Uncovering the functional diversification mechanisms of transcription factor isoforms involved in stem cell differentiation

揭示干细胞分化中转录因子亚型的功能多样化机制

• 批准号: 10458726
• 负责人: Gloria Sheynkman
• 金额: $ 40.38万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10458726
• 关键词: Alternative Splicing; Area; Binding Proteins; Biological; Biology; Cell Differentiation process; Cell Lineage; Chromatin; DNA Binding; Data; Development; Disease; Ectopic Expression; Event; Exhibits; FOXP1 gene; Gene Activation; Genes; Goals; Human; Investigation; Knowledge; Length; Libraries; Messenger RNA; Molecular; Pathologic; Pathway interactions; Phenotype; Physiological; Play; Protein Isoforms; Proteins; Proteome; Proteomics; Regenerative Medicine; Research; Role; Somatic Cell; Specificity; Systems Biology; Work; base; cofactor; embryonic stem cell; engineered stem cells; gene regulatory network; gene repression; induced pluripotent stem cell; programs; proteogenomics; screening; self-renewal; stem cell differentiation; transcription factor; transdifferentiation

Abstract Ectopic expression of transcription factors (TFs) can drive differentiation of embryonic stem cells, trans- differentiation between different somatic cell lineages, and reprogramming of somatic cells into induced pluripotent stem cells. This observation underscores the central role transcriptional factors (TFs) play in gene regulatory networks to dictate cellular phenotypes. The endogenous activities of TFs can be dramatically altered through mechanisms such as alternative splicing of TF messenger RNAs, in which multiple, functionally distinct protein isoforms can be produced from the same gene. Distinct TF isoforms can exhibit differential abilities to bind DNA targets, cofactors, or chromatin-associated proteins, resulting in complementary or opposing functions. For example, FOXP1 produces two isoforms that differ in their DNA-binding specificities: one activates self-renewal pathways, while the other activates differentiation pathways. On a global scale, such isoform-driven functional rewiring is emerging as a major regulatory strategy that controls differentiation and development. However, reliably detecting and discovering biologically relevant TF isoforms is challenging, particularly at the proteome-scale. To characterize TF isoforms that play a role in stem cell differentiation, my lab will apply a powerful combination of analytical and systems biology approaches to identify and experimentally characterize the functional consequences of TF isoforms. This project will involve several interlinked areas, including goals to: 1) discover and quantify full-length TF isoforms associated with differentiated cell states, 2) find TF isoform “drivers” of differentiation, and 3) uncover the mechanism(s) by which TF isoforms exhibit different functions. Our approach will use proteogenomic analysis that integrates long-read sequencing and MS-based proteomics data, high- throughput functional screening of large-scale isoform libraries, and characterization of molecular determinants (e.g., protein binding, DNA binding) and activities (gene activation or repression) that underlie differential TF isoform function. Collectively, the resulting data will enable discoveries of new general mechanisms of cellular differentiation and their drivers. This research program will provide a fundamental understanding of how TF isoforms function in regulatory networks and will contribute to the field by expanding the toolkit of TFs used in stem cell engineering applications. More broadly, this framework offers a new paradigm to enable isoform-resolved biological investigations, which could accelerate discovery of key isoform regulators in physiological and pathological states.

摘要 转录因子的异位表达可以促进胚胎干细胞的分化，反式- 不同体细胞系之间的分化和体细胞重新编程为诱导 多能干细胞。这一观察结果强调了转录因子(TF)在基因中的中心作用 控制细胞表型的调控网络。转录因子的内源性活动可以戏剧性地 通过诸如TF信使RNA的选择性剪接等机制改变，其中多个功能 同一基因可以产生不同的蛋白质异构体。不同的Tf亚型可以表现出不同的 结合DNA靶标、辅因子或染色质相关蛋白的能力，导致互补或 相反的功能。例如，Foxp1产生两种不同的异构体，它们与DNA结合的特异性不同： 一种激活自我更新途径，另一种激活分化途径。在全球范围内， 异构体驱动的功能重配正在成为一种控制分化和 发展。然而，可靠地检测和发现生物相关的转铁蛋白亚型是具有挑战性的， 尤其是在蛋白质组的范围内。 为了表征在干细胞分化中起作用的转铁蛋白亚型，我的实验室将应用一种强大的组合 分析和系统生物学方法来鉴定和实验表征功能 转铁蛋白亚型的后果。该项目将涉及几个相互关联的领域，包括目标：1)发现 并量化与分化细胞状态相关的全长Tf亚型，2)寻找Tf异构体 (3)揭示了Tf亚型发挥不同功能的机制(S)。我们的方法 将使用整合了长读测序和基于MS的蛋白质组学数据的蛋白质基因组分析， 大规模异构体文库的通过量功能筛选和分子决定簇的表征 (例如，蛋白质结合、DNA结合)和构成差异转铁蛋白的活性(基因激活或抑制) 同构函数。总的来说，由此产生的数据将使发现细胞的新的一般机制成为可能。 差异化及其驱动因素。 这项研究计划将提供对转铁蛋白异构体如何在调控中发挥作用的基本理解 并将通过扩大干细胞工程应用中使用的转化因子工具包，为该领域做出贡献。 更广泛地说，这个框架提供了一种新的范式来实现异构体分辨的生物学研究，这 可以加速发现生理和病理状态下的关键异构体调节因子。