A Positive Feedack Loop Between EBP2 And C-Myc Regulates RDNA Transcription,Cell Proliferation And Tumorigenesis

c-Myc is a key transcriptional factor that has critical functions in various biological processes such as cell growth, metabolism, angiogenesis, and DNA repair. Moreover, c-Myc is a potent oncoprotein that is frequently deregulated in many human cancers and can promote tumorigenesis in a very wide variety of tissues. Recent studies indicate that c-Myc is a universal transcriptional amplifier of expressed genes. c-Myc is mainly localized in the nucleus in cells to regulate the transactivation or repression of various genes by direct DNA binding. Except nuclear distribution, c-Myc is found in the cytosol and the nucleolus (NO) in cells. Myc-Nick, a truncated form of Myc, is localized in the cytosol and regulates cell differentiation. Nucleolar-localized c-Myc is essential for ribosome biogenesis by binding to ribosomal DNA (rDNA), activating RNA polymerase Ⅰ, and regulating the transcription of rDNA. The function of c-Myc in the NO is tightly controlled by protein degradation and its binding proteins. Yet, SCF-Fbw7 is the major reported E3 ubiquitin ligase to degrade c-Myc in the NO via ubiquitin-proteasome system. Recent study indicates that nucleophosmin (NPM) is required for the nucleolar localization of c-Myc and promotes the degradation of c-Myc in the nucleoli in a Fbw7-independent manner. However, the underlying mechanisms that regulate c-Myc localization and functions in the nucleoli remain to be investigated.ENBA1 binding protein 2 (EBP2) is originally identified as a binding protein of Epstein-Barr virus (EBV) nuclear antigen 1 that is important for EBV segregation. Yeast homolog EBP2 (Ebp2p) is an essential nucleolar protein required for pre-ribosomal RNA (rRNA) processing and ribosomal subunit assembly. In mammalian cells, EBP2 interacts with nucleostemin and is localized to the NO. Moreover, human EBP2 is associated with chromosome in mitosis. Overexpression of EBP2 has been shown to be able to promote the cell proliferation and chromosome instability of 293 cells or N1H3T3 cells. A recent study demonstrates that EBP2 is essential for the nucleolar localization of Fbw7y. However, the biological functions of mammal EBP2, such as whether it may affect the stability of Fbw7y substrates, remain not completely understood.In this study, we identified nucleolar protein ENBA1 binding protein 2 (EBP2) as a novel functional binding partner of c-Myc. Immunofluorescence assay indicted that coexpression of EBP2 markedly relocalized c-Myc from the nucleus to the nucleolus. This result was further confimed by sub-cellular fraction. Meanwhile, depletion of EBP2 reduced the nucleolar distribution of c-Myc. As it has been reported that c-Myc nucleolus location can be regulated by its binding partners, we speculate that c-Myc can interact with EBP2. And the following co-immunoprecipitation assay demonstrated that both endogenous and overexpressed c-Myc can bind to EBP2. Meanwhile, in vitro pull down assay also indicate c-Myc and EBP2 can interact directly. We furher found c-Myc and EBP2 interact sites using a serious of deletion mutants— the N-terminal of EBP2 mediate its interaction with c-Myc, while both N-terminal and C-terminal of c-Myc plays an essential role in its interaction with EBP2. And this result was confirmed by immunofluorescence assay. Next, we found that overexpress EBP2 can stable c-Myc proein, whereas delete EBP2 can lead to the reduction of c-Myc half-life. We further proved that the block of the degradation of c-Myc is a Fbw7 (F-box and WD repeat domain containing 7)-independent manner. Moreover, EBP2 is a transcriptional target of c-Myc, and overexpress c-Myc can lead to EBP mRNA increase. The further exploration indicated that c-Myc can bind to the promoter of EBP2 and positively regulate the EBP2 expression. EBP2 is upregulatd in lung cancer cells constract to normal lung epithelium cells, and EBP2 overexpressin can promote lung cancer cell H1299 proliferation and tumorigenesis in xenograft. In addition, both protein and mRNA levels of EBP2 are upregulated in human lung cancer samples and positively correlated with c-Myc expression. Functionally, EBP2 promotes c-Myc-mediated rRNA synthesis and cell proliferation. Collectively, our study indicates that EBP2 is a novel binding partner of c-Myc that regulates the function of nucleolar c-Myc, cell proliferation, and tumorigenesis via a positive feedback loop.