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

摘要 干细胞是未特化的细胞，具有分化为体内任何细胞的潜力。在 具有里程碑意义的实验在山中实验室，发现添加了四个关键的转录因子(TF)- Oct4、SOX2、c-myc和KLF4-能将分化的体细胞重新编程为诱导多能性 干细胞，证明了转铁蛋白活性在控制细胞命运中的巨大作用。一种广泛存在的机制 可调节的转铁蛋白活性是选择性剪接，因为几乎每个转铁蛋白基因都表达多个剪接。 异构体。来自同一基因的转铁蛋白亚型可以有不同的能力结合DNA靶标、辅因子或 染色质相关蛋白，导致基因调控的互补或相反的后果 网络。总体而言，替代Tf异构体的使用正在成为控制Tf异构体的主要调控策略 分化和发育；然而，转录因子在分化中精确的异构体特异性功能是 人们对此知之甚少。 母基金的目标是提供对剪接机制的基本理解 调节干细胞中转录因子的功能。在谢克曼实验室，我们正在开发一套 实现异构体特异性检测、过度表达和相互作用组图的方法。 父母赠款的核心目标是利用外源过表达的转铁蛋白亚型来询问它们的 功能，以测试单个转铁蛋白异构体诱导分化结果的能力是否充分。一个 独立但互补的方法将是测试Tf异构体是否有效 分化结果，通过减弱分化前和分化过程中TF亚型的表达。一组 已有用于功能研究的基因敲除工具，包括RNAi和CRISPR Cas9。然而，为了 精确地降低特定亚型的表达是一个更具挑战性的目标。这是因为 在表达中，只有一小部分异构体包含序列特异性的敲除 区域可以被靶向吗啡、RNAi或CRISPR结合，并且由于效果不同，可能不会产生结果 在强有力的击倒或过度表达的结果中。 在过去的一年里，基于Cas13的异构体特异转录基因敲除技术显示出了希望。 利用这些发展，以及在候选人的职业生涯中产生影响的机会 目标，我们建议申请者开发抑制特定表达的能力的方法学。 Tf亚型。该项目的目标是测试和应用新出现的基于CRISPR Cas13的技术 在干细胞模型内，用于实验测试转铁蛋白亚型的作用，具有多重化的潜力 对数千种异构体进行集体检测的筛选能力。该项目将提供一个工具包(异构体特定 Knokdown)补充了父母赠款中的过度表达筛选，并大大扩展了空间 以及可根据其在干细胞分化中的作用来询问的转铁蛋白亚型的规模。

SUMMARY Stem cells are unspecialized cells harboring the potential for differentiation into any cell within the body. In the landmark experiment in the Yamanaka lab, it was revealed the addition of four key transcription factors (TFs)— OCT4, SOX2, c-MYC and KLF4—can reprogram differentiated somatic cells back into induced pluripotent stem cells, demonstrating the outsize role of TF activities in controlling cell fate. A widespread mechanism by which TF activities can be modulated is alternative splicing, as nearly every TF gene expresses multiple splice isoforms. TF isoforms from the same gene can have different abilities to bind DNA targets, cofactors, or chromatin-associated proteins, resulting in complementary or opposing consequences to the gene regulatory network. Overall, alternative TF isoform usage is emerging as a major regulatory strategy in the control of differentiation and development; however, the precise isoform-specific functions of TFs in differentiation is poorly understood. The goal of the parent grant is to provide a fundamental understanding of mechanisms by which splicing modulates the function of TFs in stem cells. In the Sheynkman laboratory, we are developing a suite of approaches that enable isoform-specific detection, overexpression, and interactome mapping. Central to the parent grant's goals is to utilize exogenous overexpression of TF isoforms to interrogate their functions, to test for sufficiency of individual TF isoforms in their ability to induce differentiation outcomes. An independent, yet complementary, approach would be to test for the necessity of TF isoforms for effective differentiation outcomes, by attenuating expression of TF isoforms before and during differentiation. An array of tools exist for knockdown of genes for functional study, including RNAi and CRISPR Cas9. However, to precisely target for reduced expression of a particular isoforms is far more challenging. This is because for isoform-specific knockdown in expression, only a small region of the isoform containing a sequence-specific region can be targeted for morpholino, RNAi, or CRISPR binding, and due to variable efficacy, may not result in a robust knockdown or overexpression outcome. In the last year, Cas13-based technologies for isoform-specific transcript knockdown has shown promise. Leveraging these developments, along with the opportunity for making an impact in the candidate's career goals, we propose that the applicant develop methodology for the ability to attenuate expression of particular TF isoforms. The goal of this project is to test and apply newly emerging CRISPR Cas13-based technology within a stem cell model for experimentally testing the effect of TF isoforms, with the potential for multiplexed screening capability to test thousands of isoforms en masse. This project will provide a toolkit (isoform-specific knockdown) complementary to the overexpression screens in the parent grant, and greatly expand the space and scale of TF isoforms that can be interrogated in terms of their role in stem cell differentiation.