Optical control of mammalian endogenous transcription and epigenetic states.
Optical control of mammalian endogenous transcription and epigenetic states.
复制标题
作者：
基因表达的动态性质使细胞编程，稳态和环境适应的生活系统。因此，在细胞和有机体过程中的因果基因功能的解剖需要的方法，使空间和时间上精确的基因表达的调制。最近，各种微生物和植物来源的光敏蛋白已被工程改造为光遗传致动器，使许多细胞功能的高精度时空控制成为可能。然而，能够在哺乳动物内源基因组中光学调节转录的通用和稳健的技术仍然难以捉摸。在这里，我们描述了光诱导转录效应子（LITEs）的发展，光遗传学双杂交系统集成的可定制的TALE DNA结合结构域与光敏隐花色素2蛋白及其相互作用的合作伙伴CIB1从拟南芥。LITE不需要额外的外源性化学辅因子，易于定制以靶向许多内源性基因组基因座，并且可以在几分钟内可逆地激活。LITE可以包装到病毒载体中，并在遗传上靶向探测特定的细胞群。我们已经将该系统应用于原代小鼠神经元以及清醒小鼠的大脑中，以介导哺乳动物内源性基因表达的可逆调节以及靶向表观遗传染色质修饰。LITE系统建立了内源性细胞过程的光遗传学控制的新模式，并能够直接测试正常生物过程和疾病状态中遗传和表观遗传调节的因果作用。
The dynamic nature of gene expression enables cellular programming, homeostasis, and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high precision spatiotemporal control of many cellular functions. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here, we describe the development of Light-Inducible Transcriptional Effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical co-factors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of awake mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.