Controlling epigenetic states and nuclear architecture in the brain

描述(申请人提供)：意识到表观遗传对基因表达的控制可以覆盖DNA中编码的调控信息，这为使用表观遗传修饰物在体内稳定地改变基因表达程序提供了令人兴奋的机会。然而，我们在以时间调节的方式改变受限细胞类型中特定靶基因的表观遗传状态的能力有限，这一愿望受到了挑战。为此，我们建议将新的遗传方法与创新的生化工具相结合，这些方法在体内提供严格的时空控制，允许以序列特定的方式靶向特定的基因组座位。我们将修改我们之前设计的一种名为Tango的特定神经元群体的诱导标记方法，以控制合成TALL(转录激活子样效应器)的表达--融合蛋白将与目标基因组位点结合并改变其表观遗传学特性。作为这些原理验证实验的模型，我们将使用遗传、表观遗传和生物化学易处理的小鼠嗅觉系统。我们以前发现，嗅觉感觉神经元(OSN)中嗅觉受体(OR)基因的单基因和单等位基因的表达是受表观遗传调控的，无论是在翻译后组蛋白修饰的水平上，还是在活跃和沉默的OR等位基因的核组织和分布水平上。因此，我们建议使用探戈系统的变体在特定的OSN亚群中以诱导的方式表达具有OR基因特异性的TALE融合蛋白及其调节增强子。这样，我们将改变活跃或沉默ORs的表观遗传状态，并诱导它们重新定位到不同的核区域，目标是稳定地改变它们的表达模式。这种化学或光学控制的表观遗传操作策略将直接适用于小鼠的任何其他细胞类型，并与病毒传递方法兼容，这将使我们的方法适用于未来对人类疾病的治疗干预。

DESCRIPTION (provided by applicant): The realization that epigenetic control of gene expression can override regulatory information encoded in DNA provides the exciting opportunity to stably alter gene expression programs in vivo with the use of epigenetic modifiers. However, this aspiration is challenged by limitations in our ability to alter the epigenetic state of specific target genes in restricted cell types in a temporally regulated fashio. For this reason, we propose to combine novel genetic approaches that afford tight spatiotemporal control in vivo with innovative biochemical tools that allow the targeting of specific genomic loci in a sequence-specific manner. We will modify an assay that we previously designed for the inducible labeling of specific neuronal populations, named Tango, towards the controlled expression of synthetic TALE (Transcription Activator Like Effectors)-fusion proteins that will bind to target genomic loci and alter their epigenetic properties. As a model for these proof-of-principle experiments we will use the genetically, epigenetically and biochemically tractable mouse olfactory system. As we previously showed, the monogenic and monoallelic expression of olfactory receptor (OR) genes in olfactory sensory neurons (OSNs) is epigenetically regulated, both at the level of post-translational histone modifications and at the level of nuclear organization and distribution of active and silent OR alleles. Therefore, we propose to express TALE-fusion proteins with specificity for OR genes and their regulating enhancers in an inducible fashion in specific OSN subpopulations using variations of the TANGO system. This way we will alter the epigenetic state of active or silent ORs, and induce their re-positioning to distinct nuclear territories with the goal of stably altering their expresson pattern. This strategy of chemically or optically controlled epigenetic manipulations will be directly applicable to any other cell type in the mouse, and compatible with viral delivery methods that will make our approach applicable to future therapeutic interventions for human disease.