(PQ#2) Transposable element-mediated gene regulation in KSHV-associated cancer

人类免疫缺陷病毒(PLWH)携带者中的获得性免疫缺陷综合征(AIDS) 与多种不同恶性肿瘤的风险增加有关。这其中最常见的癌症 人群为卡波西氏肉瘤(KS)和非霍奇金淋巴瘤，如原发性渗出性淋巴瘤 (贝利)。虽然KS是一种内皮细胞起源的病变，但PEL是B细胞起源的。KS和PEL的病因 是致癌的DNA病毒，卡波西肉瘤相关疱疹病毒(KSHV)。KSHV显示两个截然不同的 病毒生命周期的各个阶段，潜伏期和裂解感染，这两个阶段都是发展到和 KSHV相关癌症的维持。感染过程中细胞基因表达的重塑 在潜伏期的建立以及进入溶血期的过程中起着重要作用。因此，破译 KSHV感染过程中的基因调控机制将识别参与肿瘤形成的过程 以及确定治疗开发的潜在靶点。转座元件(TES)是 能够或在某个时间能够在基因组中从一个位置移动到另一个位置的基因序列 最近对哺乳动物细胞遗传调控格局的分析揭示了TES在广泛的 一系列的调控过程，包括转录调控。而TE调控元件的功能 在干细胞生物学、TES在传染病和癌症中的作用等领域获得了赞赏， 例如在PLWH中的情况尚不清楚。我们的研究调查了这个未知的问题，这是挑衅性问题的基础 2(PQ#2)。利用5‘端RNA测序技术，我们绘制了转录起始图谱 潜伏的和裂解的PEL细胞，并发现了TES的广泛转录。此外，我们还发现了TES 转录自带注释的基因调控元件，如增强子和基因启动子，以及 一些转录的TES与KSHV编码的主要转录因子RTA结合。值得注意的是，一个有针对性的 CRISPR筛选发现了一个RTA结合的TE衍生增强子，它显著影响KSHV裂解再激活 在PEL中。与科学文献中的观察结果一起，我们未发表的数据形成了科学前提 进行拟议的研究。我们的中心假设是TE衍生的基因调控元件是中心 它们的调节在致病过程中是必不可少的。为了检验这一假设，我们 提出了一系列综合实验，旨在确定TES和KSHV之间的相互作用。在AIM 1，我们将确定RTA结合的TES在PEL中的功能。在目标2中，我们将确定KSHV感染是如何 内皮细胞影响TE的表达及TE衍生的调控元件对病毒的影响 肿瘤发生所需的生命周期和细胞途径。这些研究预计将于 确定TES如何促进PLWH中KSHV相关癌症的发生。我们的发现将代表着根本 对致癌DNA病毒如何利用细胞遗传调控格局导致疾病的新见解。

Acquired immunodeficiency syndrome (AIDS) in people living with human immunodeficiency virus (PLWH) is associated with an increased risk for a number of different malignances. The most common cancers within this population are Kaposi's sarcoma (KS) and non-Hodgkin's lymphomas, such as primary effusion lymphoma (PEL). While KS is an endothelial cell-derived lesion PEL is of B cell origin. The etiological agent of KS and PEL is the oncogenic DNA virus, Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV displays two distinct phases of its viral lifecycle, latency and lytic infection, and both are required for the progression to and maintenance of KSHV-associated cancers. The remodeling of cellular gene expression during infection plays an essential role in the establishment of latency as well as progression to the lytic phase. Thus, deciphering the gene regulatory mechanisms operating during KSHV infection will identify processes involved in tumorigenesis in PLWH as well as identify potential targets for therapeutic development. Transposable elements (TEs) are genetic sequences that can, or at one time could, move around the genome from one location to another and recent analyses of the genetic regulatory landscape of mammalian cells have revealed TEs function in a wide range of regulatory processes, including transcriptional regulation. While the function of TE regulatory elements has gained appreciation in areas such as stem cell biology, the role of TEs in infectious diseases and cancer, such as in PLWH is not known. Our study investigates this unknown which is the basis of provocative question 2 (PQ#2). Leveraging 5'-end RNA sequencing technology we have mapped the transcription initiation landscape of latent and lytic PEL cells and have identified wide-spread transcription of TEs. Moreover, we discovered TEs transcribed from within annotated gene regulatory elements such as enhancers and gene promoters, and that some transcribed TEs are bound by the main KSHV-encoded transcription factor, RTA. Remarkably, a targeted CRISPR screen identified an RTA-bound TE-derived enhancer that significantly affects KSHV lytic reactivation in PEL. Together with observations from the scientific literature our unpublished data form the scientific premise for the proposed studies. Our central hypothesis is that TE-derived gene regulatory elements are at the center of the host-KSHV battle and that their modulation is essential for pathogenesis. To test this hypothesis, we propose an integrated series of experiments aimed at determining the interplay between TEs and KSHV. In Aim 1, we will determine the function of RTA-bound TEs in PEL. In Aim 2, we will determine how KSHV infection of endothelial cells influences TE expression and the consequences of TE-derived regulatory elements on the viral lifecycle as well as cellular pathways required for tumorigenesis. Completion of these studies is expected to determine how TEs contribute to KSHV-associated cancer in PLWH. Our findings will represent fundamental new insights into how oncogenic DNA viruses co-opt the cellular genetic regulatory landscape to cause disease.