooperatively repress FOXO3a-dependent PUMA and GADD45a gene expression, Nucleic Acids Res. 39 (2011) 9498-9507. [31] J. Yu, L. Zhang, PUMA, a potent killer with or without p53, Oncogene 27 (2009) Suppl 1:S71-83.
[32] B. N. Jeon, M. K. Kim, J. H. Yoon, M. Y. Kim, H. An, H. J. Noh, W. I. Choi, D. I. Koh, M. W. Hur, Two ZNF509 (ZBTB49) isoforms induce cell-cycle arrest by activating transcription of p21/CDKN1A and RB upon exposure to genotoxic stress, Nucleic Acids Res. 42 (2014) 11447-11461. [33] A. Shevchenko, H. Tomas, J. Havlis, J. V. Olsen, M. Mann, In-gel digestion for mass spectrometric characterization of proteins and proteomes, Nat. Protoc. 1 (2006) 28562860. [34] J. V. Olsen, J. C. Schwartz, J. Griep-Raming, M. L. Nielsen, E. Damoc, E. Denisov, O. Lange, P. Remes, D. Taylor, M. Splendore, E. R. Wouters, M. Senko, A. Makarov, M. 19
ACCEPTED MANUSCRIPT
Mann, S. Horning, A dual pressure linear ion trap Orbitrap instrument with very high
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sequencing speed, Mol. Cell. Proteomics 8 (2009) 2759-2769. [35] W. S. El-Deiry, Regulation of p53 downstream genes, Sem. Cancer Biol. 8 (1998) 345-
RI
357.
[36] T. Riley, E. Sontag, P. Chen, A. Levine, Transcriptional control of human p53-
SC
regulated genes, Nat. Rev. Mol. Cell Biol. 9 (2008) 402-412.
Funct. Genomic Proteomic 6 (2007) 8-18.
NU
[37] J. A. Costoya, Functional analysis of the role of POK transcriptional repressors, Brief.
[38] K. F. Kelly, J. M. Daniel, POZ for effect-POZ-ZF transcription factors in cancer and
MA
development, Trends Cell Biol. 16 (2006) 578-587.
[39] W. P. Roos, A. D. Thomas, B. Kaina, DNA damage and the balance between survival and death in cancer biology, Nat. Rev. Cancer 16 (2016) 20-33.
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[40] K. T. Beiging, S. S. Mello, L. D. Attardi, Unravelling mechanisms of p53-mediated
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tumour suppression, Nat. Rev. Cancer 14 (2014) 359-370. [41] D. C. Huang, L. A. O'Reilly, A. Strasser, S. Cory, The anti-apoptosis function of Bcl-2
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can be genetically separated from its inhibitory effect on cell cycle entry, EMBO J. 16 (1997) 4628-4638.
[42] T. Yoshii, T. Fukumori, Y. Honjo, H. Inohara, H. R. Kim, A. Raz, Galectin-3 phosphorylation is required for its anti-apoptotic function and cell cycle arrest, J. Biol. Chem. 277 (2002) 6852-6857. [43] M. T. Piccolo, S. Crispi, The dual role played by p21 may influence the apoptotic or anti-apoptotic fate in cancer, Journal of Cancer Research Updates 1 (2012) 189-202. [44] Z. Andrysik, J. Kim, A. C. Tan, J. M. Espinosa, A genetic screen identifies TCF3/E2A and TRIAP1 as pathway-specific regulators of the cellular response to p53 activation, Cell Rep. 3 (2013) 1346-1354.
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Figure legends
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Fig. 1. ZNF509S1 decreases etoposide-induced apoptosis in HCT116 p53+/+ cells. (A) Structures of ZNF509L and ZNF509S1. (B) MTT assays of cells grown for 0-4 days.
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HCT116 p53+/+ and HCT116 p53-/- cells were transfected with ZNF509S1 expression
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vector or siZNF509 RNA. Control cells were cotransfected with pcDNA3.0 vector or negative siRNA. After 1 day of culture, the cells were treated with high-dose etoposide (50
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M) and analyzed for cell growth. Data shown are the averages of three independent assays. Error bars are too small to seen in the figure. (C) Microscopic images of the MTT-assayed
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cells (B) at 4 days. (D) Flow cytometry of apoptosis. Cells treated as in (B) were stained with FITC-annexin V, and analyzed by flow cytometry. The assay was repeated three times,
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and a representative result is shown.
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Fig. 2. Transcriptional activation of PUMA or pGL13-Luc, by p53 or etoposide treatment, is repressed by ZNF509S1. (A) ZNF509S1 represses transcriptional activation of pG13-Luc
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by p53. p53 and/or ZNF509S1 expression vectors, and a reporter plasmid, were transiently cotransfected into HCT116 p53-/- cells and luciferase activity was measured. Data shown are the averages of three independent assays. Error bars represent standard deviations. (B, C) qRT-PCR (B) and western blot (C) analyses. HCT116 p53+/+ cells transfected with ZNF509S1 expression vector or siZNF509 were treated with etoposide and analyzed for mRNA and protein expression of ZNF509S1, p53, CDKN1A, BAX, and PUMA. GAPDH, control; *, p<0.01; n.s., not significant. Band intensities were analyzed by MultiGauge image analyzer (FUJI film).
Fig. 3. ZNF509S1 inhibits binding of p53 to the PUMA promoter via direct interaction with p53. (A) Co-immunoprecipitation of ZNF509S1 and p53. Cell lysates prepared from HCT116 p53+/+ cells transfected with FLAG-ZNF509S1 expression vector were immunoprecipitated using anti-FLAG antibody and analyzed by western blot using the indicated antibodies. (B) In vitro GST-fusion protein pull-down assays. Recombinant GST 21
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or GST-ZNF509S1 was incubated with [35S]-methionine-labeled p53 polypeptide fragments, pulled down, and resolved by 15% SDS-PAGE. (C) ChIP-qPCR assay of ZNF509S1 and
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p53 binding to the PUMA promoter. HCT116 p53+/+ cells transfected with FLAG-
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ZNF509S1 expression vector were treated with etoposide and immunoprecipitated with an anti-FLAG or p53 antibody. Arrows at p53REs, locations of qChIP-PCR primers. The assay
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was repeated three times. (D) Oligonucleotide pull-down assay of ZNF509S1 and p53 binding to the p53-binding elements (p53RE1 and -2) of the PUMA promoter. HCT116
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p53+/+ cells were transfected with FLAG-ZNF509S1 expression vector, and extracts were incubated with biotinylated double-stranded oligonucleotides, and analyzed as described in
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the methods section. IP, immunoprecipitation; DNA-P, oligonucleotide pull-down assay; GAPDH, control.
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Fig. 4. ZNF509S1 modulates p53K382 acetylation by p300. (A) ZNF509S1 interacts with
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p53 and decreases p53 acetylation. HCT116 p53+/+ cells transfected with FLAGZNF509S1 expression vector were treated with etoposide and the cell lysates
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immunoprecipitated, using an anti-p53 antibody and analyzed by western blot using the indicated antibodies. (B) Mass spectrometry (MS) analysis of a p53 peptide acetylated at K382 by p300. “Kac,” acetylated lysine residue. “Mox,” oxidated methionine. An extracted ion chromatogram (XIC) of the acetylated peptide 382-K(Ac)LM(ox)FKTEGPDSD-393 (m/z: 713.66, charge 2+). K382 acetylation levels were quantified by comparing XICs of the acetylated peptides in three different in vitro reactions (p53 only, p53 + p300, and p53 + p300 + ZNF509S1). An XIC of the precursor peptides was plotted with 10-ppm of mass windows using Xcalibur. X axis, retention time; y axis, relative signal intensity. (C) Western blot analysis. HCT116 p53+/+ cells transfected with ZNF509S1 expression vector or siZNF509 were treated with etoposide, and analyzed by western blot using an acetylated p53K382-specific antibody. IP, immunoprecipitation; GAPDH, control.
Fig. 5. ZNF509S1 represses PUMA gene transcription through inhibition of p53K382 acetylation. (A) Transcriptional regulation of pG13-Luc by p53, ZNF509S1, or the 22
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deacetyl-mimic p53K382R and. HCT116 p53-/- cells were transiently co-transfected with
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wild-type p53 or p53K382R and/or ZNF509S1 expression vectors and luciferase activity was measured. Data shown are the averages of three independent assays. Error bars represent standard deviations. (B, C) qRT-PCR (B) and western blot (C) analyses of
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endogenous PUMA gene and protein expression. HCT116 p53-/- cells were transiently
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cotransfected with wild-type p53 or p53K382R and/or ZNF509S1 expression vectors. Transcriptional activation of endogenous PUMA by p53K382R is weaker than p53WT and
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is not altered by ectopic ZNF509S1. (D) Flow cytometry of apoptosis. Cells transfected as in B and C were stained with FITC-annexin V, and analyzed by flow cytometry. GAPDH,
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control; *, p<0.01; n.s., not significant.
Fig. 6. ZNF509S1 inhibits p53 binding to the PUMA promoter through inhibition of
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p53K382 acetylation. (A) ChIP-qPCR assay of p53, p53K382R, and ZNF509S1 binding to
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the PUMA promoter. HCT116 p53-/- cells were cotransfected with various combinations of p53, p53K382R, and FLAG-ZNF509S1 expression vectors, and immunoprecipitated with
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anti-FLAG or p53 antibody. Arrows at p53REs, locations of qChIP-PCR primers. Data shown are the averages of three independent assays. Error bars represent standard deviations. (B) Comparison of p53 binding consensus sequences, p53RE1 and p53RE2. Arrows, quarter site of p53RE. Two half sites can be separated by 0~13 bp spacer. p53RE1 of PUMA has a 1 bp spacer. R, A or G; W, A or T; Y, C or T. (C) Oligonucleotide pull-down assays of p53, p53K382R, and ZNF509S1 binding to two p53-binding response elements of the PUMA promoter. HCT116 p53-/- cells were transfected as above, and lysed cell extracts were incubated with biotinylated double-stranded oligonucleotides and analyzed as described in methods. IP, immunoprecipitation; DNA-P, oligonucleotide pull-down assay; GAPDH. control.
Fig. 7. Hypothetical model of PUMA transcription repression by ZNF509S1. (A) p53 lysine 382 is acetylated by p300 upon DNA damage. Ac-p53K382 binds to p53RE1 within the PUMA promoter and increases PUMA transcription. However, ZNF509S1 inhibits p53 23
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K382 acetylation by p300. This acetylation inhibition significantly decreases wild type p53
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transcriptional activation of the PUMA promoter, subsequently decreasing apoptotic cell death. (B) Scheme of the regulation of cell cycle arrest and apoptosis by transcriptional
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regulation of p21/CDKN1A, RB and PUMA by ZNF509S1 and ZNF509L., transcription activation; , transcription repression or inhibition at protein level. thin lines, regulatory
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events under mild DNA damage; thick lines, regulatory events under severe DNA damage.
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S1874-9399(17)30096-2 doi:10.1016/j.bbagrm.2017.07.008 BBAGRM 1170
To appear in:
BBA - Gene Regulatory Mechanisms
Received date: Revised date: Accepted date:
15 March 2017 20 June 2017 26 July 2017
Please cite this article as: Bu-Nam Jeon, Jae-Hyeon Yoon, Dohyun Han, MinKyeong Kim, Youngsoo Kim, Seo-Hyun Choi, Jiyang Song, Kyung-Sup Kim, Kunhong Kim, Man-Wook Hur, ZNF509S1 downregulates PUMA by inhibiting p53K382 acetylation and p53-DNA binding, BBA - Gene Regulatory Mechanisms (2017), doi:10.1016/j.bbagrm.2017.07.008
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ZNF509S1 downregulates PUMA by inhibiting p53K382
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acetylation and p53-DNA binding
Bu-Nam Jeona, Jae-Hyeon Yoona, Dohyun Hanb, Min-Kyeong Kima, Youngsoo Kimb, Seo-
a
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Hyun Choia, Jiyang Songa, Kyung-Sup Kima, Kunhong Kima, and Man-Wook Hura,* Brain Korea 21 Plus Project for Medical Science, Severance Biomedical Research Institute,
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Department of Biochemistry and Molecular Biology, Yonsei University School of Medicine, b
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50-1, Yonsei-Ro, SeoDaeMoon-Ku, Seoul 03722, Republic of Korea Department of Biomedical Sciences and Biomedical Engineering, Seoul National
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University College of Medicine, Seoul 110-799, Republic of Korea
Running title: ZNF509S1 represses PUMA gene transcription
*Corresponding author Man-Wook Hur, Ph.D. Department of Biochemistry and Molecular Biology Yonsei University School of Medicine 50-1, Yonsei-Ro, SeoDaeMoon-Ku, Seoul 03722, Republic of Korea Tel: 82-2-2228-1678 Fax: 82-2-312-5041 E-mail: [email protected] 1
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Abstract
Expression of the POK family protein ZNF509L, and -its S1 isoform, is induced by p53
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upon exposure to genotoxic stress. Due to alternative splicing of the ZNF509 primary transcript, ZNF509S1 lacks the 6 zinc-fingers and C-terminus of ZNF509L, resulting in
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only one zinc-finger. ZNF509L and -S1 inhibit cell proliferation by activating p21/CDKN1A and RB transcription, respectively. When cells are exposed to severe DNA
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damage, p53 activates PUMA (p53-upregulated modulator of apoptosis) transcription. Interestingly, apoptosis due to transcriptional activation of PUMA by p53 is attenuated by
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ZNF509S1. Thus we investigated the molecular mechanism(s) underlying the transcriptional attenuation and anti-apoptotic effects of ZNF509S1. We show that ZNF509S1 modulation of p53 activity is important in PUMA gene transcription by
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modulating post-translational modification of p53 by p300. ZNF509S1 directly interacts
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with p53 and inhibits p300-mediated acetylation of p53 lysine K382, with deacetylation of p53 K382 leading to decreased DNA binding at the p53 response element 1 of the PUMA
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promoter. ZNF509S1 may play a role not only in cell cycle arrest, by activating RB expression, but also in rescuing cells from apoptotic death by repressing PUMA expression in cells exposed to severe DNA damage.
Highlights
• ZNF509S1 decreases apoptosis induced by severe DNA damage only in the presence of p53. • ZNF509S1 represses PUMA gene transcription induced by etoposide. • ZNF509S1 inhibits binding of p53 to the PUMA promoter via direct interaction with p53. • ZNF509S1 modulates acetylation of p53 K382 by p300.
Keywords: ZNF509S1 (ZBTB49), p53, PUMA, Post-translational modification, Apoptosis
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1. Introduction
The tumor suppressor p53 coordinates a regulatory network that supervises and
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responds to a variety of stress signals, including DNA damage, oncogenic activation, telomere erosion, ribosomal stress, loss of cell-cell or cell-matrix adhesion, and hypoxia [1,
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2]. Such coordination occurs by p53 regulation of genes affecting many important cellular processes, including cell cycle arrest, DNA repair, apoptosis, autophagy, senescence,
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metabolism, and oncogenesis [3-6]. p53 exerts irreplaceable anti-tumorigenic function at homeostasis and has been referred to as ‘the guardian of the genome’ [7]. Regardless of the
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type of stress, the final outcome of p53 activation is either cell survival, through cell cycle arrest and DNA repair, or cell death, but the mechanism leading to the choice between these outcomes has not been fully elucidated.
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Normally, p53 levels are kept low by HDM2, an E3 ubiquitin ligase that is regulated by
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p53 at the transcriptional level [8]. In response to a variety of cellular stresses, p53 expression is induced and activated and/or stabilized by post-translational modifications
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(PTMs) [5]. These PTMs have been shown to control p53 transcriptional activity, selection of growth inhibitory, DNA repair, or apoptotic gene targets, and other biological functions, in response to diverse cellular stresses [2]. PTM of p53 is highly complex. Its N-terminus is heavily phosphorylated, whereas its C-terminal domain (CTD) can be methylated, phosphorylated, acetylated, neddylated, ubiquitinated, and/or sumoylated [9, 10, and references therein).
In particular, acetylation is critical for p53 downstream gene transactivation. This PTM increases p53 protein stability, binding to specific promoter sequences, association with other proteins, and is required for specific checkpoint responses to DNA damage and activated oncogenes [5, 10]. Histone acetyltransferase (HAT) proteins such as p300/CBP, PCAF, and Tip60 have all been identified to acetylate p53 lysine residues, predominantly within its CTD. p300/CBP acetylates p53 at K373 and K382, while PCAF acetylates K320, and Tip60 acetylates K120 [11-14]. These modifications by HATs increase p53 stability and sequence-specific DNA-binding activity, both in vitro and in vivo, possibly due to 3
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conformational changes [15, 16]. Acetylated p53 can bind promoter sequences and recruit
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specific coactivators, mediate the transcriptional “activating” acetylation of histone H3 and H4 [17, 18]. Post-translational modification of p53 is a dynamic process that relays
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signaling to target gene expression.
PUMA, a primary p53 target gene, is critical to apoptosis. PUMA interacts with anti-
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apoptotic Bcl-2 family members such as Bcl-xL, Bcl-2, Mcl-1, Bcl-w, and A1, by inhibiting their interaction with the pro-apoptotic molecules Bax and Bak [19-21]. When
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the inhibitory binding is removed, Bax is translocated to, and elicits, mitochondrial dysfunction, resulting in release of the mitochondrial apoptotic proteins cytochrome C,
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SMAC, and apoptosis-inducing factor (AIF), eventually leading to caspase proteinase activation and cell death [19, 22-24]. PUMA is normally expressed at a very low level [25], but is rapidly induced in response to a wide range of stresses. p53 is a major transcription
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factor activating PUMA gene expression in cells exposed to DNA damage [25, 26]. p53
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acetylation at K382, by p300/CBP, increases PUMA gene transcription [27]. Within hours of DNA damage, acetylated p53 is recruited to two p53-responsive elements in the PUMA
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promoter, causing further protein acetylation, of histones H3 and H4, and PUMA transcriptional activation [25-27]. p53 and other transcription regulators, including p73, FoxO3a, E2F1, c-Myc, C/EBP, CREB, c-Jun, and Sp1 can also induce PUMA gene expression. Conversely, proteins such as CTCF, Scratch2, MYSM1, MYC, and Slug repress PUMA [28-30]. However, the molecular mechanism of how activated PUMA gene expression is ‘turned off’ in heavily damaged cells remains elusive [31]. Recently, we characterized four novel members of the POK family transcription factors, ZNF509 isoforms (ZNF509L, -S1, -S2, and -S3), generated by alternative splicing of the ZNF509 primary transcript [32, Fig. 1A], showing their induction by p53 following DNA damage. Moreover, these isoforms regulate two critical genes controlling cell proliferation. ZNF509L binds to the p21/CDKN1A promoter either alone or by interacting with MIZ-1 to recruit the co-activator p300 and activate p21/CDKN1A transcription. ZNF509S1, however, does not affect p21/CDKN1A transcription, but it does bind to the distal RB promoter to interact and interfere with the MIZF repressor, resulting in derepression of RB. 4
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Immunohistochemical analysis revealed that ZNF509 is highly expressed in normal
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epithelial cells, but was completely repressed in tumor tissues of the colon, lung, and skin, indicating a possible role as a tumor suppressor. We investigated this possible novel
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function of ZNF509S1 in cell cycle regulation and/or apoptosis, and found that ZNF509S1 induced not only cell cycle arrest, but also inhibited apoptosis, enabling cells to survive by
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repressing PUMA gene transcription, despite severe DNA damage, such as that caused by
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the topoisomerase inhibitor etoposide.
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2. Materials and methods
2.1. Cell culture
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HEK293, HCT116 p53+/+, and HCT116 p53-/- cells were cultured in media recommended
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by ATCC (Manassas, VA, USA).
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2.2. Plasmids, antibodies, and reagents
pcDNA3.1-p53, pcDNA3.1-p53K382R, and pcDNA3.0-FLAG-ZNF509S1 constructs were prepared by cloning cDNA fragments into pcDNA3.0 or pcDNA3.1 (Invitrogen, Carlsbad, CA, USA). To prepare recombinant GST-POZ ZNF509 protein, a cDNA fragment encoding the ZNF509 POZ (a.a. 25~121) domain was cloned into pGEX4T3 (Amersham Biosciences, NJ, USA). All plasmid constructs were verified by sequencing. The following antibodies were used: GAPDH (FL-335, sc-25778), p53 (DO-1, sc-126), p21 (C-19, sc-397), BAX (N20, sc-493), (Ac)-Lysine (AKL5C1, sc-32268), all from Santa Cruz Biotechnology (Santa Cruz, CA, USA), PUMA (ab33906), from Abcam (Cambridge, MA, USA), (Ac)-p53K382 (#2525), from Cell Signaling Technology (Danvers, MA, USA), and FLAG-tag (F3165), from Sigma (St. Louis, MO, USA). To obtain a rabbit polyclonal antibody against ZNF509, a white rabbit was immunized by subcutaneous injection with a recombinant GST-POZ polypeptide (a.a., 25~121) eight times, at 2-week intervals. Blood was then collected, 5
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incubated at 37°C for 90 min, and centrifuged. The supernatant was incubated with Affi-
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Gel 10 beads cross-linked to a recombinant ZNF509 POZ domain (Bio-Rad, Hercules, CA, USA.). The precipitated beads were washed with PBS, and the antibody was eluted (1.0 M
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2.3. Transient transfection and transcription assay
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Tris pH 7.6). Most of the chemical reagents were purchased from Sigma.
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pG13-Luc, pcDNA3.0-p53, pcDNA3.0-p53K382R, pcDNA3.0-ZNF509S1 and pCMVLacZ expression vectors in various combinations were transiently cotransfected into
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HCT116 p53-/- cells. After 24 h of incubation, cells were harvested and analyzed for luciferase activity. Reporter activity was normalized with cotransfected β-galactosidase activity for transfection efficiency. Independent assays were repeated three times in
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2.4. MTT assay
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triplicate.
To investigate the effect of ZNF509S1 on cell growth following etoposide treatment, cells were grown in 24-well dishes to 30~50% confluency, and then incubated for 4 days. The cell growth of each sample was determined by measuring the conversion of the tetrazolium salt MTT to formazan. After 0, 1, 2, 3 and 4 days of cell culture at 37°C, 20 l of MTT in PBS (2 mg/ml) was added to each well. After incubating for 4 hrs at 37°C, the supernatant was discarded and the precipitate was dissolved wit 0.5 ml of DMSO. Plates were then read on a microplate reader at 540 nm (Molecular Devices Corp., Sunnyvale, CA, USA). Independent assays were repeated twice in triplicate.
2.5. Flow cytometry cell cycle analysis
Cells were washed with PBS containing 1% horse serum, and stained with a FITC-annexin V apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA) at RT for 30 min in 6
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the dark. DNA content, cell cycle profiles, and forward scatter profiles were determined using a Becton Dickinson LSRII flow cytometer (San Jose, CA, USA), as analyzed by
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FlowJoTM software (Tree Star, Inc., Ashland, OR, USA). The assay was repeated three
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2.6. Quantitative real-time RT-PCR (qRT-PCR)
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times, and a representative result is shown.
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Total RNA was isolated from cells using TRIzol reagent (Invitrogen). cDNA was prepared using total RNA, random hexamers, and Superscript reverse transcriptase II (Promega,
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Madison, WI, USA). qRT-PCR was conducted in an ABI PRISM 7300 RT-PCR System using a SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA), with gene-specific primers (see Supplementary Table). GAPDH mRNA was used as a control
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2.7. Western blot analysis
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for normalization. Independent assays were repeated twice in triplicate.
Cells were washed, pelleted, and resuspended in RIPA buffer supplemented with protease inhibitors. Cell extracts were separated by 12% SDS-PAGE gel electrophoresis, transferred to Immun-Blot™ PVDF (polyvinylidene difluoride) membranes (Bio-Rad) and blocked with 5% skim milk (BD Biosciences) or BSA (bovine serum albumin). Blotted membranes were then incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies conjugated to HRP (horseradish peroxidase) (Thermo Scientific, Rockford, IL, USA), at RT for 2 h. Protein bands were visualized by ECL (enhanced chemiluminescence) solution (Thermo Scientific). GAPDH protein was used as a normalization control. Intensities of western blot bands were analyzed by MultiGauge image analysis program 3.0 (Fuji Film). The assay was repeated twice.
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2.8. GST-fusion protein purification and GST pull-down assays: in vitro transcription and
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translation (TNT) of p53 polypeptides
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Recombinant GST and GST-ZNF509S1 fusion proteins were prepared from E. coli BL21 (DE3) cells by glutathione-agarose 4-bead affinity chromatography (Peptron, Daejeon,
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Korea). [35S]-methionin-labeled p53 polypeptides were prepared using an in vitro TNT kit (Promega). GST and GST-fusion ZNF509S1 protein-agarose beads were incubated with
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[35S]-labeled p53 polypeptides in HEMG buffer, centrifuged, and the pellets then washed and separated by 12% SDS-PAGE. The gels were then exposed to X-ray film (Kodak,
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Rochester, NY, USA), to detect the GST-fused protein.
2.9. Immunoprecipitation (IP)
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Cell lysates were precleared, and supernatants incubated overnight with antibodies at 4°C,
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followed by incubation with protein A/G agarose beads. Beads were collected, washed, and resuspended in equal volumes of 5x SDS loading buffer. Immunoprecipitated proteins were
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separated by 12% SDS-PAGE and analyzed by western blotting, as described above. IgG was used as a negative control for IP.
2.10. Oligonucleotide pull-down assay
Oligonucleotide probes (see Supplementary Table) were annealed by heating at 95°C for 5 min, and cooled slowly to room temperature. Cells were lysed in HKMG buffer and the extracts incubated with 1 g of biotinylated double-stranded oligonucleotide for 16 h. The mixtures were further incubated with NeutrAvidin-agarose beads and precipitated by centrifugation. The precipitate was analyzed by western blot using antibodies against FLAG or p53. The ZNF509S1 3’ UTR region was used as a negative control. The assay was repeated twice.
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2.11. Chromatin immunoprecipitation (ChIP) assay
DNA-protein interactions with ZNF509S1, p53, or p53K382R, at the endogenous PUMA
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gene promoter, were analyzed by antibody-to-chromatin pull-down and standard ChIP assay protocols, as reported elsewhere [32]. Oligonucleotide primers sets were designed to
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amplify the promoter regions of interest (see Supplementary Table). The assay was
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repeated three times.
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2.12. In-gel trypsin digestion and LC-MS/MS analysis of p53
Excised gel pieces were destained, dehydrated, and rehydrated. Dried gel pieces were reduced with 10 mM DTT and then alkylated with 50 mM iodoacetamide (IAA). The gel
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pieces were then digested overnight with sequencing-grade modified trypsin (Promega) at
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an enzyme-to-protein ratio of 1:100 w/w (%). After overnight digestion, the peptides were sequentially extracted from the gel pieces and desalted using a C18 stage Tip. Desalted
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peptides were subjected to liquid chromatography tandem mass spectrometry analysis using a 75 m I.D. 15 cm C18 column on an EasyLC (Proxeon, Odense, Denmark) interfaced to a high-throughput tandem mass spectrometer (LTQ Velos ion trap MS, Thermo Scientific, Waltham, MA, USA). Data was acquired using a data-dependent Top10 method. For each cycle, one full scan MS survey spectra (m/z 300-2000) was followed by up to 10 MS/MS in the LTQ Velos for the most intense ions. Each sample was analyzed in triplicate. The raw data acquired was processed on a SEQUEST Sorcerer 2 platform (Sage-N Research, Milpitas, CA, USA). All MS/MS data were searched with a target-decoy database searching strategy against a composite database containing the International Protein Index (IPI) Human v3.74 database. The search included cysteine carbamidomethylation as a fixed modification and acetylation of lysine and oxidation of methionine as variable modifications. Identified acetyl-peptides were filtered and validated using Scaffold 3 proteomics analysis software (Proteome Software Inc., Portland, OR, USA). Details are available upon request and reported elsewhere [33, 34]. 9
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2. 13. Statistical analysis
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Student’s t-test was used for all statistical comparisons.
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3. Results
3.1 ZNF509S1 decreases apoptosis induced by severe DNA damage only in the presence of
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p53
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Recently, we showed that ZNF509S1 activates RB gene transcription by interacting with the MIZF (HINFP) repressor, resulting in RB derepression and inhibition of cell proliferation [32]. Similarly, p53-target proteins are induced by genotoxic stresses, cell
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cycle arrest, and can also induce apoptosis or senescence. Therefore, we investigated
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whether ZNF509S1 can affect proliferation of HCT16 p53+/+ and HCT116 p53-/- cells treated with etoposide dosage (50 M), reported to cause severe DNA damage. MTT assays
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showed that cells transfected with a ZNF509S1 expression vector were growth-inhibited one day prior to etoposide treatment, as previously reported [32]. After etoposide treatment, cells gradually arrested growth and began to show apoptosis. Interestingly, after 2 days of cell culture, cells with ectopic ZNF509S1 expression showed a relatively weak decrease in cell numbers, compared to controls. In contrast, cells transfected with siZNF509 RNA grew faster than control cells, from day 2 and beyond, of etoposide exposure. Interestingly, HCT116 p53-/- cells showed proliferation patterns similar to those of HCT116 p53+/+ cells, with regard to ZNF509S1 expression, over a 24-hr period. However, ZNF509S1 expression did not elicit similar effects, and cell numbers decreased similarly over a 2-4 day period of etoposide treatment (Fig. 1B). Microscopic examination of the HCT116 p53+/+ cells transfected with ZNF509S1 expression vector or siZNF509 RNA also showed that ZNF509S1 increased cell survival at day 4 after etoposide treatment, while knockdown of ZNF509S1 decreased cell survival. In contrast, HCT116 p53-/- cells transfected with ZNF509S1 expression vector or siZNF509 10
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RNA showed similar cell survival pattern at day 4 of etoposide treatment, suggesting
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ZNF509S1’s effect on cell survival requires p53 (Fig. 1C). Previously, we reported that ZNF509L could activate p21/CDKN1A transcription [32].
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Therefore, we examined whether ZNF509L could also affect cell survival. MTT assays showed ZNF509L to inhibit cell proliferation 24-hr prior to etoposide treatment. However,
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ZNF509L had no effect on cell viability of HCT116 p53+/+ or p53-/- cells exposed to 50 M etoposide (Supplementary Figs. S1A and B).
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We next tested, by flow cytometry, whether ZNF509S1 affects apoptosis of cells treated with high dosage etoposide. In HCT116 p53+/+ cells, flow cytometry analysis showed that
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ZNF509S1 decreased apoptotic cell numbers (annexin V-stained), compared to the control, after day 4 of etoposide treatment (from 53.3% to 24.9%). Analogously, knockdown of ZNF509S1 increased apoptosis (from 53.3% to 74.8%). In contrast, HCT116 p53-/- cells
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transfected with ZNF509S1 expression or siZNF509 RNA vectors showed apoptotic cell
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numbers similar to those seen following high-dose etoposide (Fig. 1D; Supplementary Fig. S2). These results suggest that ZNF509S1 increases cell survival by decreasing apoptotic
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death of cells treated with high-dose etoposide.
3.2. ZNF509S1 represses PUMA gene transcription induced by etoposide
DNA damage by etoposide treatment similarly increased expression of p53 target genes controlling cell cycle and apoptosis, including CDKN1A, BAX, and PUMA [35, 36]. We first tested whether ZNF509S1 affected transcriptional activity of p53. p53 and/or ZNF509S1 expression vectors, and pG13-Luc p53 reporter plasmids were transiently cotransfected into HCT116 p53-/- cells. ZNF509S1 effectively decreased transcription activation of reporter gene by p53 (Fig. 2A). ZNF509S1 potently repressed endogenous PUMA gene transcription induction, and knockdown of ZNF509S1 significantly increased PUMA gene transcription, by etoposide treatment, with little effect on CDKN1A and BAX expression (Fig. 2B and C). These data suggest that ZNF509S1 decreases PUMA gene transcription activation by p53, which may inhibit apoptotic cell death in cells exposed to 11
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high-dose etoposide.
3.3. ZNF509S1 inhibits binding of p53 to the PUMA promoter via direct interaction with
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p53
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Because ZNF509S1 decreased transcriptional activation of PUMA by p53, ZNF509S1 protein-protein interaction might negatively affect p53 activities important for downstream
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gene transcriptional activation. Co-immunoprecipitation and in vitro GST-fusion protein pull-down assays showed that ZNF509S1 and the DNA-binding domain of p53 interact
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directly (Fig. 3A and B). ChIP assays of HCT116 p53+/+ cells transfected with ZNF509S1 expression vector showed endogenous p53 binding to the PUMA promoter was low, but remained similar in the presence or absence of ZNF509S1. However, robust p53 binding to
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the PUMA promoter upon etoposide treatment, was decreased by ectopic ZNF509S1
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expression. Interestingly, ZNF509S1 alone did not bind to the p53-binding element within the promoter of the PUMA gene (Fig. 3C). Oligonucleotide pull-down assays also showed
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that ZNF509S1 alone did not bind to the two p53REs, but did strongly decrease p53 binding to p53RE1, and weakly to p53RE2 (Fig. 3D).
3.4. ZNF509S1 modulates acetylation of p53 K382 by p300
As described above, ZNF509S1 interacts with p53 to inhibit p53 binding to the p53RE1 of the PUMA promoter. Because ZNF509S1 did not affect TP53 transcription or protein expression, ZNF509S1 might affect p53’s functional activity important to DNA binding. We thus investigated whether ZNF509S1 regulates acetylation of p53 C-terminus lysine residues, which are important for p53 DNA-binding activity upon DNA damage. In HCT116 p53+/+ cells transfected with ZNF509S1 and then treated with etoposide, ZNF509S1 inhibited acetylation of the p53 protein (Fig. 4A). Furthermore, p53 acetylation of in vitro p53 acetylation reaction mixtures (p53 only; p53 + p300; p53 + p300 + ZNF509S1) was analyzed by LC-MS/MS. p53 K382 acetylation 12
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levels were quantified by comparing extracted ion chromatograms (XICs) of the acetylated
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peptides in the three different in vitro reactions, showing that ZNF509S1 inhibited acetylation of p53K382 by p300 (Fig. 4B; Supplementary Figs. S3 and S4). Western blot
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analysis of HCT116 p53+/+ cell extracts with ectopic ZNF509S1 expression decreased p53K382 acetylation induced by etoposide, while knockdown of ZNF509S1 increased
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p53K382 acetylation. These data support in vitro p53 acetylation data revealed by LC-
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MS/MS (Fig. 4C).
3.5. ZNF509S1 represses transcription of PUMA by inhibition of p53K382 acetylation and
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p53 binding
Having revealed that ZNF509S1 inhibits acetylation of p53K382 by p300, we next
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investigated the functional significance of acetylation inhibition. We prepared p53K382R, a
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mimic of deacetylated p53K382, and tested whether it could regulate transcription of PUMA and/or a pG13-Luc transcriptional reporter. In HCT116 p53-/- cells, transcriptional
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activation of pG13-Luc or PUMA by wild type p53 was repressed by ZNF509S1, possibly by deacetylation of p53K382. However, while transcriptional activation of pG13-Luc or PUMA was activated by p53K382R, the activation was relatively weak, compared to p53WT. Similarly, coexpression of ZNF509S1 did not further affect the reporter or PUMA gene transcription activation by p53K382R, suggesting inhibition of acetylation at p53K382 by ZNF509S1 may be sufficient to repress PUMA gene activation by p53 (Fig. 5A-C). By flow cytometry, we tested whether ZNF509S1 affects apoptosis of the cells transfected wild-type p53 or p53K382R expression vector. In HCT116 p53-/- cells, apoptotic cell death by ectopic p53 was attenuated by co-expressed ZNF509S1 (from 28.1% to 10.2%). In contrast, ZNF509S1 only weakly affected apoptosis by p53K382R (Fig. 5D; Supplementary Fig. S5). ChIP and oligonucleotide pull-down assays of HCT116 p53-/- cells transfected with various combinations of p53, p53K382R, and/or ZNF509S1 expression vectors, were performed to investigate whether inhibition of acetylation of p53 K382 affected PUMA 13
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promoter binding, both in vivo and in vitro. That assay showed that DNA binding activity of
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wild type p53, on p53RE1, was considerably decreased by ZNF509S1. As expected, compared to wild type p53, p53K382R showed relatively weak promoter DNA binding
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activity. Moreover, p53K382R-to-DNA binding to the PUMA p53RE1 was not further affected by coexpressed ZNF509S1 (Fig. 6A). Oligonucleotide pull-down assays showed
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differences in p53-binding events at p53RE1 and p53RE2, which could not be revealed by ChIP, due to proximity of the two sites. ZNF509S1 inhibited binding of wild type p53 to
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p53RE1, but the deacetyl-mimic p53K382R showed only weak binding that was not affected by ZNF509S1. However, p53 or p53K382R binding to p53RE2 was not inhibited
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significantly by ZNF509S1 (Fig. 6B). These results suggest that ZNF509S1 affects p53 binding to the PUMA promoter p53RE1 by inhibiting acetylation of p53K382 by p300,
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4. Discussion
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severe cellular DNA damage.
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resulting in PUMA downregulation and attenuation of apoptotic cell death, caused by
Previously, we identified ZNF509S1 to be induced by p53, inhibiting cell proliferation by upregulating the tumor suppressor RB. While ZNF509S1 did not bind DNA directly, it interacted with MIZF to displace a corepressor and activate the RB promoter [32]. ZNF509S1 is a p53 downstream target gene that reciprocally, interacts with p53 (Fig. 7A). Moreover, ZNF509S1 inhibited p53-binding activity through blocking p53K382 acetylation, following severe DNA damage. p53 activity is regulated by many post-translational modifications, including ubiquitylation, phosphorylation, acetylation, sumoylation, methylation, and neddylation [9, 10, and references therein]. These distinct PTMs then dictate p53 response to diverse cellular signals. While the regulation and effects of p53 ubiquitination and phosphorylation have been extensively studied, less is known about other p53 PTMs, including acetylation. Acetylation of one or more lysines can have profound functional effects by altering protein conformation and/or interactions with other proteins [5, 10]. Several C-terminal lysines of 14
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p53 (K370, K372, K373, K381, and K382) were hypothesized to be acetylated by various
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histone acetyltransferases, including PCAF, p300/CBP, Tip60, and GCN5. It has been postulated that p53 acetylation increases DNA binding activity and consequently, transcriptional activation of p53 target genes [11-14]. Acetylation of one or more lysine
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residues of p53 (the so-called “acetylation code”) confers DNA binding specificity, and
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appears to be critical for cell fate decisions, by triggering expression of a set of p53 target genes regulating cell cycle arrest, apoptosis, senescence, DNA repair, autophagy, etc [3].
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For example, p300-mediated p53 acetylation of K382, in cancer cells exposed to DNA damage, resulted in transcriptional activation of both apoptotic and cell cycle arrest genes
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like PUMA and CDKN1A [11, 31]. Our investigation into ZNF509S1 revealed an interesting mechanism that could attenuate apoptotic cell death determined by p300mediated p53K382 acetylation. While ZNF509S1 lacked direct or indirect binding to p53-
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response elements within the PUMA promoter, it specifically inhibited p53 binding to the
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PUMA promoter by interacting with p53 and inhibiting its acetylation at K382, an event important for apoptotic p53 target gene expression.
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DNA damage induces cell cycle arrest, DNA repair, and irreversible events such as senescence and apoptosis. p53, in damaged cells, decides which fate to take, depending on the severity of DNA damage [5, 35, 36]. Incomplete repair of damaged DNA prior to replication can result in the accumulation of genetic mutations. Consequently, cells in cell cycle arrest must block the function of DNA damage-induced, apoptosis-related proteins, until DNA repair is complete [39, 40]. Several proteins were shown to arrest the cell cycle and inhibit apoptosis. Bcl-2 not only inhibits cell cycle progression, but also restrains apoptosis [41]. Galectin-3 also modulates BCL2 anti-apoptotic activity, and cell cycle arrest, by downregulation of cyclin A and upregulation of cyclin D1, p21, and p27, in response to a loss of cell-substrate interactions via BCL2 phosphorylation [42]. p21 (CDKN1A) functions as a negative regulator of cell cycle progression, and also induces cellular senescence and repression of pro-apoptotic genes [43]. In addition, the transcriptional regulator TCF3/E2A is required for full p21 induction upon p53 activation, and also acts as a repressor of PUMA expression [44]. Although these reports suggested that the above15
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mentioned proteins could separate cell cycle arrest from scenescence or apoptotic cell death,
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detailed mechanisms need yet to be demonstrated. Our current study reveals a unique mechanism regarding how ZNF509S1 arrests cell proliferation by activating RB
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transcription, and rescuing cells from apoptotic death by repressing PUMA transcription, in cells exposed to severe DNA damage (Fig. 7B). These features of ZNF509S1 may be
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important in tumor suppressor function. Our finding may also provide an example of how molecular programs can influence a regulatory protein that induces cell cycle arrest, while
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also inhibiting apoptotic cell death, thereby driving cell fate to cell cycle arrest (and thus,
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survival).
Author contributions
B.-N.J. and M.-W.H. designed and performed experiments, analyzed data and wrote the
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Funding
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and K.K. analyzed data.
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manuscript; J.-H.Y., D.H., M.-K.K., Y.K., S.-H.C., and J.S. performed experiments. K.-S.K.,
This work was supported by JeonRack-HooSok Grant 2016R1E1A1A02921938 (to M.-W. H), Do-Yak Research Grant 2011-0028817 (to M.-W.H.), and MRC Research Grant 20110030086 (to M.-W.H.) from the National Research Foundation of Korea (NRF) of the Korean Government (MSIP).
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References
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[1] H. F. Horn, K. H. Vousden, Coping with stress: multiple ways to activate p53, Oncogene 26 (2007) 1306-1316.
RI
[2] B. Gu, W. G. Zhu, Surf the post-translational modification metwork of p53 regulation, Int. J. Biol. Sci. 8 (2012) 672-684.
SC
[3] K. H. Vousden, C. Prives, Blinded by the light: The growing complexity of p53, Cell 137 (2009) 413-431.
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[4] A. J. Levine, M. Oren, The first 30 years of p53: Growing ever more complex, Nat. Rev. Cancer 9 (2009) 749-758.
MA
[5] S. M. Reed, D. E. Quelle, p53 acetylation: regulation and consequences, Cancers 7 (2014) 30-69.
[6] C. F. Cheok, C. S. Verma, J. Baselga, D. P. Lane, Translating p53 into the clinic, Nat.
D
Rev. Clin. Oncol. 8 (2011) 25-37.
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[7] D. P. Lane, p53, Guardian of the genome, Nature 358 (1992) 15-16. [8] M. H. Kubbutat, S. N. Jones, K. H. Vousden, Regulation of p53 stability by Mdm2,
AC CE P
Nature 387 (1997) 299-303.
[9] A. M. Bode, Z. Dong, Post-translational modification of p53 in tumorigenesis, Nat. Rev. Cancer 4 (2004) 793-805.
[10] L. Feng, T. Lin, H. Uranishi, W. Gu, Y. Xu, Functional analysis of the roles of posttranslational modifications at the p53 C terminus in regulating p53 stability and activity, Mol. Cell. Biol. 25 (2005) 5389-5395. [11] W. Gu, X. L. Shi, R. G. Roeder, Synergistic activation of transcription by CBP and p53, Nature 387 (1997) 819-823. [12] N. A. Barlev, L. Liu, N. H. Chehab, K. Mansfield, K. G. Harris, T. D. Halazonetis, S. L. Berger,
Acetylation
of
p53
activates
transcription
through
recruitment
of
coactivators/histone acetyltransferases, Mol. Cell 8 (2001) 1243-1254. [13] L. Liu, D. M. Scolnick, R. C. Trievel, H. B. Zhang, R. Marmorstein, T. D. Halazonetis, S. L. Berger, p53 sites acetylated in vitro by PCAF and p300 are acetylated in vivo in response to DNA damage, Mol. Cell. Biol. 19 (1999) 1202-1209. 17
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[14] Y. Tang, J. Luo, W. Zhang, W. Gu, Tip60-dependent acetylation of p53 modulates the
PT
decision between cell-cycle arrest and apoptosis, Mol. Cell 24 (2006) 827-839. [15] W. Gu, R. G. Roeder, Activation of p53 sequence-specific DNA binding by acetylation
RI
of the p53 C-terminal domain, Cell 90 (1997) 595-606.
[16] J. Luo, M. Li, Y. Tang, M. Laszkowska, R. G. Roeder, W. Gu, Acetylation of p53
SC
augments its site-specific DNA binding both in vitro and in vivo, Proc. Natl. Acad. Sci. USA 101 (2004) 2259-2264.
NU
[17] R. A. Henry, Y. M. Kuo, A. J. Andrews, Differences in specificity and selectivity between CBP and p300 acetylation of histone H3 and H3/H4, Biochemistry 52 (2013)
MA
5746-5759.
[18] L. Xu, Y. Chen, Q. Song, D. Xu, Y. Wang, D. Ma, PDCD5 interacts with Tip60 and functions as a cooperator in acetyltransferase activity and DNA damage-induced
D
apoptosis, Neoplasia 11 (2009) 345-354.
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[19] J. Yu, Z. Wang, K. W. Kinzler, B. Vogelstein, L. Zhang, PUMA mediates the apoptotic
1936.
AC CE P
response to p53 in colorectal cancer cells, Proc. Natl. Acad. Sci. USA 100 (2003) 1931-
[20] J. E. Chipuk, T. Moldoveanu, F. Llambi, M. J. Parsons, D. R. Green, The BCL-2 family reunion, Mol. Cell 37 (2010) 299-310. [21] J. R. Jeffers, E. Parganas, Y. Lee, C. Yang, J. Wang, J. Brennan, K. H. MacLean, J. Han, T. Chittenden, J. N. Ihle, P. J. McKinnon, J. L. Cleveland, G. P. Zambetti, Puma is an essential mediator of p53-dependent and -independent apoptotic pathways, Cancer Cell 4 (2003) 321-328. [22] K. S. Yee, K. H. Vousden, Contribution of membrane localization to the apoptotic activity of PUMA, Apoptosis 13 (2008) 87-95. [23] L. Ming, P. Wang, A. Bank, J. Yu, L. Zhang, PUMA dissociated Bax and Bcl-XL to induce apoptosis in colon cancer cells, J. Biol. Chem. 28 (2006) 16034-16042. [24] J. E. Chipuk, L. Bouchier-Hayes, T. Kuwana, D. D. Newmayer, D. R. Green, PUMA couples the nucler and cytoplasmic proapoptotic function of p53, Science 309 (2005) 1732-1735. 18
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[25] J. Yu, L. Zhang, P. M. Hwang, K. W. Kinzler, B. Vogelstein, PUMA induces the rapid
PT
apoptosis of colorectal cancer cells, Mol. Cell 7 (2001) 673-682. [26] P. Wang, J. Yu, L. Zhang, The nuclear function of p53 is required for PUMA-mediated
RI
apoptosis induced by DNA damage, Proc. Natl. Acad. Sci. USA 104 (2007) 4054-4059. [27] N. G. Iyer, S. F. Chin, H. Ozdag, Y. Daigo, D. E. Hu, M. Cariati, K. Brindle, S.
SC
Aparicio, C. Caldas, p300 regulates p53-dependent apoptosis after DNA damage in colorectal cancer cells by modulation of PUMA and p21 levels, Proc. Natl. Acad. Sci.
NU
USA 101 (2004) 7386-7391.
[28] E. Rodríguez-Aznar, M. A. Nieto, Repression of Puma by scratch2 is required for
MA
neuronal survival during embryonic development, Cell Death Differ. 18 (2011) 11961207.
[29] J. I. Belle, J. C. Petrov, D. Langlais, F. Robert, R. Cencic, S. Shen, J. Pelletier, P. Gros,
D
A. Nijnik, Repression of p53-target gene Bbc3/PUMA by MYSM1 is essential for the
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survival of hematopoietic multipotent progenitors and contributes to stem cell maintenance, Cell Death Differ. 23 (2016) 759-775.
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[30] S. Amente, J. Zhang, M. L. Lavadera, L. Lania, E. V. Avvedimento, B. Majello, Myc and PI3K/AKT signaling cooperatively repress FOXO3a-dependent PUMA and GADD45a gene expression, Nucleic Acids Res. 39 (2011) 9498-9507. [31] J. Yu, L. Zhang, PUMA, a potent killer with or without p53, Oncogene 27 (2009) Suppl 1:S71-83.
[32] B. N. Jeon, M. K. Kim, J. H. Yoon, M. Y. Kim, H. An, H. J. Noh, W. I. Choi, D. I. Koh, M. W. Hur, Two ZNF509 (ZBTB49) isoforms induce cell-cycle arrest by activating transcription of p21/CDKN1A and RB upon exposure to genotoxic stress, Nucleic Acids Res. 42 (2014) 11447-11461. [33] A. Shevchenko, H. Tomas, J. Havlis, J. V. Olsen, M. Mann, In-gel digestion for mass spectrometric characterization of proteins and proteomes, Nat. Protoc. 1 (2006) 28562860. [34] J. V. Olsen, J. C. Schwartz, J. Griep-Raming, M. L. Nielsen, E. Damoc, E. Denisov, O. Lange, P. Remes, D. Taylor, M. Splendore, E. R. Wouters, M. Senko, A. Makarov, M. 19
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Mann, S. Horning, A dual pressure linear ion trap Orbitrap instrument with very high
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sequencing speed, Mol. Cell. Proteomics 8 (2009) 2759-2769. [35] W. S. El-Deiry, Regulation of p53 downstream genes, Sem. Cancer Biol. 8 (1998) 345-
RI
357.
[36] T. Riley, E. Sontag, P. Chen, A. Levine, Transcriptional control of human p53-
SC
regulated genes, Nat. Rev. Mol. Cell Biol. 9 (2008) 402-412.
Funct. Genomic Proteomic 6 (2007) 8-18.
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[37] J. A. Costoya, Functional analysis of the role of POK transcriptional repressors, Brief.
[38] K. F. Kelly, J. M. Daniel, POZ for effect-POZ-ZF transcription factors in cancer and
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development, Trends Cell Biol. 16 (2006) 578-587.
[39] W. P. Roos, A. D. Thomas, B. Kaina, DNA damage and the balance between survival and death in cancer biology, Nat. Rev. Cancer 16 (2016) 20-33.
D
[40] K. T. Beiging, S. S. Mello, L. D. Attardi, Unravelling mechanisms of p53-mediated
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tumour suppression, Nat. Rev. Cancer 14 (2014) 359-370. [41] D. C. Huang, L. A. O'Reilly, A. Strasser, S. Cory, The anti-apoptosis function of Bcl-2
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can be genetically separated from its inhibitory effect on cell cycle entry, EMBO J. 16 (1997) 4628-4638.
[42] T. Yoshii, T. Fukumori, Y. Honjo, H. Inohara, H. R. Kim, A. Raz, Galectin-3 phosphorylation is required for its anti-apoptotic function and cell cycle arrest, J. Biol. Chem. 277 (2002) 6852-6857. [43] M. T. Piccolo, S. Crispi, The dual role played by p21 may influence the apoptotic or anti-apoptotic fate in cancer, Journal of Cancer Research Updates 1 (2012) 189-202. [44] Z. Andrysik, J. Kim, A. C. Tan, J. M. Espinosa, A genetic screen identifies TCF3/E2A and TRIAP1 as pathway-specific regulators of the cellular response to p53 activation, Cell Rep. 3 (2013) 1346-1354.
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Figure legends
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Fig. 1. ZNF509S1 decreases etoposide-induced apoptosis in HCT116 p53+/+ cells. (A) Structures of ZNF509L and ZNF509S1. (B) MTT assays of cells grown for 0-4 days.
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HCT116 p53+/+ and HCT116 p53-/- cells were transfected with ZNF509S1 expression
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vector or siZNF509 RNA. Control cells were cotransfected with pcDNA3.0 vector or negative siRNA. After 1 day of culture, the cells were treated with high-dose etoposide (50
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M) and analyzed for cell growth. Data shown are the averages of three independent assays. Error bars are too small to seen in the figure. (C) Microscopic images of the MTT-assayed
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cells (B) at 4 days. (D) Flow cytometry of apoptosis. Cells treated as in (B) were stained with FITC-annexin V, and analyzed by flow cytometry. The assay was repeated three times,
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and a representative result is shown.
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Fig. 2. Transcriptional activation of PUMA or pGL13-Luc, by p53 or etoposide treatment, is repressed by ZNF509S1. (A) ZNF509S1 represses transcriptional activation of pG13-Luc
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by p53. p53 and/or ZNF509S1 expression vectors, and a reporter plasmid, were transiently cotransfected into HCT116 p53-/- cells and luciferase activity was measured. Data shown are the averages of three independent assays. Error bars represent standard deviations. (B, C) qRT-PCR (B) and western blot (C) analyses. HCT116 p53+/+ cells transfected with ZNF509S1 expression vector or siZNF509 were treated with etoposide and analyzed for mRNA and protein expression of ZNF509S1, p53, CDKN1A, BAX, and PUMA. GAPDH, control; *, p<0.01; n.s., not significant. Band intensities were analyzed by MultiGauge image analyzer (FUJI film).
Fig. 3. ZNF509S1 inhibits binding of p53 to the PUMA promoter via direct interaction with p53. (A) Co-immunoprecipitation of ZNF509S1 and p53. Cell lysates prepared from HCT116 p53+/+ cells transfected with FLAG-ZNF509S1 expression vector were immunoprecipitated using anti-FLAG antibody and analyzed by western blot using the indicated antibodies. (B) In vitro GST-fusion protein pull-down assays. Recombinant GST 21
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or GST-ZNF509S1 was incubated with [35S]-methionine-labeled p53 polypeptide fragments, pulled down, and resolved by 15% SDS-PAGE. (C) ChIP-qPCR assay of ZNF509S1 and
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p53 binding to the PUMA promoter. HCT116 p53+/+ cells transfected with FLAG-
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ZNF509S1 expression vector were treated with etoposide and immunoprecipitated with an anti-FLAG or p53 antibody. Arrows at p53REs, locations of qChIP-PCR primers. The assay
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was repeated three times. (D) Oligonucleotide pull-down assay of ZNF509S1 and p53 binding to the p53-binding elements (p53RE1 and -2) of the PUMA promoter. HCT116
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p53+/+ cells were transfected with FLAG-ZNF509S1 expression vector, and extracts were incubated with biotinylated double-stranded oligonucleotides, and analyzed as described in
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the methods section. IP, immunoprecipitation; DNA-P, oligonucleotide pull-down assay; GAPDH, control.
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Fig. 4. ZNF509S1 modulates p53K382 acetylation by p300. (A) ZNF509S1 interacts with
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p53 and decreases p53 acetylation. HCT116 p53+/+ cells transfected with FLAGZNF509S1 expression vector were treated with etoposide and the cell lysates
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immunoprecipitated, using an anti-p53 antibody and analyzed by western blot using the indicated antibodies. (B) Mass spectrometry (MS) analysis of a p53 peptide acetylated at K382 by p300. “Kac,” acetylated lysine residue. “Mox,” oxidated methionine. An extracted ion chromatogram (XIC) of the acetylated peptide 382-K(Ac)LM(ox)FKTEGPDSD-393 (m/z: 713.66, charge 2+). K382 acetylation levels were quantified by comparing XICs of the acetylated peptides in three different in vitro reactions (p53 only, p53 + p300, and p53 + p300 + ZNF509S1). An XIC of the precursor peptides was plotted with 10-ppm of mass windows using Xcalibur. X axis, retention time; y axis, relative signal intensity. (C) Western blot analysis. HCT116 p53+/+ cells transfected with ZNF509S1 expression vector or siZNF509 were treated with etoposide, and analyzed by western blot using an acetylated p53K382-specific antibody. IP, immunoprecipitation; GAPDH, control.
Fig. 5. ZNF509S1 represses PUMA gene transcription through inhibition of p53K382 acetylation. (A) Transcriptional regulation of pG13-Luc by p53, ZNF509S1, or the 22
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deacetyl-mimic p53K382R and. HCT116 p53-/- cells were transiently co-transfected with
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wild-type p53 or p53K382R and/or ZNF509S1 expression vectors and luciferase activity was measured. Data shown are the averages of three independent assays. Error bars represent standard deviations. (B, C) qRT-PCR (B) and western blot (C) analyses of
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endogenous PUMA gene and protein expression. HCT116 p53-/- cells were transiently
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cotransfected with wild-type p53 or p53K382R and/or ZNF509S1 expression vectors. Transcriptional activation of endogenous PUMA by p53K382R is weaker than p53WT and
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is not altered by ectopic ZNF509S1. (D) Flow cytometry of apoptosis. Cells transfected as in B and C were stained with FITC-annexin V, and analyzed by flow cytometry. GAPDH,
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control; *, p<0.01; n.s., not significant.
Fig. 6. ZNF509S1 inhibits p53 binding to the PUMA promoter through inhibition of
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p53K382 acetylation. (A) ChIP-qPCR assay of p53, p53K382R, and ZNF509S1 binding to
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the PUMA promoter. HCT116 p53-/- cells were cotransfected with various combinations of p53, p53K382R, and FLAG-ZNF509S1 expression vectors, and immunoprecipitated with
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anti-FLAG or p53 antibody. Arrows at p53REs, locations of qChIP-PCR primers. Data shown are the averages of three independent assays. Error bars represent standard deviations. (B) Comparison of p53 binding consensus sequences, p53RE1 and p53RE2. Arrows, quarter site of p53RE. Two half sites can be separated by 0~13 bp spacer. p53RE1 of PUMA has a 1 bp spacer. R, A or G; W, A or T; Y, C or T. (C) Oligonucleotide pull-down assays of p53, p53K382R, and ZNF509S1 binding to two p53-binding response elements of the PUMA promoter. HCT116 p53-/- cells were transfected as above, and lysed cell extracts were incubated with biotinylated double-stranded oligonucleotides and analyzed as described in methods. IP, immunoprecipitation; DNA-P, oligonucleotide pull-down assay; GAPDH. control.
Fig. 7. Hypothetical model of PUMA transcription repression by ZNF509S1. (A) p53 lysine 382 is acetylated by p300 upon DNA damage. Ac-p53K382 binds to p53RE1 within the PUMA promoter and increases PUMA transcription. However, ZNF509S1 inhibits p53 23
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K382 acetylation by p300. This acetylation inhibition significantly decreases wild type p53
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transcriptional activation of the PUMA promoter, subsequently decreasing apoptotic cell death. (B) Scheme of the regulation of cell cycle arrest and apoptosis by transcriptional
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regulation of p21/CDKN1A, RB and PUMA by ZNF509S1 and ZNF509L., transcription activation; , transcription repression or inhibition at protein level. thin lines, regulatory
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