Dynamic association of transcription initiation proteins with chromatin at single-molecule resolution in living yeast

真核生物基因控制的一个中心范式假定，序列特异性转录因子搜索并 在拥挤的核环境中定位它们的基因组靶点，通常以组合和合作的方式， 染色质中的调节DNA元件，以募集和指导转录前起始的有序组装 由一般转录因子和RNA聚合酶组成的复合物。虽然身份和功能 在已知的数百种转录相关蛋白中，关于转录相关蛋白的时间尺度的信息很少。 它们在转录过程中的作用，以及染色质结构的调节影响， 对转录蛋白动力学的重塑和修饰。这项提议旨在检验以下假设： 细胞核中转录起始蛋白的染色质结合在活细胞中以快速动力学发生，并且 染色质重塑和修饰在调节转录蛋白动力学中起作用。我们将使用 活细胞单分子成像在模式生物芽殖酵母监测扩散行为 高时空分辨率的转录起始蛋白。这种“体内生物化学”方法与 来自ChIP-Seq技术，并与ChIP-Seq技术互补，ChIP-Seq技术在全基因组范围内定位转录因子占据率， 碱基对分辨率，但提供的结合动力学的信息很少。为了阐明监管贡献， 染色质结构到转录起始蛋白动力学，我们将测量它们在酵母中的迁移率 有条件地耗尽染色质重塑和修饰酶的突变体。 我们将设计和功能验证DNA构建体编码的组件代表的 一般的转录因子和主要序列特异性的DNA结合转录因子， 标记蛋白质标签（HaloTag），其允许用细胞可渗透的有机荧光团（Janelia Fluor）进行标记。直播- 单分子分辨率的荧光标记转录因子的细胞成像将测量蛋白质 扩散和区分染色质结合和无染色质的群体以及估计驻留 人口的时代。此外，我们将使用6种主要染色质重塑因子的条件性去除， 组蛋白修饰剂，以揭示转录起始蛋白在一定条件下扩散参数的变化 的染色质扰动，以告知其中几个扩散参数是受染色质控制。 通过将条件突变遗传学与活细胞单分子成像相结合， 了解染色质结构调控转录起始的动力学机制 处理和开发一种潜在的常规技术，以补充目前的全基因组分析 技术，使其他领域的酵母细胞核和染色体生物学，包括DNA复制的研究， 修复和重组。

A central paradigm for eukaryotic gene control posits that sequence-specific transcription factors search for and locate their genomic targets in a crowded nuclear environment, often acting combinatorially and cooperatively at regulatory DNA elements in chromatin to recruit and direct the ordered assembly of a transcription pre-initiation complex composed of general transcription factors and RNA polymerase. Although the identities and functions of several hundred transcription-related proteins are known, there is little information on the timescales under which they operate in the transcription process, and the regulatory influence of chromatin architecture, remodeling and modification on transcription protein kinetics. This proposal aims to test the hypothesis that chromatin binding of transcription initiation proteins in the cell nucleus occurs with rapid kinetics in live cells, and that chromatin remodeling and modification has a role in regulating transcription protein dynamics. We will use live-cell single-molecule imaging in the model organism budding yeast to monitor the diffusive behavior of transcription initiation proteins at high spatio-temporal resolution. This ‘in vivo biochemistry’ approach differs from and is complementary to ChIP-Seq techniques that map transcription factor occupancy genome-wide at base pair resolution but provide little information on binding dynamics. To elucidate regulatory contributions of chromatin architecture to transcription initiation protein dynamics, we will measure their mobilities in yeast mutants conditionally depleted for chromatin remodeling and modification enzymes. We will engineer and functionally validate DNA constructs encoding components representative of the general transcription factors and major sequence-specific DNA binding transcription factors fused to a self- labeling protein tag (HaloTag) that allows labeling with a cell-permeable organic fluorophore (Janelia Fluor). Live- cell imaging of fluorescently labeled transcription factors at single-molecule resolution will measure protein diffusion and distinguish between chromatin-bound and chromatin-free populations and estimate residence times of the bound population. Further, we will use conditional depletion of 6 major chromatin remodelers and histone modifiers to reveal changes in the diffusive parameters of transcription initiation proteins under conditions of chromatin perturbation to inform which among several diffusive parameters are subject to chromatin controls. By combining with conditional mutant genetics with live-cell single-molecule imaging, we hope to transform understanding of the kinetic mechanisms by which chromatin architecture regulates the transcription initiation process and develop a potentially routine technology complementary to current genome-wide analytical techniques to benefit other areas of yeast nuclear and chromosome biology, including studies of DNA replication, repair, and recombination.