Transcriptional Control During Erythropoiesis

描述(申请人提供)：红细胞生成是一个动态的过程，由转录因子(TF)的相对水平及其特定亚型的数量变化所支配 和翻译后修饰(PTM)。由于目前缺乏关于构成转录调控网络的蛋白质的定量数据，目前的红细胞生成模型主要基于mRNA的测量，通常不考虑特定TF、其异构体或PTM的蛋白质水平的变化。这极大地限制了对红细胞生成和其他转录调控过程的理解，例如贝珠蛋白的表达，最终影响了纠正血红蛋白紊乱的能力。长期目标是破译控制健康和疾病中红细胞生成的转录网络。 本研究的目的是建立一个基于人造血干/祖细胞(HSPC)红系分化过程中Tf蛋白水平动态变化的红细胞生成网络模型。中心假设是，转铁蛋白的相对蛋白质水平是 在分化的每个阶段建立适当的基因表达程序的一个关键参数，红细胞生成是由特定的转录因子组合的相对量的逐步变化驱动的。其基本原理是，将蛋白质组的动态和定量性质整合到红细胞生成的转录网络中将产生一个具有更高预测能力的模型，该模型将作为健康红细胞生成的基准，用于比较与红系相关的疾病状态，并将有助于识别药物以恢复正常的红细胞生成。设计了两个特定的目标：1)基于转录因子蛋白质水平的动态变化来建立红细胞生成转录网络的模型；以及2)识别在红细胞生成过程中核蛋白和磷蛋白丰度的新变化。在第一个目标下，申请者开发的一种新的靶向质谱学方法将用于在健康捐赠者的HSPC体外红细胞生成过程中的多个阶段测量TFS的绝对水平。在第二个目标下，将使用一种无偏见的蛋白质组学方法来识别在健康捐赠者的HSPC体外红系生成过程中经历其水平和/或磷酸化状态定量变化的先前未被欣赏的蛋白质。这种方法是创新的，因为它使用了新的质谱学方法来系统地识别和量化调节人类原代细胞红血球生成的因子。这项拟议的研究意义重大，因为它将阐明控制红细胞生成的复杂调控过程。最终，这些知识有可能指导新的治疗方法的设计，以重新建立贝型地中海贫血症患者适当的贝珠蛋白表达。

DESCRIPTION (provided by applicant): Erythropoiesis is a dynamic process governed by quantitative changes in the relative levels of transcription factors (TFs), their specific isoforms and post-translational modifications (PTMs). Due to the current paucity of quantitative data on the proteins that constitute the transcriptional regulatory network, current models of erythropoiesis are based primarily on mRNA measurements and do not typically consider changes in the protein levels of specific TFs, their isoforms or PTMs. This significantly limits th understanding of erythropoiesis and other transcriptionally regulated processes such as ß-globin expression, ultimately impinging on the capacity to correct hemoglobin disorders. The long-term goal is to decipher the transcriptional network that controls erythropoiesis in health and disease. The objective of this proposal is to build a network model of erythropoiesis based on dynamic changes in TF protein levels during erythroid differentiation of human hematopoietic stem/progenitor cells (HSPCs). The central hypothesis is that the relative protein levels of TFs is a critical parameter in the establishment of proper gene expression programs at each stage of differentiation, and that erythropoiesis is driven by graded changes in the relative amounts of specific combinations of TFs. The rationale is that integration of the dynamic and quantitative nature of the proteome into the transcriptional network of erythropoiesis will result in a model with improved predictive power, which will serve as a benchmark for healthy erythropoiesis against which to compare erythroid-related disease states, and will facilitate the identification o pharmacological agents to restore normal erythropoiesis. Two specific aims have been designed: 1) Model the erythropoiesis transcriptional network based on measurements of dynamic changes in the protein levels of transcription factors; and 2) Identify novel changes in abundance of nuclear proteins and phosphoproteins during erythropoiesis. Under the first aim, a novel targeted mass spectrometry approach developed by the applicants will be used to measure absolute levels of TFs at multiple stages during ex vivo erythropoiesis of HSPCs derived from healthy donors. Under the second aim, an unbiased proteomic approach will be used to identify previously unappreciated proteins that undergo quantitative changes in their levels and/or phosphorylation status during ex vivo erythropoiesis of HSPCs from healthy donors. The approach is innovative because it uses novel mass spectrometry approaches to systematically identify and quantify TFs that regulate erythropoiesis in primary human cells. The proposed research is significant because it will illuminate complex regulatory processes that control erythropoiesis. Ultimately, such knowledge has the potential to guide the design of new therapeutics to re-establish proper ß-globin expression in ß-thalassemic patients.