Spatiotemporal Control of the Epigenome via Photoactivatable Nuclear Localization

描述（由申请人提供）：摘要通过表观遗传修饰的基因激活和沉默对于确定细胞命运是重要的，并且对于多细胞生物体的适当发育是至关重要的。确定各种表观遗传标记在特定发育过程中的相对重要性可能具有挑战性，因为这些修饰通常是动态的，并且在细胞和组织类型之间变化。常见的方法，如表观遗传修饰所需的酶的基因敲除，可用于评估修饰的整体重要性，但不能提供直接的方法来测试修饰在特定基因座、细胞类型和发育时间点的重要性。在这里，我们的目标是创建一个通用的方法来控制表观遗传修饰，这是可逆的，可以在特定的时间在开发中应用到一组特定的细胞。在初步研究中，我们已经开发了一种光激活蛋白，该蛋白在黑暗中定位于细胞质，但当细胞被蓝光照射时进入细胞核。我们的假设是，这个开关，称为局域网，即光激活核穿梭机，可以用来控制必须在细胞核中才能发挥作用的蛋白质的活性。作为概念的证明，我们已经表明，我们可以使用局域网来控制酵母中转录因子的活性。在这里，我们将探索光激活的核定位是否可以用来控制组蛋白和DNA甲基化和乙酰化所需的酶和支架蛋白。为了靶向预定的基因座，我们将局域网与天然存在的和工程化的DNA结合结构域融合。我们将在哺乳动物细胞培养中测试局域网开关，并探索该开关是否可用于操纵C。通过光介导的组蛋白去乙酰化酶的控制。

DESCRIPTION (provided by applicant): Abstract Gene activation and silencing via epigenetic modifications is important for determining cell fate and is critical for the proper development of multicellular organisms. Determining the relative importance of various epigenetic markers in specific developmental processes can be challenging because these modifications are often dynamic and vary between cell and tissue types. Common approaches such as a gene knockout of an enzyme required for epigenetic modifications can be used to assess the global importance of the modification, but do not provide a straightforward approach for testing the importance of the modification at specific loci, cell types and developmental time points. Here, we aim to create a general approach for the control of epigenetic modifications that is reversible and can be applied at specific times in development to a specified set of cells. In preliminary studies we have developed a light activatable protein that localizes to the cytoplasm in the dark, but enters the nucleus when the cell is illuminated with blue light. Our hypothesis is that this switch, called LANS for Light Activatable Nuclear Shuttle, can be used to control the activity of proteins that must be in the nucleus to be functional. As proof of concept, we have shown that we can use LANS to control the activity of a transcription factor in yeast with light. Here, we wil explore if light activated nuclear localization can be used to control enzymes and scaffolding proteins required for histone and DNA methylation and acetylation. To target predetermined loci, we will fuse LANS with naturally occurring and engineered DNA binding domains. We will test the LANS switch in mammalian cell culture, and we will explore whether the switch can be used to manipulate cell fate in C. elegans via light mediated control of a histone deacetylase.