Regulation of Telomerase by NFX1

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DESCRIPTION (provided by applicant): The overall goal of this career development proposal is to assist in establishing the independent research career of the candidate in the study of tumor virus-host cell interactions and specifically of the oncogenic transformation caused by high risk human papillomavirus (HPV) infections that lead to cervical and anogenital cancers. The training environments are the laboratory of the mentor, Dr. Denise Galloway, at the Fred Hutchinson Cancer Research Center, and the University of Washington and Children's Hospital and Regional Medical Center. The specific aims of the proposed research project focus on elucidating the mechanism by which HPV activates telomerase in cellular immortalization through NFX1 and determining the role of NFX1 in normal and high-risk HPV E6-expressing cells. In high-risk HPV infection, the E6 protein upregulates expression of the catalytic subunit of telomerase, hTERT, and activates telomerase. The candidate's preliminary data has shown that a splice variant of the protein Nuclear Factor binds to the X1 box (NFX1-123) interacts with HPV 16E6. The overexpression or knockdown of NFX1-123 in HPV 16E6- expressing cells affects hTERT mRNA levels and telomerase activity, and interaction of NFX1-123 with cytoplasmic poly(A) binding proteins appears critical for this effect. The first aim of the proposed research project is to determine whether NFX1-123 functions as a classic transcriptional activator of hTERT using gel shift and chromatin immunoprecipitation assays. The second aim is to determine whether NFX1-123 acts as a post-transcriptional regulator of hTERT using RNA transfection and RNA binding assays. Both of these mechanisms of regulation may be important and are not mutually exclusive. Finally, the third aim is to determine the global effects of NFX1-123 in epithelial cells with and without HPV 16E6 expression using gene expression microarray studies and protein purification assays. Understanding the biology of high-risk HPV-mediated cellular immortalization will clarify how a tumor virus can affect a host cell's self regulation, and determining how NFX1-123 affects hTERT expression is likely critical to understanding how most cancers activate telomerase.