Transcriptional Control During Erythropoiesis

项目总结/摘要 红细胞生成是一个动态过程，受转录因子相对水平的定量变化控制。 转录因子（TF）、其特异性同种型和翻译后修饰（PTM）。由于目前缺乏 构成转录调控网络的蛋白质的定量数据，目前的erythm模型， 造血主要基于mRNA测量，通常不考虑蛋白质的变化， 特定TF、其同种型或PTM的水平。这极大地限制了对红细胞生成的理解， 其他转录调节过程，如β-珠蛋白表达，最终影响到 纠正血红蛋白紊乱。长期目标是破译控制红细胞的转录网络， 在健康和疾病中的作用。本研究的目的是建立红细胞生成的网络模型 基于人造血干细胞红系分化过程中TF蛋白水平的动态变化， 干/祖细胞（HSPCs）。核心假设是TF的相对蛋白水平是一个关键因素， 在每个分化阶段建立适当的基因表达程序， 红细胞生成由TF的特定组合的相对量的分级变化驱动。那个... 功能是将蛋白质组动态和定量性质整合到转录网络中， 红细胞生成的工作将产生具有改善的预测能力的模型，其将作为 健康的红细胞生成，以比较红细胞相关疾病状态，并将有助于识别 阳离子药理学试剂以恢复正常的红细胞生成。设计了两个具体目标：1） 基于蛋白质动态变化测量的红细胞生成转录网络建模 转录因子水平;和2）识别核蛋白和磷酸化丰度的新变化 在红细胞生成过程中。在第一个目标下，由 本申请人将用于在离体红细胞生成过程中的多个阶段测量TF的绝对水平， 来自健康供体的HSPC。在第二个目标下，将使用无偏倚的蛋白质组学方法来 鉴定以前未被识别的蛋白质，其水平和/或磷酸化水平发生定量变化， 在来自健康供体的HSPC的离体红细胞生成期间的分离状态。这种方法是创新的，因为它 使用新的质谱方法系统地鉴定和定量调节红细胞生成的TF， 在原代人类细胞中。这项研究具有重要意义，因为它将阐明复杂的调控机制。 控制红细胞生成的过程最终，这些知识有可能指导新的设计， 在β-地中海贫血患者中重新建立适当β-珠蛋白表达的治疗剂。

PROJECT SUMMARY/ABSTRACT Erythropoiesis is a dynamic process governed by quantitative changes in the relative levels of transcription fac- tors (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 eryth- ropoiesis 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 the 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 eryth- ropoiesis 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 pa- rameter 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 ra- tionale is that integration of the dynamic and quantitative nature of the proteome into the transcriptional net- work 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 identifi- cation of 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 phosphopro- teins 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 phosphoryla- tion 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 erythropoi- esis 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.