A Novel Epigenetic Gene Silencing Technology

描述(由申请人提供)：这项题为“一种新的表观遗传学基因沉默技术”的研究提案涉及广泛的挑战领域：(06)使能技术，以及具体的挑战主题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基因功能障碍引起的人类难治性疾病的新疗法带来了巨大的希望。RNA干扰(RNAi)通过小干扰RNA(SiRNA)引发的信使RNA破坏阻止基因翻译成蛋白质，在研究中产生了革命性的影响，也为新的治疗方法带来了巨大的兴奋和希望。然而，由于siRNA传递方面的巨大挑战和非靶点副作用，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包装组蛋白中的位置特异性修饰决定了基因的表达或沉默，以响应生理和环境刺激。例如，由三胸基团复合体控制的组蛋白H3赖氨酸4甲基化(H3K4me)意味着基因激活，而由Polycomb基团复合体控制的H3赖氨酸27甲基化(H3K27me)是染色质紧致导致的基因沉默的表观遗传标记。虽然在哺乳动物的基因转录控制中是有效的，但三胸或多梳复合体以高度协调的方式通过多种蛋白质发挥作用。因此，为了研究或治疗疾病的目的，将三胸或多梳蛋白用于靶向基因激活或沉默是不可行的。我们最近发现了一种以组蛋白甲基化为中心的宿主转录抑制的新病毒机制(自然细胞生物学，2008)。我们发现小球藻病毒使用一种病毒蛋白(称为vSET)来特异性地修饰感染细胞中的主体组蛋白H3赖氨酸27，从而切换宿主转录机制以进行病毒复制。鉴于这种表观遗传沉默机制在真核生物中是保守的，我们发现vSET可以有效地抑制人类细胞中Polycomb靶基因的转录表达，如Hox基因。由于vSET对组蛋白H3在赖氨酸27位的甲基化具有很好的活性，原则上可以通过融合到基因序列特异性识别区域而发展成为一种选择性的表观遗传基因沉默技术，这是我们提出的研究目标。我们期待这一新的基因沉默技术填补了在转录水平控制基因沉默的重要技术空白，作为人类生物学和疾病基因组研究的有力工具，将产生广泛的影响。翻译后的组蛋白修饰决定了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得到了很好的认识，但为了研究或疾病治疗的目的，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，我们建议开发一种新的组蛋白赖氨酸甲基化导向的基因沉默技术，该技术基于我们最近发现的一种新的病毒基因沉默机制，我们预计这将是一种新的变革性工具，既可以用于机械性基因组生物学研究，也可以用于未来开发新的表观遗传疗法来治疗难治性人类疾病。

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.