Collaborative Research: Tools 4 Cells: Developing Next Generation Methods for Studying Cytoskeletal Factors in the Cell Nucleus

近年来，人们已经清楚地认识到，基因在细胞核中的定位对它们的开启和关闭非常重要。然而，人们对基因定位是如何调控的知之甚少。该项目的目标是了解肌动蛋白，一种在组织细胞质中起核心作用的蛋白质，如何也参与基因组的组织和基因表达的控制。这就需要新颖的工具来精确地干扰细胞核中的肌动蛋白，以避免由于其细胞质部分的破坏而产生的混淆效应。这些工具包括核肌动蛋白丝的光活化稳定剂和选择性降解核肌动蛋白的系统。总之，这些工具将能够对核肌动蛋白进行精细控制，而不会影响其在细胞其他部分的功能。该项目的广泛影响包括其固有的优点，因为所有有核细胞都可能含有肌动蛋白，并且开发的工具将传播给该领域的其他研究人员，并有望广泛影响我们对控制基因组定位和基因使用的基本机制的理解。此外，芝加哥大学是一个少数族裔服务机构，芝加哥大学参与了许多努力，从STEM学科中代表性不足的群体中招收学生。该项目将保持这一传统，并通过让这些群体的学生大量参与科学，为加强STEM劳动力做出贡献。以肌动蛋白为基础的细胞骨架因子，对确定细胞形状至关重要，也存在于细胞核中，在那里它们与转录和染色质组织有关。揭示这些效应背后的机制是复杂的挑战，特别是针对核池的细胞骨架因子的动态扰动。现有的技术引入了工件，并且不能在快速的时间尺度上进行操作。在这个项目中，将开发新的、更锋利的工具。其中包括LILAC的适应性，一种光激活探针，用于跟踪和/或稳定细胞核中的肌动蛋白丝，而不会干扰细胞质肌动蛋白。反之，为了精确控制核肌动蛋白降解，生长素诱导降解（AID）标签将融合到内源性β -肌动蛋白上，并与强核定位的TIR1泛素连接酶结合。总之，这些克隆细胞系中肌动蛋白缺失（degron depletion）和功能获得（gain-of-function， LILAC）的核肌动蛋白诱导操作将有助于发现肌动蛋白动力学对核过程的直接影响。这些工具将被广泛地描述并用于研究肌动蛋白在核组织中的作用。高分辨率染色质构象捕获（Micro-C）将用于揭示核肌动蛋白动力学对精细染色质重组的影响。经过校准的ChIP-seq和PRO-seq将对染色质状态的相关变化和对新生转录的功能影响产生互补的见解。将在活细胞和固定细胞中使用定量超分辨率显微镜研究调整核肌动蛋白动力学对纳米尺度Pol II聚集的直接影响。该项目由分子和细胞生物科学部的遗传机制项目共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过基金会的智力价值和更广泛的影响审查标准进行了评估，认为值得支持。

In recent years it has become clear that the positioning of genes in the cell nucleus is important for turning them on and off. However, little is known about how gene positioning is regulated. The goal of this project is to understand how actin, a protein that plays a central role in organizing the cytoplasm, is also involved in the organization of the genome and the control of gene expression. This requires novel tools for the precise perturbation of actin only in the cell nucleus to avoid any confounding effects due to disruptions of its cytoplasmic fraction. These tools include a light-activatable stabilizer of nuclear actin filaments and a system to selectively degrade nuclear actin. Together, these tools will enable fine control over nuclear actin without impacting its functions in other parts of the cell. The Broader Impacts of this project include its intrinsic merit as all nucleated cells likely contain actin and the developed tools will be disseminated to other researchers in the field and are expected to broadly impact our understanding of basic mechanisms that control genome positioning and gene usage. Further, UIC is a minority serving institution and the University of Chicago participates in numerous efforts to recruit students from groups underrepresented in STEM disciplines. This project will maintain this tradition and contribute to strengthening the STEM workforce by heavily involving students from these groups in science.Actin-based cytoskeletal factors, essential for defining cell shape, are also present in the nucleus, where they have been linked to transcription and chromatin organization. Uncovering the mechanisms behind these effects is complicated by the challenges of specifically targeting the nuclear pool of cytoskeletal factors for dynamic perturbation. Existing techniques introduce artifacts and are not manipulable on rapid timescales. In this project, new and sharper tools will be developed. These include an adaptation of LILAC, a photoactivated probe, to track and/or stabilize actin filaments in the nucleus, without perturbations to cytoplasmic actin. Vice versa, to enable precise temporal control of nuclear actin degradation, an auxin inducible degron (AID) tag will be fused to endogenous beta-actin and combined with a strongly nuclear-localized TIR1 ubiquitin ligase. Together, these loss- (degron depletion) and gain-of-function (LILAC) nuclear actin inducible manipulations in clonal cell lines will enable the discovery of direct effects of actin dynamics on nuclear processes. These tools will be broadly characterized and used to investigate the role of actin in nuclear organization. High-resolution chromatin conformation capture (Micro-C) will be used to reveal the impact of nuclear actin dynamics on fine-scale chromatin reorganization. Calibrated ChIP-seq and PRO-seq will generate complementary insights into associated changes in chromatin state and functional impacts on nascent transcription. Direct effects of tuning nuclear actin dynamics on Pol II clustering at the nanometer scale will be investigated using quantitative superresolution microscopy in live and fixed cells.The project is co-funded by the Genetic Mechanisms program in the Division of Molecular and Cellular BiosciencesThis award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.