The Role of H3.3 histone variant in the pathogenesis of oral Kaposi's Sarcoma

摘要 卡波西肉瘤相关疱疹病毒(KSHV)是卡波西肉瘤(KS)的病原体。 和两种淋巴增生性疾病：原发性渗出性淋巴瘤(PEL)和多中心Castleman病 疾病(MCD)。KS是口腔癌中最常见的人类免疫缺陷病毒(HIV)感染者 病人。口腔是病毒通过唾液传播的主要场所。尽管这一事件的发生率 最近，由于联合抗逆转录病毒疗法(CART)，美国艾滋病-KS病例有所减少 研究表明，长期治疗的艾滋病患者会复发口服KS。而KS肿瘤细胞 大多是潜伏感染，并表达有限数量的病毒基因，牙龈上皮细胞 口腔主动复制KSHV，这对a)在B细胞中播种潜伏库非常重要 在初次感染后和b)在口腔内脱落并通过唾液传播。表观遗传学和 染色质结构在转录和复制的调控中起着核心作用。 去调节、异常沉积和组蛋白变体的突变，如H3.3、CENP-A和 H2A.Z及其相关的伴侣蛋白与多种人类癌症有关 包括头部和颈部。在新感染后，KSHV基因组迅速与 核小体，获得特定的表观遗传修饰，将病毒Episome分割成 转录活性结构域和沉默结构域。而多个KSHV表观遗传标记 在淋巴样细胞、上皮细胞和内皮细胞中被定位，突起和潜在的宿主- 病毒相互作用导致潜伏期允许性上皮体的形成仍然很差 明白了。鉴于KSHV潜伏期相关核抗原(LANA)与 染色质重构体和H3.3组蛋白伴侣蛋白Daxx，Hira和Dek，WE 假设H3.3的沉积在建立和维持过程中起重要作用 KSHV潜伏期。我们的初步数据表明，H3.3在KSHV上的沉积可以 在长期感染细胞的上染色体上从头感染后早期被检测到。此外，我们 证明了通过基因干扰H3.3分子伴侣途径Hira和Daxx CRISPR/Cas9导致KSHV潜伏期控制显著变化，与 表观遗传状态。我们进一步假设潜伏期相关基因产物包括 LANA调节和/或去调节组蛋白伴侣蛋白通路 延迟。为了直接解决这一假设，我们建议对组蛋白H3.3进行机械研究 组蛋白翻译后修饰的沉积和形成，目的是 了解它们与病毒基因表达和潜伏/裂解的关系 复制。这些研究的一个主要方面是调查病毒基因产物如何与 并调节组蛋白变异的伴侣通路。此外，我们还使用了人类口腔原代上皮 比转化的上皮细胞系更具裂解性的细胞系以表征表观遗传学 导致其裂解表型的变化。重要的是，通过与Dr. 唐纳德·科恩，牙医学院，我们建议在体内建立口腔KS的表观基因组 福尔马林固定和冰冻的肿瘤样本。总体目标是研究组蛋白是如何 变异沉积以一种特定细胞类型的方式决定潜伏感染和裂解感染。结果是 可能指向针对KSHV的口服KS的新治疗靶点。

Abstract Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), and two lymphoproliferative diseases: primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). KS is the most common oral cancer in human immunodeficiency virus (HIV)-infected patients. The oral cavity is the major site for virus transmission via saliva. Although the incidence of AIDS-KS cases has been reduced in the US due to combination antiretroviral therapy (cART), recent studies suggest recurrence of oral KS in long-term treated AIDS patients. While KS tumor cells are mostly latently infected, and express a limited number of viral genes, gingival epithelial cells in the oral cavity actively replicate KSHV which is important for a) seeding the latency reservoir in B cells after primary infection and b) shedding in the oral cavity and transmission via saliva. Epigenetics and chromatin structure play a central role in the regulation of both transcription and replication. Deregulation, aberrant deposition, and mutations of histone variants such as H3.3, CENP-A, and H2A.Z and their associated chaperones have been implicated in multiple human cancers including head and neck. After de novo infection, KSHV genomes rapidly associate with nucleosomes which acquire specific epigenetic modifications that partition the viral episome into transcriptionally active and silenced domains. While multiple KSHV epigenetic marks have been mapped in lymphoid, epithelial and endothelial cells, the processes and potential host- viral interactions leading to the formation of latency permissive episomes are still poorly understood. Given that the KSHV latency-associated nuclear antigen (LANA) interacts with both chromatin remodelers and H3.3 histone chaperones Daxx, HIRA, and DEK, we hypothesize that H3.3 deposition plays an important role in the establishment and maintenance of KSHV latency. Our preliminary data demonstrate that H3.3 deposition on KSHV episomes can be detected early after de novo infection on episomes of long-term infected cells. Moreover, we demonstrated that genetically disrupting the H3.3 chaperone pathways HIRA and Daxx by CRISPR/Cas9 leads to marked changes in KSHV latency control, associated with alterations in the epigenetic status. We further hypothesize that latency-associated gene products including LANA modulate and or de-regulate histone chaperone pathways during de novo infection and latency. To directly address this hypothesis we propose to mechanistically study histone H3.3 deposition and the formation of histone post translational modifications (PTMs), with the goal of understanding their relationship with respect to viral gene expression and latent/lytic replication. A main aspect of these studies is to investigate how viral gene products interact with and modulate histone variant chaperone pathways. In addition, we use human oral primary epithelial cells which are more lytic than transformed epithelial cell lines to characterize epigenetic changes responsible for their lytic phenotype. Importantly, through a collaboration with Dr. Donald Cohen, College of Dentistry, we propose to establish in vivo epigenomes of oral KS in both formalin-fixed and snap frozen tumor samples. The overall goal is to investigate how histone variant deposition determines latent and lytic infection in a cell type-specific manner. The results may point to KSHV-specific novel therapeutic targets for oral KS.