Controlling epigenetic states and nuclear architecture in the brain

描述（由申请人提供）：认识到基因表达的表观遗传控制可以覆盖DNA编码的调控信息，这为使用表观遗传修饰剂稳定地改变体内基因表达程序提供了令人兴奋的机会。然而，这一愿望受到我们以暂时调节的方式改变受限制细胞类型中特定靶基因表观遗传状态的能力的限制的挑战。出于这个原因，我们建议结合新颖的遗传方法，提供严格的体内时空控制与创新的生化工具，允许以序列特异性的方式靶向特定的基因组位点。我们将修改我们之前设计的用于特定神经元群体诱导标记的实验，命名为Tango，以控制合成TALE（转录激活因子样效应器）的表达-融合蛋白将结合目标基因组位点并改变其表观遗传特性。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上易于处理的小鼠嗅觉系统。如前所述，嗅觉感觉神经元（OSNs）中嗅觉受体（OR）基因的单基因和单等位基因表达受表观遗传调控，既在翻译后组蛋白修饰水平上，也在核组织和活性或沉默OR等位基因的分布水平上。因此，我们建议使用TANGO系统的变体，在特定的OSN亚群中以诱导的方式表达具有OR基因及其调节增强子特异性的tale融合蛋白。通过这种方式，我们将改变活跃或沉默的or的表观遗传状态，并诱导它们重新定位到不同的核区域，以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.