Regulation of Telomerase by NFX1

描述(由申请人提供)：本职业发展建议的总体目标是帮助候选人在肿瘤病毒与宿主细胞相互作用的研究方面建立独立的研究生涯，特别是研究由高危人类乳头瘤病毒(HPV)感染导致宫颈癌和肛门癌的致癌转化。培训环境是导师丹尼斯·加洛韦博士在弗雷德·哈钦森癌症研究中心的实验室，以及华盛顿大学儿童医院和地区医学中心。这项拟议的研究项目的具体目标是阐明HPV通过NFX1激活端粒酶在细胞永生化中的机制，并确定NFX1在正常和高危HPVE6表达细胞中的作用。在高危HPV感染中，E6蛋白上调端粒酶催化亚单位hTERT的表达，并激活端粒酶。候选人的初步数据显示，核因子与X1盒(NFX1-123)结合的蛋白质的剪接变体与HPV16E6相互作用。在表达HPV16E6的细胞中，NFX1-123的过表达或下调会影响hTERT的mRNA水平和端粒酶活性，而NFX1-123与细胞质Poly(A)结合蛋白的相互作用似乎是这一作用的关键。该研究项目的第一个目的是通过凝胶移位和染色质免疫沉淀分析来确定NFX1-123是否作为hTERT的经典转录激活剂发挥作用。第二个目的是通过RNA转染和RNA结合实验来确定NFX1-123是否作为hTERT的转录后调节因子。这两种监管机制可能都很重要，但并不是相互排斥的。最后，第三个目标是通过基因表达微阵列研究和蛋白质纯化分析来确定NFX1-123在表达HPV16E6和不表达HPV16E6的上皮细胞中的整体效应。了解高危HPV介导的细胞永生化的生物学将澄清肿瘤病毒如何影响宿主细胞的自我调节，并确定NFX1-123如何影响hTERT表达，这可能是理解大多数癌症如何激活端粒酶的关键。

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.