Transcriptional Control During Erythropoiesis

红细胞生成是一个由转录因子相对水平的数量变化控制的动态过程。 Tors(TFS)。由于目前缺乏关于构成转录调控的蛋白质的定量数据。 乳房网络(TRN)，大多数红细胞生成模型主要基于mRNA的测量，而不是 通常考虑特定转录因子的蛋白质水平的变化。这极大地限制了人们对 红血球生成和其他转录调控的过程，如贝珠蛋白的表达，最终影响 关于纠正血红蛋白紊乱的能力。我们的长期目标是破译控制一切的TRN- 健康和疾病中的轮回发生。该建议目标是显著扩展我们针对小区的TRN模型 动态批量和单细胞TF蛋白丰度测量相结合的红细胞生成过程中的命运决定 与其他转录相关组学数据进行比较。中心假说是相对蛋白质水平 在整个过程中，TFs是建立适当的基因表达程序的关键参数 分化，而红细胞生成是由特定结合的相对量的分级变化驱动的- 关于TF的评论。其基本原理是将Tf蛋白质组的动态和定量性质整合到 扩大的红细胞生成TRN将导致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，用于比较红系相关疾病的状态，并将有助于- 对恢复正常红细胞生成的药物进行鉴定。三个具体目标是 设计：1)红细胞生成过程中Tf蛋白质组的绝对定量；2)确定循序渐进的程度 单个细胞中多个转录因子丰度的变化启动并逐渐加强细胞命运决定 以及3)红系生成TRN的计算分析、建模和验证。 对于第一个目标，将使用高通量定量质谱学(MS)方法来测量 健康供者HSPC体外红细胞生成过程中Tf蛋白质组的绝对水平。对于 第二个目标是将互补细胞周期和靶向MS蛋白质组学方法结合起来估计转铁蛋白 单细胞蛋白质丰度，与其他单细胞组学技术一起测量基因的变化。 体外红细胞生成过程中的抑制和转铁蛋白基因组结合。在第三个目标下，建立了人民币汇率的TRN模型。 Esis将利用Tf蛋白丰度的测量和其他与转录相关的组学数据来建立。 将对参与转录调控的转录因子进行功能验证- 根据我们最近的结果进行了假冒。这种方法是创新的，因为它使用了一种新颖的单一 用细胞和批量蛋白质组学方法定量检测原始人红细胞生成过程中大量的转录因子 单元格，并将数据用于集成的TRN建模。这项拟议的研究意义重大，因为它将照亮- Nate控制红细胞生成的复杂调控过程。归根结底，这种知识有可能 指导设计新的治疗方法，以重建贝型地中海贫血症患者正确的矿化珠蛋白表达。

Erythropoiesis is a dynamic process governed by quantitative changes in the relative levels of transcription fac- tors (TFs). Due to the current paucity of quantitative data on the proteins that constitute the transcriptional regu- latory network (TRN), most models of erythropoiesis are based primarily on mRNA measurements and do not typically consider changes in the protein levels of specific TFs. 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 TRN that controls eryth- ropoiesis in health and disease. The objective of this proposal is to significantly expand our TRN model for cell fate decision during erythropoiesis by integrating dynamic bulk and single cell TF protein abundance measure- ments with other transcription-relevant -omics data. The central hypothesis is that the relative protein levels of TFs are critical parameters in the establishment of proper gene expression programs during the continuum of differentiation, and that erythropoiesis is driven by graded changes in the relative amounts of specific combina- tions of TFs. The rationale is that integration of the dynamic and quantitative nature of the TF proteome into an expanded TRN 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 facili- tate the identification of pharmacological agents to restore normal erythropoiesis. Three specific aims have been designed: 1) Absolute quantification of the TF proteome during erythropoiesis; 2) Determine how gradual changes in the abundance of multiple TFs in single cells initiate and progressively reinforce cell fate decisions along the erythroid trajectory; and 3) Computational analysis, modeling and validation of the erythropoiesis TRN. For the first aim, a high throughput quantitative mass spectrometry (MS) approach will be used to measure absolute levels of the TF proteome during ex vivo erythropoiesis of HSPCs derived from healthy donors. For the second aim, complementary CyTOF and targeted-MS proteomic approaches will be combined to estimate TF protein abundances in single cells, with other single cell –omics technologies to measure changes in gene ex- pression and TF genomic binding during ex vivo erythropoiesis. Under the third aim, TRN models of erythropoi- esis will be built utilizing measurements of TF protein abundances, and other transcription-relevant –omics data. Functional validation will be performed for TFs that have been implicated in transcriptional control during eryth- ropoiesis based on our recent results. The approach is innovative because it uses a novel combination of single cell and bulk proteomics methodologies to quantify large numbers of TFs during erythropoiesis in primary human cells and uses the data for integrative TRN modeling. The proposed research is significant because it will illumi- nate 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.