Decoding the role of transcription factor isoforms

项目总结 这项建议的总体目标是系统地表征转录因子的作用。 通过利用高通量的实验基因组学方法获得同种异构体。转录因子(TF) 是基因表达的主要调节者，因此在各种生物过程中发挥关键作用， 包括细胞生长和分化、生物体发育和对环境刺激的反应。这个 据估计，人类基因组含有约1600个TF基因；然而，这些~1600个TF中的大多数表达为 由来自同一基因座的选择性剪接的mRNAs编码的一系列蛋白质亚型。虽然是一个 已知少数替代的转铁蛋白亚型在细胞中扮演着重要的(和独特的)功能角色， 绝大多数--数以千计的蛋白质--仍然完全没有特征，新的转铁蛋白亚型 继续被发现。因此，解码tf异构体的作用是在系统水平上理解 基因调控。在这里，我的目标是通过利用新的RNA靶向来解码Tf亚型的功能 CRISPR/Cas13d系统。Cas13d最近作为一种精确的、程序化的和高效的酶出现在 用于系统地击倒RNA--克服了现有方法所表现出的许多限制 整体扰乱异构体。我将使用Cas13d在一次单次测试中摧毁数千个Tf亚型 实验，首次在全基因组范围内将细胞表型与转铁蛋白亚型联系起来。我会用乳房 癌症作为一个模型系统，因为细胞表型，如细胞生长，在生物学上与 癌症，以及少数替代的转铁蛋白亚型已被证明在乳腺癌中发挥重要作用。 在目标1中，我将使用Cas13d建立一个针对特定异构体的敲除框架。这就做 开发一种算法，以编程方式设计高效的、特定于异构体的Cas13d指南RNA，并验证 他们在人类细胞系中进行了有针对性的单链基因敲除实验。在目标2中，我将系统地 以乳腺癌为模型系统，评估转铁蛋白亚型对细胞生长的影响。这就做 执行基于Cas13d的池化筛选，以确定有注释和未注释的Tf亚型 在乳腺癌细胞生长中具有重要的生物学作用。通过完成这项提案，我将开发出小说 可以用来超越僵化的“以基因为中心”的框架，迈向“异构体”的技术。 水平“框架，它更准确地捕捉到人类基因组编码的深层复杂性。 此外，我将阐明转铁蛋白亚型在乳腺癌中所起的作用，这将优先考虑 未来的机械学研究。在此期间，我将进一步完善我在生物信息学方面的专业知识，同时 与之相辅相成的是实验高通量功能基因组学方面的新培训。最终，我的目标是 运行我自己的独立研究小组，采用计算和实验相结合的方式 探索人类基因组的奥秘及其在发育和疾病中的作用的方法。

PROJECT SUMMARY The overall goal of this proposal is to systematically characterize the role of transcription factor isoforms by leveraging high-throughput experimental genomics approaches. Transcription factors (TFs) are master regulators of gene expression and as such play key roles in a variety of biological processes, including cell growth and differentiation, organismal development, and response to environmental stimuli. The human genome is estimated to harbor ~1600 TF genes; however, most of these ~1600 TFs are expressed as a series of protein isoforms encoded by alternatively spliced mRNAs arising from the same locus. Though a handful of alternative TF isoforms are known to play functionally important (and distinct) roles in the cell, the overwhelming majority—thousands of proteins—remain entirely uncharacterized, and new TF isoforms continue to be discovered. Thus, decoding the roles of TF isoforms is key to a systems-level understanding of gene regulation. Here, I aim to decode the functions of TF isoforms by leveraging the novel RNA-targeting CRISPR/Cas13d system. Cas13d has recently emerged as a precise, programmatic, and efficient enzyme to use for systematic knockdown of RNA—overcoming many of the limitations exhibited by existing approaches to perturb isoforms en masse. I will employ Cas13d to knock down thousands of TF isoforms in a single experiment, linking, for the first time, cellular phenotypes to TF isoforms, genome-wide. I will use breast cancer as a model system, as cellular phenotypes such as cell growth are highly biologically relevant to cancer, and a handful of alternative TF isoforms have been shown to play important roles in breast cancer. In Aim 1, I will establish a framework for isoform-specific knockdowns using Cas13d. I will develop an algorithm to programmatically design efficient, isoform-specific Cas13d guide RNAs, and validate them using targeted, singleplex knockdown experiments in human cell lines. In Aim 2, I will systematically assess the effects of TF isoforms on cellular growth, using breast cancer as a model system. I will perform a Cas13d-based pooled screen to identify TF isoforms—both annotated and unannotated—that play biologically important roles in breast cancer cell growth. By completing this proposal, I will develop novel technologies that can be employed to move beyond a rigid “gene-centric” framework and towards an “isoform- level” framework, which more accurately captures the deep complexity encoded in the human genome. Moreover, I will shed light on the role that TF isoforms play in breast cancer, which will prioritize candidates for future mechanistic studies. During this Fellowship, I will further refine my expertise in bioinformatics while complementing it with new training in experimental, high-throughput functional genomics. Ultimately, I aim to run my own independent research group that employs a combination of computational and experimental approaches to probe the mysteries of the human genome and their roles in development and disease.