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HBV core promoter mutations promote cellular proliferation through E2F1-mediated upregulation of S-phase kinase-associated protein 2 transcription.

ABSTRACT: Hepatitis B virus (HBV) core promoter (CP) mutations have been associated with an increased risk of hepatocellular carcinoma (HCC) in clinical studies. We previously reported that a combination of CP mutations seen in HCC patients, expressed in HBx gene, increased SKP2 (S-phase kinase-associated protein 2) expression, thereby promoting cellular proliferation. Here, we investigate the possible mechanisms by which CP mutations upregulate SKP2.We used immunoblotting and ATPlite assay to validate the effect of CP mutations in full-length HBV genome on cell cycle regulator levels and cell proliferation. Activation of SKP2 mRNA was assessed by quantitative real-time PCR in primary human hepatocytes (PHH) and HCC cell lines. Effect of CP mutations on SKP2 promoter activity was determined by luciferase assay. Target regulation of E2F1 on SKP2 was analyzed by siRNAs.CP mutations in full-length HBV genome upregulated SKP2 expression, thereby downregulating cell cycle inhibitors and accelerating cellular proliferation. CP mutations enhanced SKP2 promoter activity but had no effect on SKP2 protein stability. Mapping of the SKP2 promoter identified a region necessary for activation by CP mutations that contains an E2F1 response element. Knocking down E2F1 reduced the effects of CP mutations on SKP2 and cellular proliferation. The effect of CP mutations on E2F1 might be mediated through hyperphosphorylation of RB.HBV CP mutations enhance SKP2 transcription by activating the E2F1 transcription factor and in turn downregulate cell cycle inhibitors, thus providing a potential mechanism for an association between CP mutations and HCC.

SUBMITTER: Huang Y

PROVIDER: S-EPMC3898818 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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HBV core promoter mutations promote cellular proliferation through E2F1-mediated upregulation of S-phase kinase-associated protein 2 transcription.

Huang Yuehua Y Tai Andrew W AW Tong Shuping S Lok Anna S F AS

Journal of hepatology 20130121 6

<h4>Background & aims</h4>Hepatitis B virus (HBV) core promoter (CP) mutations have been associated with an increased risk of hepatocellular carcinoma (HCC) in clinical studies. We previously reported that a combination of CP mutations seen in HCC patients, expressed in HBx gene, increased SKP2 (S-phase kinase-associated protein 2) expression, thereby promoting cellular proliferation. Here, we investigate the possible mechanisms by which CP mutations upregulate SKP2.<h4>Methods</h4>We used immun ...[more]

PMID: 23348237

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Project description:The motif ACTAYRNNNCCCR (Y being C or T, R being A or G, and N any nucleotide), called M4, was discovered as a putative cis-regulatory element, present 520 times in human promoter regions. Of these, 317 (61 %) are conserved within promoter sequences of four related organisms: human, mouse, rat, and dog. Recent genome-wide studies have described M4 as a transcription factor (TF) binding site for THAP11 that does often overlap with SBS (STAF Binding Site) a second core-promoter associated TF binding module, which associates with the TFs STAF/ZNF143 and RBP-J. Human M4-promoter genes show enhanced expression in cells of hematopoietic origin, especially in B lymphoblasts and peripheral blood B and T cells. Apart from RBP-J that is well known to recruit ICN1 (the intracellular transcriptional mediator of activated Notch1), the functional role of the hyperconserved M4 cis-element in the context of transcriptional regulation of M4-genes in lymphoid cells remains poorly defined.Here, we present a quantitative proteomic investigation of the M4 motif TF binding landscape in lymphoid cell lines that is further validated by ChIP experiments and functional assays. Our data strongly suggest that THAP11 and Ikaros interact directly, while NFKB1 (NF-kappa B p50) and HCF-1 are binding indirectly to M4-promoters in vitro and in living cells. Further analysis reveals that M4 is a bipartite composite cis-element, which is recognized by THAP11 via binding to the ACTAYR sequence module, thereby promoting ternary complex formation with HCF-1. Similarly, Ikaros binds to the CCCR module of the M4 motif and this interaction is crucial for recruiting NFKB1 to M4 harboring genes. Transient reporter assays in HEK293 and loss-of-function experiments in Molt4 T cells unequivocally demonstrate that binding of Ikaros and/or THAP11 to M4 bearing promoters is functionally important and therefore biologically relevant. Accordingly, this study validates our SILAC-based DNA protein interaction screening methodology as a valuable surrogate for a bona fide reverse ChIP technology.The M4 motif (ACTAYRNNNCCCR) is a functional regulatory bipartite cis-element, which engages a THAP11/HCF-1 complex via binding to the ACTAYR module, while the CCCRRNRNRC subsequence part constitutes a binding platform for Ikaros and NFKB1.

Dataset Information

HBV core promoter mutations promote cellular proliferation through E2F1-mediated upregulation of S-phase kinase-associated protein 2 transcription.

Publications

HBV core promoter mutations promote cellular proliferation through E2F1-mediated upregulation of S-phase kinase-associated protein 2 transcription.

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