Uncovering Transcriptional Regulation of a Master Hematopoietic Transcription Factor at Single Molecule Resolution

摘要 基因表达，包括从最初的基因激活到最终的蛋白质折叠的一系列反应， 对于任何正在研究的细胞群体来说，这都是一个固有的嘈杂过程。因为流程中的每一步都要遵守 独立的监管压力，细胞之间的微小差异，这些监管机构的水平可以产生 大量的转录异质性，然后可能传播到大量的功能多样性。它是 因此，要理解复杂的多细胞组织是如何忠实地发展的具有挑战性的 必须协调表达的基因才能建立细胞特性。主要调控转录因子 (Tf)是这一目的论困境的拟议解决方案。这些分子已被证明可以控制 正常细胞功能所需的基因队列，并达到适当的水平和化学计量比 在正常组织发育过程中，不同的转铁蛋白之间似乎对命运的决定至关重要。此外， 这些因子在表达或功能上的失控似乎在恶性疾病中起着重要作用 转型。Tf在高度分化的分化网络--造血中得到了广泛的研究。 这有力地和动态地产生了一系列功能不同的血细胞群体，负责 止血、气体交换和免疫功能。为了实现这种复杂的细胞输出， 造血分化被认为是随着一系列结节命运的决定而发生的 低电势干细胞室和祖细胞室(HSPC)，每个都有不同的基因表达程序 由特遣部队管理。因此，了解HSPC如何获得这些转移因子的适当剂量和活性 对我们理解稳态血液分化至关重要，并可能暴露出新的治疗窗口 血液病。然而，使这些努力复杂化的是，发现HSPC在功能上和 转录异质性，这限制了该领域揭示转铁蛋白的明确调控的能力 基于整体测量。该项目旨在通过以下方法量化这种异质性的起源 单分子、定量技术揭示主造血细胞的调控和表达 Tf，PU.1。我们的建议是：(1)确定PU1mRNA和蛋白质的产生是如何动态变化的 用RNAFISH/IF和TO(2)独立检测小鼠单个原代HSPC分化过程中 高度保守的顺式调控元件(URE)如何控制PU1的速率、幅度和动态 抄写。我们的初步发现表明，我们的实验方法不仅是可行的，它还 已经揭示了关于PU1mRNA合成的有趣发现，这些发现以前是未知的。使用这些 工具和复杂的分析技术，这项提议将提供最高的分辨率，定量 一种主要调控转录因子在原发HSPC中的调控和活性的研究。我们 预计我们的方法将为调节分子范式提供新的和基本的见解 造血和白血病的发生。

ABSTRACT Gene expression, which encompasses a series of reactions from initial gene activation to final protein folding, is an inherently noisy process for any cell population under study. As each step in the process is subject to independent regulatory pressures, small between cell differences in the levels of these regulators can produce substantial transcriptional heterogeneity, which may then propagate into substantial functional diversity. It is therefore challenging to understand how complex multi-cellular tissues faithfully develop given the number of genes that must be coordinately expressed to establish cellular identity. Master regulatory transcription factors (TF) are the proposed solution to this teleological dilemma. These molecules have been shown to control cohorts of genes required for normal cell function, and achieving the appropriate level and stoichiometry between different TF appears to be critical for fate decisions during normal tissue development. Moreover, the deregulation in either the expression or function of these factors appears to play a substantial role in malignant transformation. TF have been extensively studied in hematopoiesis, the highly arborized differentiation network that robustly and dynamically produces a spectrum of functionally distinct blood cell populations responsible for hemostasis, gas exchange, and immune function. In order to achieve this complex cellular output, hematopoietic differentiation is postulated to occur as a series of nodal fate decisions in increasingly oligopotent stem and progenitor cell compartments (HSPC), each with distinct gene expression programs governed by TF. Understanding how HSPC achieve the appropriate dose and activity of these TF is therefore vital to our understanding of steady state blood differentiation and may expose novel therapeutic windows in hematological disease. Complicating these efforts, however, is the finding that HSPC are functionally and transcriptionally heterogeneous, which have limited the field's ability to uncover definitive regulation of TF based on ensemble measurements. This project is intended to quantify the origins of that heterogeneity with single molecule, quantitative techniques to uncover the regulation and expression of a master hematopoietic TF, PU.1. Our proposal is to (1) determine how PU.1 mRNA and protein production is dynamically changed during differentiation in single primary HSPC from mice by RNA FISH/IF and to (2) independently measure how a highly conserved cis regulatory element (URE) controls the rate, magnitude, and dynamics of PU.1 transcription. Our preliminary findings have indicated that not only is our experimental approach feasible, it has already revealed intriguing findings about PU.1 mRNA synthesis that were previously unknown. Using these tools and sophisticated analytical techniques, this proposal will provide the highest resolution, quantitative study to date of the regulation and activity of a master regulatory transcription factor in primary HSPC. We anticipate that our approach will provide novel and fundamental insight into the molecular paradigms regulating hematopoiesis and leukemogenesis.