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（TF）的异位表达可以驱动胚胎干细胞的分化， 不同体细胞谱系之间的分化，以及将体细胞重编程为诱导的 多能干细胞。这一观察结果强调了转录因子（TF）在基因表达中的核心作用。 调控网络来决定细胞表型。TF的内源性活性可以显著地 通过TF信使RNA的选择性剪接等机制改变，其中多个功能性 不同的蛋白质同种型可以由相同的基因产生。不同的TF亚型可以表现出不同的 结合DNA靶点、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产生两种在DNA结合特异性方面不同的同种型： 一个激活自我更新途径，而另一个激活分化途径。在全球范围内， 同种型驱动的功能性重新布线正在成为控制分化和 发展然而，可靠地检测和发现生物学相关的TF亚型是具有挑战性的， 特别是在蛋白质组水平上。 为了表征在干细胞分化中发挥作用的TF亚型，我的实验室将采用一种强大的组合， 的分析和系统生物学方法，以确定和实验表征的功能 TF异构体的后果。该项目将涉及几个相互关联的领域，包括以下目标：1）发现 并定量与分化细胞状态相关的全长TF同种型，2）找到TF同种型的“驱动因子”， 分化，和3）揭示TF同种型表现出不同功能的机制。我们的方法 将使用蛋白质基因组学分析，整合长读测序和基于MS的蛋白质组学数据， 大规模同种型文库的通量功能筛选，以及分子决定簇的表征 (e.g.,蛋白质结合、DNA结合）和作为差异TF基础的活性（基因激活或抑制 同种型功能总的来说，所得到的数据将使发现新的一般机制的细胞 差异化及其驱动因素。 这项研究计划将提供一个基本的了解如何TF亚型的功能，在调节 网络，并将通过扩大干细胞工程应用中使用的TF工具包为该领域做出贡献。 更广泛地说，这个框架提供了一个新的范式，使异构体分辨生物学研究， 可以加速发现生理和病理状态下的关键亚型调节剂。