Decoding the role of transcription factor isoforms

解码转录因子亚型的作用

• 批准号: 10651610
• 负责人: Kaia Mattioli
• 金额: $ 3.86万
• 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/10651610
• 关键词: Affect; Algorithms; Alternative Splicing; Binding; Bioinformatics; Biological Models; Biological Process; Biology; Breast Cancer Cell; CRISPR screen; Cancer Cell Growth; Cell Differentiation process; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complement; DNA; Development; 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; 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; 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 该提案的总体目标是系统地表征转录因子的作用 通过利用高通量实验基因组学方法的同工型。转录因子（TFS） 是基因表达的主要调节剂，并且此类在多种生物过程中起关键作用， 包括细胞生长和分化，有机发育以及对环境刺激的反应。 据估计，人类基因组具有〜1600 TF基因；但是，大多数〜1600 TF表示为 由同一基因座引起的替代剪接的mRNA编码的一系列蛋白质同工型。虽然 众所周知，少数替代TF同工型在细胞中扮演着功能重要（且不同的）角色， 绝大多数（成千上万种蛋白质）完全没有特征，新的TF同工型 继续被发现。那，解码TF同工型的角色是系统级别理解的关键 基因调节。在这里，我的目标是通过利用新型RNA靶向来解码TF同工型的功能 CRISPR/CAS13D系统。 CAS13D最近成为一种精确，程序化和有效的酶 用于系统敲低RNA的使用 - 包括现有方法暴露的许多限制 大批扰动同工型。我将使用CAS13D在一个单一中击倒数千种TF同工型 实验，首次将细胞表型与TF同工型（全基因组）联系起来。我会用乳房 癌症作为模型系统，作为细胞表型，例如细胞生长在生物学上与 癌症和少数替代TF同工型已显示出在乳腺癌中起重要作用。 在AIM 1中，我将使用CAS13D建立一个针对同工型特异性敲低的框架。我会 开发一种算法，以编程设计有效，同工型特异性CAS13D指南RNA，并验证 他们使用人类细胞系中的靶向单重敲低实验。在AIM 2中，我会系统地 使用乳腺癌作为模型系统，评估TF同工型对细胞生长的影响。我会 执行基于CAS13D的合并屏幕，以识别播放的TF同工型（包括注释和未被宣布） 生物学上重要的作用在乳腺癌细胞生长中。通过完成此建议，我将开发小说 可以雇用以超越刚性“以基因为中心”的框架而朝着“同工型 - 水平”框架，更准确地捕获了人类基因组中编码的深层复杂性。 此外，我将阐明TF同工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。在此奖学金期间，我将进一步完善我的生物信息学专业知识 通过实验，高通量功能基因组学中的新培训来补充它。最终，我的目标是 运行我自己的独立研究小组，该研究小组采用了计算和实验的组合 探究人类基因组及其在发育和疾病中的作用的方法。