Regulation of Telomerase by NFX1

描述（由申请人提供）：该职业发展计划的总体目标是协助建立候选人在肿瘤病毒-宿主细胞相互作用研究中的独立研究生涯，特别是由高危人乳头瘤病毒（HPV）感染引起的致癌转化，导致宫颈癌和肛门生殖器癌。培训环境是导师Denise Galloway博士在Fred哈钦森癌症研究中心的实验室，以及华盛顿大学和儿童医院和区域医疗中心。拟议研究项目的具体目标集中在阐明HPV通过NFX 1在细胞永生化中激活端粒酶的机制，并确定NFX 1在正常和高危HPV E6表达细胞中的作用。在高危型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。候选人的初步数据表明，蛋白质核因子结合X1盒（NFX 1 -123）的剪接变体与HPV 16 E6相互作用。在表达HPV 16 E6的细胞中NFX 1 -123的过表达或敲低影响hTERT mRNA水平和端粒酶活性，NFX 1 -123与细胞质poly（A）结合蛋白的相互作用似乎对这种作用至关重要。该研究项目的第一个目的是使用凝胶移位和染色质免疫沉淀分析来确定NFX 1 -123是否作为hTERT的经典转录激活因子发挥作用。第二个目的是使用RNA转染和RNA结合测定来确定NFX 1 -123是否作为hTERT的转录后调节因子。这两种调节机制可能都很重要，而且并不相互排斥。最后，第三个目的是使用基因表达微阵列研究和蛋白质纯化测定来确定NFX 1 -123在有和没有HPV 16 E6表达的上皮细胞中的总体作用。了解高危HPV介导的细胞永生化的生物学将阐明肿瘤病毒如何影响宿主细胞的自我调节，并且确定NFX 1 -123如何影响hTERT表达对于了解大多数癌症如何激活端粒酶可能至关重要。

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.