New insight into the significance of KLF4 PARylation in genome stability, carcinogenesis, and therapy
2020; Springer Nature; Volume: 12; Issue: 12 Linguagem: Inglês
10.15252/emmm.202012391
ISSN1757-4684
AutoresZhuan Zhou, Furong Huang, Indira H. Shrivastava, Rui Zhu, Aiping Luo, Michael O. Hottiger, İvet Bahar, Zhihua Liu, Massimo Cristofanilli, Yong Wan,
Tópico(s)Chronic Myeloid Leukemia Treatments
ResumoArticle24 November 2020Open Access Source DataTransparent process New insight into the significance of KLF4 PARylation in genome stability, carcinogenesis, and therapy Zhuan Zhou orcid.org/0000-0002-8693-3084 Department of Obstetrics and Gynecology, Department of Pharmacology, The Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL, USAThese authors contributed equally to this work Search for more papers by this author Furong Huang State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Indira Shrivastava Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USAThese authors contributed equally to this work Search for more papers by this author Rui Zhu State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Search for more papers by this author Aiping Luo State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Search for more papers by this author Michael Hottiger orcid.org/0000-0002-7323-2270 Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland Search for more papers by this author Ivet Bahar Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Search for more papers by this author Zhihua Liu State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Search for more papers by this author Massimo Cristofanilli Lynn Sage Breast Cancer Program, Department of Medicine-Hematology and Oncology, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Search for more papers by this author Yong Wan Corresponding Author [email protected] Department of Obstetrics and Gynecology, Department of Pharmacology, The Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Search for more papers by this author Zhuan Zhou orcid.org/0000-0002-8693-3084 Department of Obstetrics and Gynecology, Department of Pharmacology, The Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL, USAThese authors contributed equally to this work Search for more papers by this author Furong Huang State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaThese authors contributed equally to this work Search for more papers by this author Indira Shrivastava Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USAThese authors contributed equally to this work Search for more papers by this author Rui Zhu State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Search for more papers by this author Aiping Luo State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Search for more papers by this author Michael Hottiger orcid.org/0000-0002-7323-2270 Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland Search for more papers by this author Ivet Bahar Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Search for more papers by this author Zhihua Liu State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Search for more papers by this author Massimo Cristofanilli Lynn Sage Breast Cancer Program, Department of Medicine-Hematology and Oncology, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Search for more papers by this author Yong Wan Corresponding Author [email protected] Department of Obstetrics and Gynecology, Department of Pharmacology, The Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL, USA Search for more papers by this author Author Information Zhuan Zhou1, Furong Huang2, Indira Shrivastava3, Rui Zhu2, Aiping Luo2, Michael Hottiger4, Ivet Bahar3, Zhihua Liu2, Massimo Cristofanilli5 and Yong Wan *,1 1Department of Obstetrics and Gynecology, Department of Pharmacology, The Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 2State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China 3Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 4Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland 5Lynn Sage Breast Cancer Program, Department of Medicine-Hematology and Oncology, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL, USA *Corresponding author. Tel: +312-503-2769; Fax: +312-503-0095; E-mail: [email protected] EMBO Mol Med (2020)12:e12391https://doi.org/10.15252/emmm.202012391 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract KLF4 plays a critical role in determining cell fate responding to various stresses or oncogenic signaling. Here, we demonstrated that KLF4 is tightly regulated by poly(ADP-ribosyl)ation (PARylation). We revealed the subcellular compartmentation for KLF4 is orchestrated by PARP1-mediated PARylation. We identified that PARylation of KLF4 is critical to govern KLF4 transcriptional activity through recruiting KLF4 from soluble nucleus to the chromatin. We mapped molecular motifs on KLF4 and PARP1 that facilitate their interaction and unveiled the pivotal role of the PBZ domain YYR motif (Y430, Y451 and R452) on KLF4 in enabling PARP1-mediated PARylation of KLF4. Disruption of KLF4 PARylation results in failure in DNA damage response. Depletion of KLF4 by RNA interference or interference with PARP1 function by KLF4YYR/AAA (a PARylation-deficient mutant) significantly sensitizes breast cancer cells to PARP inhibitors. We further demonstrated the role of KLF4 in modulating homologous recombination through regulating BRCA1 transcription. Our work points to the synergism between KLF4 and PARP1 in tumorigenesis and cancer therapy, which provides a potential new therapeutic strategy for killing BRCA1-proficient triple-negative breast cancer cells. SYNOPSIS This study reveals a novel role for KLF4 PARylation in DNA damage response in cancer, and reports a synergy between targeting KLF4 and PARP1 in the treatment of triple negative breast cancer (TNBC). PARP1 is a binding partner of KLF4 protein in response to DNA damage. Structure-based modeling elucidates the mechanism of interaction between KLF4 and PARP1, and unveils that the YYR motif of KLF4 enables its PARylation. PARP1-mediated PARylation of KLF4 promotes its recruitment to the chromatin, which facilitates KLF4-mediated transcriptional function. KLF4 PARylation is required for KLF4-mediated DNA damage response, while KLF4-involved regulation of BRCA1-homologous recombination (HR) is independent of PARylation. Combination of KLF4 inactivation and PARP1 blockade leads to a synergistic killing of BRCA1-proficient TNBC tumor. The paper explained Problem Results from recent "The Cancer Genome Atlas" and pathophysiological studies have unveiled the critical role of KLF4 in genomic integrity, breast carcinogenesis, and drug resistance. In response to oncogenic signaling as well as genotoxic stress, KLF4 is posttranslationally modified, such as in ubiquitylation and methylation. However, the posttranslational modifications orchestrate KLF4 in DNA damage response and DNA repair and its underlying mechanism remains unclear. Results We discovered that the function of KLF4 is tightly regulated by poly (ADP-ribosyl)ation (PARylation) for cellular compartmentation. The PBZ domain YYR motif (Y430, Y451 and R452) on KLF4 that enables PARP1-mediated PARylation, which recruits KLF4 from the nucleus to the chromatin and facilitates KLF4-mediated transcriptional function on p21 and Bax, promotes DNA damage response. KLF4 also modulates homologous recombination through regulating BRCA1 transcription, which is independent of PARylation. Combination of KLF4 inactivation and PARP1 inhibition leads to synthetic lethality that provides a novel strategy to kill BRCA1-proficient triple-negative breast cancer tumors. Impact Our study reveals the PARylation-dependent KLF4 function in DNA damage response and PARylation-independent function in homologous recombination. Finding a functional interaction between KLF4 and PARP1 in DNA damage response and KLF4-BRCA1 in homologous recombination unveils a novel approach to induce synthetic lethality that further leverages PARP inhibitors specifically to benefit BRCA-proficient TNBC patients. Introduction Krüppel-like factor 4 (KLF4, GKLF) plays a pivotal role in orchestrating a variety of cellular processes, including cell cycle control, genome stability, signal transduction, stem cell expansion, and immune response (Rowland & Peeper, 2006; Tetreault et al, 2013; Ghaleb & Yang, 2017). Results from recent pathophysiological and "The Cancer Genome Atlas (TCGA)" studies have unveiled an oncogenic property for KLF4 in breast carcinogenesis (Fletcher et al, 2011; Li et al, 2013; Hu et al, 2015), although its underlying mechanism in breast tumor initiation and invasion remains unclear. KLF4 acts as a transcriptional factor and regulates various biological functions by either activating or inhibiting a network of genes involved in developmental and etiological events (Gamper et al, 2012; Tetreault et al, 2013). To our surprise, recent physiological studies provide an ambivalent view of KLF4 with regard to oncogenesis as either a tissue-specific tumor suppressor or an oncogenic factor with an unknown mechanism (Rowland & Peeper, 2006; Tetreault et al, 2013). Depending on tumor type, KLF4 is thought to be a tumor suppressor in gastrointestinal, esophageal, lung, and pancreatic cancers (Evans & Liu, 2008; Hung et al, 2013; Wei et al, 2016), while it acts as an oncogenic player in breast and squamous cell carcinoma (Foster et al, 1999; Foster et al, 2000; Pandya et al, 2004; Foster et al, 2005; Rowland et al, 2005; Dong et al, 2014). Despite the knowledge about its role in gastrointestinal and pancreatic cancers, the process through which abnormal accumulation of KLF4 that promotes malignant transformation in mammary glands and skin remains unclear (Evans & Liu, 2008; Tetreault et al, 2013). Our recent work has demonstrated that in response to oncogenic signaling as well as genotoxic stress, KLF4 undergoes posttranslationally modified such as in ubiquitination and methylation (Evans et al, 2007; Meng et al, 2009; Hu & Wan, 2011; Hu et al, 2015; Tian et al, 2015; Zhou et al, 2017a). We have discovered that the interplay between ubiquitin ligase VHL/VBC and arginine methyltransferase PRMT5 governs KLF4 protein stability (Hu et al, 2015). To further explore whether posttranslational modifications orchestrate KLF4 protein trafficking, in particular within the cytosol, from the cytosol to the nucleus, and from the nucleus to the chromatin, we have searched for new posttranslational modifiers that are involved in the above critical cellular processes. Here, we report the novel finding that (ADP-ribosyl)ation (PARylation) of KLF4 by Poly [ADP-ribose] polymerase 1 (PARP1) regulates KLF4 chromatin recruitment following DNA damage response, which provides potential novel strategies for targeted cancer therapy. PARP1 is the enzyme responsible for PARylation, which catalyzes the covalent transfer of mono- or oligomeric ADP-ribose groups from NAD+ to target proteins (Kim et al, 2005). Among the seven members of the PARP family, PARP1 plays a key role in multiple DNA damage response pathways and governs genome stability. Upon DNA damage, PARP1 is rapidly recruited to the altered DNA damage lesion sites, where its catalytic activity increases by a hundred-fold, resulting in the conjugation of long branched PAR chains (Jackson & Bartek, 2009; De Vos et al, 2012; Wang et al, 2012; Yazinski et al, 2017). Recent works have shown a series of critical DNA damage-related and DNA-repair proteins that are modified by PARylation, including histones, topoisomerase, DNA protein kinase (DNA-PK), XRCC1, mitotic recombination 11 (MRE11), and ataxia telangiectasia-mutated (ATM) (Wang et al, 2012; Wei & Yu, 2016). Similar to protein phosphorylation and ubiquitylation, PARylation is a reversible process, wherein the conjugation of the PAR polymer can be countered by two enzymes, including poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose hydrolase ARH3 through hydrolyzing the PAR chain (Leung, 2014). For mouse genetic studies on the impact of PARP1 in both tumor suppression and oncogenesis, different methodologies and physiological context have shown differing results (Yelamos et al, 2011; Schiewer & Knudsen, 2014). While ablation of PARP1 linking to mammary tumor formation (de Murcia et al, 1997; Tong et al, 2007) suggests its tumor suppressing effect, the oncogenic role of PARP1 was implied due to the correlation between its abnormal accumulation and poor prognoses in various types of cancers, including breast, uterine, lung, ovarian, colorectal, and skin cancers (Ossovskaya et al, 2010). Although considerable attention has been paid to elucidate the biochemical mechanism by which functional proteins are modulated by PARylation, how exactly the impaired PARP1 drives oncogenesis remains unclear. Specifically, while the overlapping physiological impact for both PARP1 and KLF4 is observed in many aspects, such as developmental control, stem cell self-renewal, DNA damage response/DNA repair, and tumorigenesis, the functional mechanism between PARP1 and KLF4 and its physiological consequences have not yet been adequately explored. The present identification of DNA damage-induced PARylation of KLF4 fills a critical gap in the knowledge of the impact of PARP1 and KLF4 in oncogenesis and on therapeutics. Development of PARP inhibitors, including olaparib, rucaparib, niraparib, and talazoparib, has provided a method to treat triple-negative breast cancer (TNBC) patients with BRCAness, who mimic BRCA1 or BRCA2 loss and are deficient with regard to homologous recombination (HR), based on their synthetic lethal effect (McCann & Hurvitz, 2018; Papadimitriou et al, 2018). However, the application of the PARP inhibitors is limited to a small fraction of TNBC patients who bear the BRCA1/2 mutation (Papadimitriou et al, 2018; Sulai & Tan, 2018). Unfortunately, approximately 80% of TNBC patients who have normal BRCA function are not responsive to PARP inhibitors due to their normal HR function (Hartman et al, 2012; Greenup et al, 2013; Papadimitriou et al, 2018). Thus, a new strategy that blocks BRCA-mediated HR could potentially expand the therapeutic value of PARP inhibitors to benefit BRCA-proficient TNBC patients (Johnson et al, 2016). Here, we identified PARP1 as a previously undocumented posttranslational modifier of KLF4 in genome stability through regulating KLF4 transcriptional function by dictating its recruitment to the chromatin, ensuring DNA damage response. We further unveiled a newly discovered function of KLF4 in modulating HR through regulating BRCA1 in a PARylation-independent manner. Finding a functional interaction between KLF4 and PARP1 in DNA damage response and DNA repair unveils a novel approach to induce synthetic lethality that further leverages PARP inhibitors specifically to benefit BRCA-proficient TNBC patients. Indeed, inactivation of KLF4 or depletion of KLF4 in preclinical models significantly sensitizes BRCA-proficient TNBC breast cancer tumor to PARP inhibitors. Therefore, our findings demonstrate a potential novel therapeutic strategy to target BRCA-proficient TNBC breast cancer by exploiting the synergism of KLF4 and PARP1. Results Establishment of KLF4loxp/loxp coupled AAV7-Cre inducible mouse to determine the impact of KLF4 in governing genome stability and tumorigenesis Previous results using a cultured-cell model obtained by us and others demonstrated the critical role of KLF4 in governing DNA damage response and DNA repair; otherwise, deregulation could lead to genome instability and tumorigenesis (Yoon et al, 2003; Yoon & Yang, 2004; Yoon et al, 2005; Ghaleb et al, 2007). It has been a technical challenge in the field to determine the relevance of KLF4-mediated genome stability in cancer formation due to KLF4's ambivalent role in tumorigenesis as either a tissue-specific tumor suppressor or an oncogene. In order to dissect the impact of KLF4 in genome stability, carcinogenesis, and therapeutics, we have established KLF4 loxp/loxp mice followed by engineering conditional knockout of KLF4 in intestinal tissue by utilizing an adeno-associated virus-Cre inducible system (Polyak et al, 2008; Zincarelli et al, 2008) (Fig 1A). We then exposed mice with conditional knockout of KLF4 to γ-radiation (Fig 1A) and subsequently detected the relevance of KLF4 ablation to genome stability by measuring morphological damage of the intestine, mouse tolerance to genotoxic stress, and DNA damage status in the intestine (Talmasov et al, 2015). Figure 1. Establishment of KLF4loxp/loxp coupled AAV-Cre inducible mouse to determine the impact of KLF4 on governing genome stability and tumorigenesis A. Schematic diagram of establishment of KLF4loxp/loxp coupled AAV7-Cre inducible mouse. B. Genotyping of KLF4loxp/loxp coupled AAV-Cre inducible mouse. 2 × 1011 particles of AAV7-Cre-mCherry or AAV7-mCherry were intraperitoneally injected into KLF4loxp/loxp mice (6–8 weeks). Five weeks later, mice tails were cut and collected for DNA extraction and PCR analysis. PCR results were analyzed by 1% agarose gel. C, D. Validation of inducible knockout of KLF4 in mouse intestine. Five weeks after injection of AAV7-Cre-mCherry, mouse intestine was removed and followed by the preparation of tissue section. The KLF4 expression in the intestine was then detected by Western blot (C) and immunohistochemistry (D). Scale bars, 100 μm. E. Kaplan–Meier survival curves of KLF4loxp/loxp mice with intraperitoneal injection of AAV7-Cre-mCherry or AAV7-mCherry followed by the treatment with 8-Gy (total-body) γ-irradiation 5 weeks after then. AAV7-mCherry, n = 10 per group; AAV7-Cre-mCherry, n = 12 per group. P = 0.0023, log-rank test. F. Histological analysis of intestinal epithelium of KLF4loxp/loxp mice with AAV7-mCherry or AAV7-Cre-mCherry intraperitoneal injection followed by 8-Gy (total-body) γ-irradiation 5 weeks after then. Tissues were collected from the sham mice and mice at different time after exposure to γ-irradiation. Scale bars, 100 μm. G, H. Immunofluorescent staining of γ-H2AX, 53BP1, and cleaved caspase-3 in the intestinal epithelium of KLF4 loxp/loxp mice with injection of AAV7-mCherry or AAV7-Cre-mCherry followed by treatment of γ-irradiation. Tissues were collected from sham mice and mice at different time after exposure to γ-irradiation and then staining with indicated antibodies. (G) Quantification of γ-H2AX, 53BP1 and active caspase-3-positive cells based on the Immunofluorescent staining results presented in (H). Data are mean ± SEM; n = 4 per group; P = 0.0023 (r-H2AX), P = 7.9 × 10−4 (53BP1) P = 5.5 × 10−3 (active caspase-3). One-way ANOVA was used for the statistical analysis. Scale bars, 60 μm. Download figure Download PowerPoint We have tested various AAV serotypes with green fluorescent protein (GFP) reporter in vivo by intraperitoneal injection and found that AAV7 exhibits the strongest intestinal tissue tropism among other AAV serotypes (AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9) (Appendix Fig S1A and B). The intraperitoneal injection of AAV7 could deliver GFP reporter to the gastroenterology system, including the stomach, duodenum, and jejunum/ileum (Appendix Fig S1A and C). Therefore, we applied the AAV7-Cre-mCherry to deliver Cre expressed in the gastroenterology system to establish adult inducible KLF4 knockout in KLF4loxp/loxp mice. As shown in Fig 1B–D, intraperitoneal injection of AAV7-Cre-mCherry into KLF4loxp/loxp mice induces significant local KLF4 knockout in intestinal tissue. We have observed that, after mice were subjected to 8 Gy total-body γ-radiation, ablation of KLF4 causes shorter survival times as compared to KLF4 loxp/loxp mice (Fig 1E). Results from histological analysis of intestinal epithelium of KLF4 loxp/loxp mice with either AAV7-mCherry or AAV7-Cre-mCherry delivery showed profound intestinal tissue damage at 96 h postirradiation after KLF4 deletion (Fig 1F), while no significant distinction is observed pre-irradiation or 6 h postirradiation. Furthermore, at 96 h postirradiation, the intestinal epithelium in KLF4 loxp/loxp/ AAV7-Cre-mCherry mice developed deep-set crypts and damaged intestinal mucosal structures with focal villus edema, indicating severe damage of the intestinal epithelium (Fig 1F) (Potten et al, 1990; Talmasov et al, 2015). Moreover, immunofluorescent staining of the intestinal epithelium with critical DNA damage/DNA repair and apoptosis markers, including p53, p-CHK2/Thr68, γ-H2AX, 53BP1, and cleaved caspase-3, indicated increased DNA damage (γ-H2AX, 53BP1) and apoptosis (cleaved caspase-3) in the duodenum of KLF4 loxp/loxp mice with AAV7-Cre-mCherry in comparison with AAV7-mCherry injection (Fig 1G and H, and Appendix Fig S1D and E). Taken together, our results of intestine-specific inducible ablation of KLF4 showed the important role of KLF4 in genome stability through modulating DNA damage response and DNA repair. KLF4 orchestrates DNA damage response and DNA repair To further study the role of KLF4 in DNA damage response and DNA repair, we have measured the effect of KLF4 knockout on chromosomal instability, DNA-double strain break, aneuploidy, alteration of mitotic index, and HR and non-homologous end joining using KLF4 knockout mouse embryonic fibroblasts (MEFs) as well as U2OS cells (Hagos et al, 2009; El-Karim et al, 2013). As shown in Appendix Fig S2A–E, genetic ablation of KLF4 leads to significant chromosomal breaking, as measured by metaphase karyotype analysis (Elenbaas et al, 2001). KLF4 deletion causes obvious accumulation of 53BP1 and γ-H2AX foci compared with wild-type MEF cells, which indicated a failure in DNA damage repair/response (Appendix Fig S2F–H; Harper & Elledge, 2007). Moreover, we observed that loss of KLF4 in MEFs resulted in increased aneuploidy cells (> 4n) and pH3-positive cells (Appendix Fig S2I and J; Hu et al, 2015). To assess the role of KLF4 in HR and non-homologous end joining, we engineered stable KLF4 knockdown based on U2OS cells, followed by performing HR and non-homologous end joining (NHEJ) assays in U2OS-GFP-EJ5 and U2OS-DR-GFP cells (Wei et al, 2015). As shown in Appendix Fig S2K and L, deletion of KLF4 led to significant defects in DNA repair of HR, while no significant difference was observed in NHEJ between KLF4 knockdown and wild-type cells. Taken together, our data suggest that KLF4 is a critical player, and its dysfunction affects both DNA damage response and DNA repair. Identification of KLF4 PARylation by PARP1 in KLF4-mediated DNA damage response The above genetic and physiological analyses suggest a pivotal role for KLF4 in governing genome stability by regulating cellular response to DNA damage and damage lesion repair. While the fact that KLF4 governs transactivation of its downstream targeting genes has been partially explained, not much is known about how KLF4 is regulated in response to genotoxic stress. We recently reported that KLF4 is a fast turnover protein with its protein stability governed by an interplay between VHL-mediated ubiquitylation and PRMT5-mediated methylation (Hu et al, 2015). To search for other proteins that might regulate KLF4 function in genome stability and carcinogenesis, we took a biochemical approach to identify new KLF4-interacting proteins, especially for interactions enhanced in response to DNA damage signal, using cells expressing tagged KLF4 to isolate KLF4 protein complexes by tandem immune-purification (Gamper et al, 2012; Hu et al, 2015). Our efforts led to the identification of interaction between KLF4 and PARP1 using mass spectrometric analyses (Fig 2A–C and Appendix Fig S3A and Table S1). Our purification indicates, while basal interaction between KLF4 and PARP1 is detected, the capacity of PARP1 to bind to KLF4 increases several-fold after cellular exposure to γ-radiation, suggesting the potentially critical role of PARP1 in regulating KLF4 in genome stability. Figure 2. Identification of KLF4 PARylation by PARP1 in KLF4-mediated DNA damage response A. Purification of KLF4 protein complex in the presence and absence of DNA damage based on TAP-KLF4 stable expression cells (U2OS). Proteins that interacted with KLF4 were purified from U2OS cells expressing FLAG and HA-tagged KLF4 in the absence and presence of 5 Gy radiation at 4h after the treatment. The accumulated bind induced in response to radiation was isolated for mass spectrometry analysis. PARP1 was identified as a binding partner for KLF4. B. The sequences of mass spectrometry analysis for identification of PARP1 (P09874) to be an interacting partner of KLF4. The identified peptides were labeled in yellow. C. The purified complex was further confirmed by Western blot detected by FLAG-KLF4, PARP1(lane 1). The interaction between KLF4 and PARP1 was significantly increased in response to γ-radiation detected by pulldown experiment (lanes 3 and 4). D. Validation of interaction between ectopically expressed KLF4 and PARP1. 293T cells were transfected with FLAG-KLF4 and Myc-PARP. Whole cell lysates or IP complex pulled down by anti-FLAG antibody were analyzed by Western blotting. E. Validation of interaction between endogenous KLF4 and PARP1 in cytosolic lysate and nuclear lysate using immunoprecipitation and Western blotting in MDA-MB-231 cells. The Histone 3 (H3) is the control for nuclear portion, and actin is the control for cytosol portion. F. Co-immunoprecipitation of PARP1 with endogenous KLF4 is independent on DNA. The DNA binding inhibitor EtBr was added to the MDA-MB-231 cell lyses followed by immunoprecipitation of KLF4 complex. G, H. Co-localization analysis for endogenous PARP1 and KLF4 (G), or ectopic expressed GFP-PARP1 and DsRed-KLF4 (H) in MDA-MB-231 cells. PARP1 and KLF4 are co-localized in the nucleus, and this co-localization is increased by in response to radiation. Scale bars, 5 μm. I. Validation of the interaction between endogenous KLF4 and PARP1 by in situ proximity ligation assay (PLA). No positive staining in KLF4/Rabbit IgG antibody PLA assay. Scale bar, 100 µm. The right panel shows the blow-up. J. KLF4 was poly(ADP-ribosyl)ated. KLF4 was immunoprecipitated and detected by anti-PAR and anti-KLF4 antibodies in MDA-MB-231 cells. K. The PARylation of KLF4 was increased after exposure to DNA damage. KLF4 complex was purified using immunoprecipitation from MDA-MB-231 cells that were treated with 5 µM doxorubicin and were then collected at different times (1, 2, 4 and 8 h). PARylation was detected by using antibody against PAR. L. Loss of PARP1 attenuates KLF4 PARylation. PARP1+/+ and PARP1−/− MEFs cells were transfected with FLAG-KLF4 followed by 4h after 5 Gy radiation, and then the PARylation of KLF4 was detected by pulldown. M. PARP1 inhibitors decrease KLF4 PARylation. U2OS cells were pretreated with 10 µM various PARP1 inhibitor niraparib, olaparib, and rucaparib for 1hr followed by exposure to 10 Gy radiation for 4 h. KLF4 was pulled down, and the PARylation was detected. Download figure Download PowerPoint The interaction of KLF4 and PARP1 was further confirmed by immunoprecipitation of endogenous KLF4 complex followed by immune-blotting of PARP1 (Fig 2D) or by determining complexes of overexpressed tagged KLF4 precipitated with anti
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