Decoding the role of transcription factor isoforms

解码转录因子亚型的作用

• 批准号: 10386594
• 负责人: Kaia Mattioli
• 金额: $ 7.03万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10386594
• 关键词: Affect; Algorithms; Alternative Splicing; Binding; Bioinformatics; Biological Models; Biological Process; Biology; Breast Cancer Cell; CRISPR screen; Cancer Cell Growth; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complement; DNA; Development; Differentiation and Growth; Disease; Enzymes; Exhibits; Fellowship; Foundations; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genomic approach; Genomics; Goals; Guide RNA; Human Cell Line; Human Genome; Individual; Light; Link; Malignant Neoplasms; Messenger RNA; Phenotype; Play; Property; Protein Isoforms; Proteins; RNA; Regulator Genes; Research; Role; Running; Screening Result; Series; Stimulus; System; Techniques; Time; Training; Transcript; Translating; Untranslated RNA; Work; base; cell growth; design; developmental disease; experimental study; functional genomics; genome-wide; human disease; knock-down; malignant breast neoplasm; mammalian genome; new technology; novel; response; skills; software development; transcription factor; transcriptome sequencing

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.

项目摘要 这个建议的总体目标是系统地描述转录因子的作用 通过利用高通量实验基因组学方法来研究异构体。转录因子（TF） 是基因表达的主要调节因子，因此在多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。的 据估计，人类基因组含有约1600个TF基因;然而，这约1600个TF中的大多数表达为 一系列由来自同一基因座的选择性剪接mRNA编码的蛋白质同种型。虽然 已知少数可选择的TF同种型在细胞中发挥重要的（和不同的）功能作用， 绝大多数--成千上万的蛋白质--仍然完全没有特征， 继续被发现。因此，解码TF异构体的作用是系统水平理解 基因调控在这里，我的目标是通过利用新的RNA靶向技术来解码TF亚型的功能， CRISPR/Cas 13 d系统。Cas 13 d最近已经成为一种精确、程序化和有效的酶， 用于RNA的系统敲除-克服现有方法所表现出的许多限制 干扰异构体的能力。我将使用Cas 13 d在一个单一的基因组中敲除数千种TF亚型， 实验，连接，第一次，细胞表型TF异构体，全基因组。我会用乳房 癌症作为一个模型系统，因为细胞表型，如细胞生长是高度生物学相关的， 癌症，和少数替代TF同种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Cas 13 d建立亚型特异性敲除的框架。我会 开发一种算法，以编程方式设计高效的、亚型特异性的Cas 13 d指导RNA，并验证 他们使用有针对性的，单重敲除实验在人类细胞系。在目标2中，我将系统地 评估TF亚型对细胞生长的影响，使用乳腺癌作为模型系统。我会 进行基于Cas 13 d的合并筛选，以鉴定TF亚型-注释和未注释- 在乳腺癌细胞生长中具有重要的生物学作用。通过完成这份提案，我将开发新的 可以用来超越严格的“以基因为中心”的框架，走向“同种型”的技术， 这是一个“水平”框架，它更准确地捕捉了人类基因组中编码的深层复杂性。 此外，我将阐明TF亚型在乳腺癌中的作用，这将优先考虑候选人。 未来的机械研究。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， 在实验性的、高通量的功能基因组学方面进行新的培训。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法， 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。