A Novel Epigenetic Gene Silencing Technology

描述（由申请人提供）：本研究提案题为“一种新型表观遗传基因沉默技术”，涉及广泛的挑战领域：（06）使能技术和特定的挑战主题，01-OD-107：表观基因组修饰的功能调节。调控基因表达的能力为基因组生物学研究和由基因功能障碍引起的难治性人类疾病的新治疗带来了巨大的希望。RNA干扰（RNAi）通过小干扰RNA（siRNA）触发的信使RNA破坏来阻断基因翻译为蛋白质，在研究中产生了变革性的影响，也为新疗法带来了巨大的兴奋和希望。然而，由于siRNA递送和脱靶副作用的艰巨挑战，RNAi基因沉默技术尚未产生新的治疗方法。另一方面，表观遗传学领域的最新进展表明，染色体DNA包装组蛋白中的位点特异性修饰决定了响应生理和环境刺激的基因表达或沉默。例如，由Trithorax组复合物指导的组蛋白H3赖氨酸4甲基化（H3 K4 me）表示基因激活，而由Polycomb组复合物控制的H3赖氨酸27甲基化（H3 K27 me）是由染色质压缩引起的基因沉默的表观遗传标记。虽然在哺乳动物基因转录控制中有效，但三胸或多梳组复合物以高度协调的方式通过多个蛋白质起作用。因此，为了研究或治疗性疾病治疗的目的，指导Trithorax或Polycomb蛋白用于靶向基因激活或沉默是不可行的。我们最近发现了以组蛋白甲基化为中心的宿主转录抑制的新病毒机制（Nature Cell Biology，2008）。我们表明，该病毒使用的病毒蛋白（称为vSET），以修改特定的主机组蛋白H3赖氨酸27在感染的细胞，从而切换宿主转录机制的病毒复制。鉴于这种表观遗传沉默机制在真核生物中是保守的，我们表明vSET可以有效地抑制人类细胞中Polycomb靶基因（如HOX基因）的转录表达。由于其对组蛋白H3在赖氨酸27处的甲基化的精细活性，vSET原则上可以通过将其自身融合到基因序列特异性识别结构域而发展成选择性表观遗传基因沉默技术，这是我们提出的研究的目标。我们期待这种新的基因沉默技术，填补了一个重要的技术空白，控制基因沉默在转录水平上，将有广泛的影响，作为一个强大的工具，在人类生物学和疾病的基因组研究。翻译后组蛋白修饰决定人类基因的转录激活或沉默。虽然组蛋白修饰在基因调控中的重要性已被充分认识，但为了研究或疾病治疗的目的而调节组蛋白修饰酶的功能仍然是一个重大挑战。为了应对这一科学挑战，我们提出开发一种新的组蛋白赖氨酸甲基化指导的基因沉默技术，该技术基于我们最近发现的一种新的病毒基因沉默机制，我们预计这将成为一种新的变革性工具，用于机制基因组生物学研究和未来开发新的表观遗传疗法来治疗人类顽固性疾病。

DESCRIPTION (provided by applicant): This research proposal, entitled "A Novel Epigenetic Gene Silencing Technology", addresses the broad Challenge Area: (06) Enabling Technologies, and specific Challenge Topic, 01-OD-107: Functional Modulation of Epigenomic Modifications. The ability to modulate gene expression holds great promise for genome biology research and for novel treatments of intractable human diseases caused by the dysfunction of genes. RNA interference (RNAi) that blocks gene translation to protein by small interfering RNA (siRNA)-triggered messenger RNA destruction has had transformative impact in research and also generated enormous excitement and hope for new therapies. However, due to daunting challenges in siRNA delivery and off-target side effects, the RNAi gene silencing technology has yet to produce new therapeutics. On the other hand, recent advances in the field of epigenetics show that site-specific modifications in chromosomal DNA-packing histones dictate gene expression or silencing in response to physiological and environmental stimuli. For instance, histone H3 lysine 4 methylation (H3K4me) directed by the Trithorax group complex signifies for gene activation, whereas H3 lysine 27 methylation (H3K27me) controlled by the Polycomb group complex is an epigenetic marker for gene silencing resulting from chromatin compaction. While effective in mammalian gene transcriptional control, the Trithorax or Polycomb group complex works through multiple proteins in a highly coordinated fashion. Thus, it is not feasible to direct Trithorax or Polycomb proteins for targeted gene activation or silencing for the purpose of research or therapeutic disease treatments. We have recently discovered a novel viral mechanism for host transcriptional repression centered on histone methylation (Nature Cell Biology, 2008). We show that the chlorella virus uses a viral protein (termed vSET) to modify specifically host histone H3 lysine 27 in the infected cells, thereby switching host transcription machinery for viral replication. Given that this epigenetic silencing mechanism is conserved in eukaryotes, we show that vSET can effectively suppress transcriptional expression of Polycomb target genes in human cells such as the HOX genes. Because of its exquisite activity for methylation of histone H3 at lysine 27, vSET can in principle be developed into a selective epigenetic gene silencing technology by fusing itself to a gene sequence-specific recognition domain, which is the goal of our proposed research. We expect that this novel gene silencing technology, which fills an important technological gap of controlling gene silencing at the transcription level, will have broad impact as a powerful tool in genome research of human biology and disease. Post-translational histone modifications determine transcriptional activation or silencing of human genes. While the importance of histone modifications in gene regulation is well recognized, it remains a major challenge to modulate the functions of histone modifying enzymes for the purpose of research or disease treatments. To address this scientific challenge, we propose to develop a new histone lysine methylation-directed gene silencing technology that is based on our recent discovery of a novel viral gene silencing mechanism, which we expect will be a new transformative tool for both the mechanistic genome biology research and for future development of novel epigenetic therapies to intractable human diseases.