Transcriptional Control During Erythropoiesis

红细胞生成过程中的转录控制

基本信息

批准号：
10892619
负责人：
Marjorie Carole Brand
金额：
$ 15.1万
依托单位：
INSTITUTE FOR SYSTEMS BIOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-15 至 2024-07-31
项目状态：
已结题

项目摘要

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.

红细胞生成是一个动态过程，该过程受转录相对水平的定量变化的控制 TOR（TFS）。由于目前缺乏构成转录调节的蛋白质的定量数据大多数红细胞生成模型主要基于mRNA测量，而不是通常考虑特定TF的蛋白质水平的变化。这显着限制了对红细胞生成和其他转录调节的过程，例如β-珠蛋白的表达，最终影响纠正血红蛋白疾病的能力。长期的目标是破译控制生成的TRN 健康和疾病中的ropoiesis。该提案的目的是显着扩展我们的TRN模型通过整合动态大量和单细胞TF蛋白抽象测量 - 与其他相关的 - 组数据。中心假设是 TF是建立适当基因表达程序的关键参数分化，促红细胞生成是由特定组合的相对量的分级变化所驱动的 TFS的影响。理由是TF蛋白质组的动态和定量性质的整合到一个扩大的红细胞生成TRN将导致具有改善预测能力的模型，这将作为一个与红相关疾病状态进行比较的健康红细胞生成基准的基准，并将促进鉴定药物剂以恢复正常的红细胞生成。三个具体目标是设计：1）在红细胞生成期间对TF蛋白质组的绝对定量； 2）确定如何等级单个细胞中多个TF的抽象的变化启动并逐步增强细胞脂肪决策沿着红细胞轨迹； 3）促红细胞生成的计算分析，建模和验证。对于第一个目标，将使用高吞吐量定量质谱法（MS）方法来测量源自健康供体的HSPC的离体红细胞生成过程中TF蛋白质组的绝对水平。为了第二个目的，将组合完整的细胞和靶向MS蛋白质组学方法以估计TF 单个细胞中的蛋白质丰度，以及其他单细胞 - 组技术，以测量基因的变化在离体红行中的情况下提供和TF基因组结合。在第三个目标下，红细胞生成的TRN模型 ESI将使用TF蛋白丰度的测量以及其他转录相关的 - 组数据来构建。将对在月经期间在转录控制中暗示的TF进行功能验证基于我们最近的结果。这种方法具有创新性，因为它使用了单一的新型组合细胞和大量蛋白质组学方法在原代人中量化了红细胞生成期间大量TF的细胞和蛋白质组学方法单元格并使用数据进行集成的TRN建模。拟议的研究很重要，因为它将照亮控制红细胞生成的NATE复杂调节过程。最终，这种知识有可能指导新的治疗剂的设计，以重新建立ß-丘脑血症患者的适当β-珠蛋白表达。