p53 gain‐of‐function mutations increase Cdc7‐dependent replication initiation
2017; Springer Nature; Volume: 18; Issue: 11 Linguagem: Inglês
10.15252/embr.201643347
ISSN1469-3178
AutoresArindam Datta, Dishari Ghatak, Sumit Das, Taraswi Banerjee, Anindita Paul, Ramesh Butti, Mahadeo Gorain, Sangeeta Ghuwalewala, Anirban Roychowdhury, Sk. Kayum Alam, Pijush K. Das, Raghunath Chatterjee, Maitrayee DasGupta, Chinmay Kumar Panda, Gopal C. Kundu, Susanta Roychoudhury,
Tópico(s)Epigenetics and DNA Methylation
ResumoArticle8 September 2017free access Source DataTransparent process p53 gain-of-function mutations increase Cdc7-dependent replication initiation Arindam Datta Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Search for more papers by this author Dishari Ghatak Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Search for more papers by this author Sumit Das Laboratory of Tumor Biology, Angiogenesis and Nanomedicine Research, National Centre for Cell Science (NCCS), Pune, India Search for more papers by this author Taraswi Banerjee Laboratory of Molecular Gerontology, National Institute on Aging, NIH Biomedical Research Center, NIH, Baltimore, MD, USA Search for more papers by this author Anindita Paul Department of Biochemistry, University of Calcutta, Kolkata, India Search for more papers by this author Ramesh Butti Laboratory of Tumor Biology, Angiogenesis and Nanomedicine Research, National Centre for Cell Science (NCCS), Pune, India Search for more papers by this author Mahadeo Gorain Laboratory of Tumor Biology, Angiogenesis and Nanomedicine Research, National Centre for Cell Science (NCCS), Pune, India Search for more papers by this author Sangeeta Ghuwalewala Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Search for more papers by this author Anirban Roychowdhury Department of Oncogene Regulation, Chittaranjan National Cancer Institute, Kolkata, India Search for more papers by this author Sk Kayum Alam Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Search for more papers by this author Pijush Das Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Search for more papers by this author Raghunath Chatterjee Human Genetics Unit, Indian Statistical Institute, Kolkata, India Search for more papers by this author Maitrayee Dasgupta Department of Biochemistry, University of Calcutta, Kolkata, India Search for more papers by this author Chinmay Kumar Panda Department of Oncogene Regulation, Chittaranjan National Cancer Institute, Kolkata, India Search for more papers by this author Gopal C Kundu Laboratory of Tumor Biology, Angiogenesis and Nanomedicine Research, National Centre for Cell Science (NCCS), Pune, India Search for more papers by this author Susanta Roychoudhury Corresponding Author [email protected] orcid.org/0000-0002-4828-7755 Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Saroj Gupta Cancer Centre and Research Institute, Kolkata, India Search for more papers by this author Arindam Datta Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Search for more papers by this author Dishari Ghatak Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Search for more papers by this author Sumit Das Laboratory of Tumor Biology, Angiogenesis and Nanomedicine Research, National Centre for Cell Science (NCCS), Pune, India Search for more papers by this author Taraswi Banerjee Laboratory of Molecular Gerontology, National Institute on Aging, NIH Biomedical Research Center, NIH, Baltimore, MD, USA Search for more papers by this author Anindita Paul Department of Biochemistry, University of Calcutta, Kolkata, India Search for more papers by this author Ramesh Butti Laboratory of Tumor Biology, Angiogenesis and Nanomedicine Research, National Centre for Cell Science (NCCS), Pune, India Search for more papers by this author Mahadeo Gorain Laboratory of Tumor Biology, Angiogenesis and Nanomedicine Research, National Centre for Cell Science (NCCS), Pune, India Search for more papers by this author Sangeeta Ghuwalewala Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Search for more papers by this author Anirban Roychowdhury Department of Oncogene Regulation, Chittaranjan National Cancer Institute, Kolkata, India Search for more papers by this author Sk Kayum Alam Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Search for more papers by this author Pijush Das Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Search for more papers by this author Raghunath Chatterjee Human Genetics Unit, Indian Statistical Institute, Kolkata, India Search for more papers by this author Maitrayee Dasgupta Department of Biochemistry, University of Calcutta, Kolkata, India Search for more papers by this author Chinmay Kumar Panda Department of Oncogene Regulation, Chittaranjan National Cancer Institute, Kolkata, India Search for more papers by this author Gopal C Kundu Laboratory of Tumor Biology, Angiogenesis and Nanomedicine Research, National Centre for Cell Science (NCCS), Pune, India Search for more papers by this author Susanta Roychoudhury Corresponding Author [email protected] orcid.org/0000-0002-4828-7755 Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Saroj Gupta Cancer Centre and Research Institute, Kolkata, India Search for more papers by this author Author Information Arindam Datta1,‡, Dishari Ghatak1,‡, Sumit Das2, Taraswi Banerjee3, Anindita Paul4, Ramesh Butti2, Mahadeo Gorain2, Sangeeta Ghuwalewala1, Anirban Roychowdhury5, Sk Kayum Alam1, Pijush Das1, Raghunath Chatterjee6, Maitrayee Dasgupta4, Chinmay Kumar Panda5, Gopal C Kundu2 and Susanta Roychoudhury *,1,7 1Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India 2Laboratory of Tumor Biology, Angiogenesis and Nanomedicine Research, National Centre for Cell Science (NCCS), Pune, India 3Laboratory of Molecular Gerontology, National Institute on Aging, NIH Biomedical Research Center, NIH, Baltimore, MD, USA 4Department of Biochemistry, University of Calcutta, Kolkata, India 5Department of Oncogene Regulation, Chittaranjan National Cancer Institute, Kolkata, India 6Human Genetics Unit, Indian Statistical Institute, Kolkata, India 7Saroj Gupta Cancer Centre and Research Institute, Kolkata, India ‡These authors contributed equally to this work *Corresponding author. Tel: +91 33 24995823; Fax: +91 33 24735197; E-mail: [email protected] EMBO Rep (2017)18:2030-2050https://doi.org/10.15252/embr.201643347 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 Cancer-associated p53 missense mutants confer gain of function (GOF) and promote tumorigenesis by regulating crucial signaling pathways. However, the role of GOF mutant p53 in regulating DNA replication, a commonly altered pathway in cancer, is less explored. Here, we show that enhanced Cdc7-dependent replication initiation enables mutant p53 to confer oncogenic phenotypes. We demonstrate that mutant p53 cooperates with the oncogenic transcription factor Myb in vivo and transactivates Cdc7 in cancer cells. Moreover, mutant p53 cells exhibit enhanced levels of Dbf4, promoting the activity of Cdc7/Dbf4 complex. Chromatin enrichment of replication initiation factors and subsequent increase in origin firing confirm increased Cdc7-dependent replication initiation in mutant p53 cells. Further, knockdown of CDC7 significantly abrogates mutant p53-driven cancer phenotypes in vitro and in vivo. Importantly, high CDC7 expression significantly correlates with p53 mutational status and predicts poor clinical outcome in lung adenocarcinoma patients. Collectively, this study highlights a novel functional interaction between mutant p53 and the DNA replication pathway in cancer cells. We propose that increased Cdc7-dependent replication initiation is a hallmark of p53 gain-of-function mutations. Synopsis Cancer-associated p53 missense mutants confer gain-of-function (GOF) and promote tumorigenesis. This study shows that one hallmark of p53 GOF mutants is Myb-dependent transactivation of Cdc7, which leads to enhanced Cdc7/Dbf4-dependent replication initiation in cancer cells. Gain-of-function mutant p53 cooperates with Myb to activate CDC7 transcription in cancer cells. Increased CDC7 expression and high Dbf4 levels lead to increased replication origin firing in p53 mutant cancer cells. High CDC7 expression correlates with poor prognosis in lung adenocarcinoma patients. Introduction The majority of the human cancers harbors TP53 mutation 1. These are mostly missense mutations that result in full-length p53 proteins with altered function. The six “hot spot” residues (R175, G245, R248, R249, R273, and R282) of p53 DNA binding domain are frequently mutated in cancer 2. Besides losing tumor suppressor function, these hot spot mutants gain novel oncogenic properties, defined as mutant p53 gain of function (GOF), and have been broadly categorized as contact (R248W, R248Q, and R273H) or structural (G245S, R249S, R282H, and R175H) mutants depending on the function of the residues altered 2. Importantly, data from cell-based assays as well as from animal model experiments suggest that mutants from these two classes differ in terms of GOF phenotypes 23. For example, p63/p73 interacts with both structural and contact mutants, albeit less effectively with the latter 24. Selective gain-of-function effect also has been reported in the context of chemoresistance. Whereas mutant p53R175H has been shown to confer substantial resistance to etoposide in cultured cancer cells, mutant p53R273H showed less protective effect 5. It has been suggested that the molecular mechanism underlying GOF varies with different p53 mutants, which can be attributed to the differences in structural alterations caused by different mutations 3. Cancer-associated GOF p53 mutants promote several cancer phenotypes including increased cellular growth, invasion and metastasis, genomic instability, deregulated energy metabolism, and enhanced chemoresistance 2. By acting as an “oncogenic transcription factor”, GOF mutant p53 transactivates a number of signaling genes by cooperating with other cellular transcription factors such as Ets-2, Sp1, NF-Y, VDR, SREBP, and Nrf2 26. Although several signaling pathways involved in mutant p53 gain of functions have been identified, many are still unexplored 2. Recent study by Polotskaia et al 7 suggests that DNA replication pathway might be a crucial target of mutant p53. DNA replication is a tightly coordinated process that allows accurate duplication of the entire genome only once per cell division cycle. Errors in DNA replication lead to genomic instability and neoplastic transformation 8. Genes involved in replication are often overexpressed in cancer tissues and chromatin enrichment of pre-replicative complex (pre-RC) proteins has been reported in transformed cells compared to their normal counterparts 9. Moreover, cancer cells utilize a significantly higher number of replication origins than normal cells 10. Replication starts from thousands of replication origins distributed over the genome by the sequential and coordinated action of several replication factors 8. One such factor is Cdc7, which is frequently overexpressed in various cancer cell lines and primary tumor tissues 11. Originally isolated in budding yeast, Cdc7 is a conserved serine/threonine kinase required for replication initiation in vertebrates 12. At the G1/S transition, Cdc7 binds to its regulatory subunit protein Dbf4 to form active DDK (Dbf4-dependent kinase) complex and triggers early as well as late origin firing throughout the S-phase 13. In early G1-phase, origin recognition complex (ORC) along with Cdt1 and Cdc6 facilitates the loading of Mcm2-7 helicase on chromatin, which constitutes “licensed” pre-RCs. The individual origins are activated and “fired” in S-phase upon phosphorylation of Mcm proteins by Cdc7 and other S-phase CDKs followed by the subsequent recruitment of initiation factors Cdc45 and GINS 13. Besides its conserved role in replication initiation, Cdc7 is also involved in DNA damage response (DDR) and helps maintain genome integrity under replication stress 14. Deregulated Cdc7 activity has been implicated in advanced clinical stage, aneuploidy, survival, and chemoresistance in various cancer types, including oral cancer, ovarian cancer, colorectal cancer, melanoma, and breast cancer 15. Notably, high Cdc7 expression often correlates with p53 mutation 11. In this study, we elucidated the regulation of Cdc7 kinase by GOF mutant p53 and demonstrated Cdc7-dependent increased replication initiation in cancer cells harboring mutant p53. DNA replication was enriched as the most over-represented pathway with significant upregulation of Cdc7 kinase in GOF mutant p53-harboring lung cancer patients. We showed that GOF mutant p53 transactivates CDC7 by cooperating with oncogenic transcription factor Myb in cancer cells. In addition, mutant p53 cells showed increased level of Dbf4 protein, the regulatory subunit of Cdc7 kinase. Importantly, mutant p53-expressing non-small cell lung carcinoma (NSCLC) cells showed increased replication initiation in a Cdc7-dependent manner. We further investigated the contribution of Cdc7 kinase to mutant p53 gain of functions both in vitro and in vivo and explored its significance in predicting clinical outcome of NSCLC patients. Collectively, our results demonstrate Cdc7-dependent altered replication initiation as a novel gain-of-function property of mutant p53. Results Increased CDC7 expression in GOF mutant p53 cells Given the well-defined role of GOF mutant p53 as an oncogenic transcription factor (TF) and the high prevalence of p53 mutation in lung cancer, we explored the possible mutant p53 targetome in TCGA lung adenocarcinoma (LUAD) cohort. Functional annotation of the differentially regulated genes (fold change ≥ 1.5, P-value < 0.01) between patients with wild-type and GOF mutant p53 ranked DNA replication as the most significantly enriched pathway in LUAD patients harboring GOF mutant p53 (Fig EV1A, Datasets EV1 and EV2). We found that genes enriched in this pathway are particularly involved in the initiation step of DNA replication (Dataset EV2). Among these genes, initiation factor Cdc7 kinase, which showed significantly higher expression in patients with GOF mutant p53 (Fig 1A), had been reported to be frequently overexpressed in multiple cancer cell lines and tumor specimens with p53 mutation 11. We anticipated that GOF mutant p53 might positively regulate Cdc7 expression in cancer cells and therefore examined the effect of tumor-derived common p53 mutants on Cdc7 expression in p53-null NSCLC cell line, H1299. Compared to the control vector-infected cells (EV), increased Cdc7 expression at both RNA and protein levels was observed in mutant p53-R175H- and p53-R273H-expressing H1299 stable cells (Fig 1B and C). Similar results were obtained upon ectopic expression of these p53 mutants in H1299 as well as in colorectal cancer cell line HCT116 p53−/− (Figs 1D and E, and EV1B and C). In contrast, a small but significant decrease in CDC7 mRNA level was observed upon ectopic expression of wild-type p53 in H1299 cells (Fig 1D), suggesting that the observed upregulation of CDC7 in these cells is mutant p53 specific. Since along with Cdc7, its regulatory subunit Dbf4 is generally overexpressed in multiple human cancers, we next checked the RNA level of DBF4 in presence of GOF mutant p53 11. However, DBF4 was not enriched among the replication genes differentially regulated between TCGA patients with mutant and wild-type p53 (Dataset EV2). Also, we did not observe any significant change in DBF4 transcript level either in mutant p53-expressing H1299 stable cells (Fig 1F) or upon ectopic expression of mutant or wild-type p53 in H1299 cells (Fig 1D). Interestingly, although mRNA levels were unchanged, we detected increased level of Dbf4 protein upon stable or transient expression of mutant p53 in these cells (Fig 1G and H). To ensure that the results obtained were not due to non-physiological level of p53, we compared the levels of ectopically expressed wild-type and mutant p53 in HCT116 p53−/− cells with that seen in tumor cell lines harboring endogenous p53. The levels of exogenously expressed wild-type and mutant p53 proteins were not found to be higher than those observed in cells expressing endogenous p53 (Fig EV1D). We also observed reduced expression of Cdc7 at both mRNA and protein levels upon stable knockdown of endogenous mutant p53 in breast cancer cell line SkBr3 (GOF mutant p53R175H) and colorectal cancer cell line SW480 (GOF mutant p53R273H; Fig 1I and J). siRNA-mediated transient knockdown of mutant p53 also led to reduced Cdc7 protein levels in these cell lines (Fig EV1E). Although mRNA levels were unchanged, substantial decrease in Dbf4 protein level was observed upon stable or transient knockdown of mutant p53 in these cell lines (Fig EV1F, J and G). Since Dbf4 level and Cdc7 activity peak at S-phase and maintained till the end of mitosis, it was important to see whether the results obtained were simply due to the higher fraction of S and G2/M population of mutant p53-expressing cells. Cell cycle profiles revealed that the percentage of S and G2/M population of mutant p53-expressing H1299 cells was not higher than that of control cells (Fig EV1H). Similarly, SkBr3 as well as SW480 cells showed similar cell cycle profile upon mutant p53 knockdown (Fig EV1I and J, respectively). These results suggest that the observed increase in Dbf4 and Cdc7 levels in mutant p53-expressing cells is not merely a reflection of changes in the cell cycle pattern, rather is mutant p53 dependent. Taken together, our observations suggest that mutant p53 upregulates CDC7 expression and there is a simultaneous increase in the level of Dbf4 protein. Click here to expand this figure. Figure EV1. GOF mutant p53 targets DNA replication pathway and induces CDC7 expression in cancer cells A. IPA-generated bar plots showing canonical pathways enriched in TCGA lung adenocarcinoma patients harboring GOF mutant p53. Blue bars represent the significance of the enriched pathways. Bars above the threshold line represent pathways significantly enriched (P-value < 0.05) in patients with GOF mutant p53. For each canonical pathway, P-value calculated by Fisher's exact test is shown as −log10(P-value) along the y-axis. The horizontal red line represents the threshold with P-value 0.05. The yellow points connected by the yellow line indicate the ratio of the number of genes enriched in individual pathways and the total number of genes presented in the respective reference pathways. B. Relative mRNA expression of CDC7 upon ectopic expression of mutant p53-R175H and p53-R273H in HCT116 p53−/− cells (upper panel). Two-tailed Student's t-test: *P < 0.05, ***P < 0.001. Ectopic expression of mutant p53 was evaluated by immunoblotting (lower panel). C. Immunoblots showing Cdc7 protein levels upon ectopic expression of mutant p53-R175H (upper panel) and p53-R273H (lower panel) in increasing doses in HCT116 p53−/− cells. D. Immunoblots showing protein levels of ectopically expressed wild-type and mutant p53 in HCT116 p53−/− cells compared to that of endogenous wild-type and mutant p53 in various cancer cell lines. UT and T indicate untreated and 5-FU treated respectively. E. Immunoblots showing Cdc7 protein levels upon siRNA-mediated knockdown of endogenous mutant p53 in SkBr3 (left panel) and SW480 (right panel) cells. F. Relative mRNA expression of DBF4 upon stable knockdown of endogenous mutant p53 in SW480 and SkBr3 cells. G. Immunoblots showing Dbf4 protein levels upon siRNA-mediated knockdown of endogenous mutant p53 in SkBr3 (upper panel) and SW480 (lower panel) cells. H. Bar graph showing cell cycle profiles of control and mutant p53 expressing H1299 stable cell lines. I, J. Cell cycle profiles of SkBr3 (I) and SW480 (J) cells upon stable knockdown of endogenous mutant p53. Data information: Data represent mean ± SD of three independent experiments. β-actin served as the loading control. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Increased Cdc7 expression in cancer cells harboring GOF mutant p53 Box–whisker plot showing relative CDC7 expression (normalized read counts) in TCGA lung adenocarcinoma patients with wild-type and GOF mutant p53. The lower, middle and upper horizontal lines of the boxes indicate 25th, 50th (median) and 75th percentiles of the dataset respectively. The upper whisker represents maximum observed value below 1.5 IQR (interquartile range) and the lower whisker represents minimum observed value above 1.5 IQR. Mann–Whitney test was used to compute statistical significance. P-value is indicated. Relative mRNA expression of CDC7 in H1299 cells either harboring empty vector (EV) or stably expressing GOF mutant p53-R175H and p53-R273H (upper panel). Levels of mutant p53 protein in stable cell lines were verified by immunoblotting (lower panel). Immunoblots showing Cdc7 protein level in control and mutant p53-expressing H1299 stable cells. Relative mRNA expression of CDC7 and DBF4 upon ectopic expression of wild-type p53, mutant p53-R175H, and p53-R273H in H1299 cells (upper and middle panels, respectively). Ectopic expression of wild-type and mutant p53 was verified by immunoblotting (lower panel). Immunoblots showing Cdc7 protein levels upon ectopic expression of mutant p53-R175H (left panel) and p53-R273H (right panel) in H1299 cells. Relative mRNA expression of DBF4 in control (EV) and mutant p53-expressing H1299 stable cells. Immunoblots showing Dbf4 protein level in control and mutant p53-expressing H1299 stable cells. Immunoblots showing Dbf4 protein levels upon ectopic expression of mutant p53-R175H and p53-R273H in H1299 cells. Relative mRNA expression of CDC7 in SkBr3 (left panel) and SW480 (right panel) cells upon shRNA-mediated knockdown of endogenous mutant p53. Immunoblots showing Cdc7 and Dbf4 protein levels upon stable knockdown of mutant p53 in SkBr3 (left panel) and SW480 (right panel) cells. Data information: β-actin served as the loading control in immunoblots. Bar graphs represent mean ± SD; n ≥ 3. (B, D, F, I) Two-tailed Student's t-test: *P < 0.05; ***P < 0.001; n.s., non-significant. Source data are available online for this figure. Source Data for Figure 1 [embr201643347-sup-0008-SDataFig1.pdf] Download figure Download PowerPoint GOF mutant p53 interacts with Myb to transactivate CDC7 To investigate the underlying mechanism of regulation of Cdc7 kinase by GOF mutant p53, we measured CDC7 promoter activity in cells expressing mutant p53. Compared to control cells, increased CDC7 promoter activity was observed in mutant p53-R175H- and p53-R273H-expressing H1299 stable cell lines (Fig 2A). However, no significant change in DBF4 promoter activity was observed in presence of mutant p53 (Fig 2B). Ectopic expression of p53 mutants also elicited a dose-dependent increase in CDC7 promoter activity in H1299 cells (Fig 2C). In contrast, we observed a dose-dependent decrease in the promoter activity upon ectopic expression of wild-type p53, thereby suggesting a repressive effect of wild-type p53 on CDC7 promoter (Fig 2C). Furthermore, stable knockdown of endogenous mutant p53 in SkBr3 and SW480 cells resulted in a significant decrease in CDC7 promoter activity (Fig 2D). These results suggest that GOF p53 mutants can transactivate CDC7 promoter in cancer cells. Mutant p53 proteins are known to interact with other cellular transcription factors and thereby enhance the expression of their respective target genes 2. We therefore investigated for the possible transcription factor necessary for transactivation of CDC7 by mutant p53. Oncogenic transcription factor Myb has been reported to be a direct transcriptional activator of CDC7 1516. Indeed, when we analyzed CDC7 promoter region, we found two consensus binding sites of Myb (Figs 2E and EV2A). This prompted us to further investigate whether mutant p53 cooperates with Myb to drive CDC7 expression in cancer cells. Results from ChIP-qPCR assay in H1299/R175H and H1299/R273H cells with or without mutant p53 knockdown showed selective enrichment of CDC7 promoter sequences spanning Myb binding sites (−596 to −367 bp) in p53 immunoprecipitates (Fig 2F). On the contrary, no significant recruitment of mutant p53 was observed on distal CDC7 promoter (−2,620 to −2,414 bp), 3′UTR of CDC7 and LMNB1 (Lamin B1) promoter, thereby suggesting the specificity of the recruitment. However, we could not detect any significant recruitment of wild-type p53 on CDC7 promoter (Fig EV2B). Specific recruitment of endogenous mutant p53 on CDC7 promoter was also observed in SkBr3 and SW480 cells (Fig EV2C and D, respectively). Importantly, knockdown of Myb significantly abrogated mutant p53 recruitment on CDC7 promoter in H1299 cells, thereby suggesting the recruitment is Myb dependent (Appendix Fig S1A and Fig 2G). In support of this hypothesis, co-immunoprecipitation assays in mutant p53-expressing H1299 cells as well as in SkBr3 and SW480 cells confirmed that mutant p53 physically interacts with Myb in vivo (Fig 3A and B). We also detected wild-type p53 in Myb immunoprecipitates from HCT116 cells treated with 5-fluorouracil (5-FU; Fig 3C), which is consistent with the previous reports describing in vivo interaction between wild-type p53 and Myb 1718. We further attempted to identify the domain of mutant p53 required for the interaction with Myb. Results from co-immunoprecipitation experiments in H1299 cells co-transfected with Myb and either full-length mutant p53-R175H or its different mutant derivatives (Fig EV2E) showed reduced interaction upon deletion of a region between amino acid residues 355 and 338 of the tetramerization domain of mutant p53 (Fig 3D). The observation suggests that the tetramerization domain, which has been previously shown to mediate interaction with other factors including YAP and ETS2, might be important to form complex with Myb 1920. Next, we investigated the possible role of Myb in mediating mutant p53-driven transactivation of CDC7. Knockdown of Myb led to a significant decrease in enhanced CDC7 promoter activity in mutant p53-expressing H1299 cells (Fig 3E). Further, unlike wild-type promoter (pCDC7-LucWT), activity of the CDC7 promoter deleted of region flanking Myb binding sites (pCDC7-LucΔMyb) was not significantly increased in H1299 cells stably expressing p53-R175H (Appendix Fig S1B and C). Similar observation was obtained upon ectopic expression of this p53 mutant in these cells (Appendix Fig S1D). Deletion of Myb binding sequences also led to a significant reduction in basal CDC7 promoter activity in control H1299 cells (Appendix Fig S1C and D). To further strengthen our observations, we introduced point mutations in the individual Myb binding sites on CDC7 promoter (Constructs S1, S2, and S2+S1 in Fig 3F) and performed luciferase assay in control and mutant p53-expressing H1299 cells. Mutations in either distal (S1) or proximal (S2) Myb binding sites led to a significant reduction in basal CDC7 promoter activity in control H1299 cells (Fig 3G), further suggesting Myb as a positive regulator of CDC7 expression. Compared to the wild-type CDC7 promoter (WT), mutant promoter with either distal (S1) or proximal (S2) site mutation showed attenuated transactivation by mutant p53 (Fig 3G). Although not completely abolished, the increase in CDC7 promoter activity by mutant p53 was further reduced upon mutation in both distal and proximal Myb binding sites (S2+S1; Fig 3G). Similar observation was obtained upon ectopic expression of mutant p53-R175H in H1299 cells transfected with either wild-type (WT) or Myb site-mutated (S1, S2, and S2+S1) promoter constructs (Fig EV2F). Accordingly, the increase in CDC7 mRNA level was found to be significantly compromised upon Myb knockdown in mutant p53-expressing H1299 cells (Fig EV2G). As expected, the basal-level expression of CDC7 was also reduced in control H1299/EV cells upon Myb knockdown (Appendix Fig S1E). These data suggest an important contribution of Myb to mutant p53-driven transactivation of CDC7 promoter and also indicate that there could be some other modes of regulation exist. Figure 2. GOF mutant p53 transactivates CDC7 and is recruited to its promoter via Myb A, B. Relative CDC7 (A) and DBF4 (B) promoter activity in control and mutant p53-expressing H1299 stable cells. C. Relative CDC7 promoter activity in control vector-, wild-type p53-, GOF mutant p53-R175H-, and p53-R273H-transfected H1299 cells (upper panel). Ectopic expression of p53 was verified by immunoblotting (lower panel). D. CDC7 promoter activity upon stable knockdown of endogenous mutant p53 in SkBr3 and SW480 cells. E. Schematic representation of CDC7 promoter harboring Myb binding sites. The Myb binding sites (−387 and −588 bp) wrt transcription start site (+1) are shown. Arrows represent pair of primers used to amplify CDC7 promoter region spanning Myb consensus sequences in ChIP-qPCR assays. F. ChIP-qPCR showing mutant p53 recruitment on CDC7 promoter (−596 to −367 bp) in H1299-R175H (upper panel) and H1299-R273H (lower panel) stable cells transfected wi
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