Mechanisms of Transcriptional Control Revealed by Nascent Transcript Sequencing

新生转录本测序揭示的转录控制机制

• 批准号: 9052194
• 负责人: Lee Stirling Churchman
• 金额: $ 51.97万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9052194
• 关键词: Address; Affect; Affinity; Animal Model; Area; Biological Process; Cell Line; Cell physiology; Cells; Chromatin Structure; Complex; DNA Sequence; Data; Data Set; Defect; Development; Diagnosis; Disease; Event; Foundations; Future; Gene Expression; Genes; Genetic Transcription; Genome; Genomic approach; Genomics; Goals; Health; Heart; Human; Human Biology; Human Cell Line; Human Genome; Investigation; Knowledge; Learning; Maps; Messenger RNA; Methodology; Methods; Mission; Modeling; Molecular; Monitor; Nucleotides; Organism; Pattern; Physiological; Process; Protocols documentation; Public Health; Publishing; RNA Polymerase II; Regulation; Research; Resolution; Role; Saccharomyces cerevisiae; Sampling; Sequence Analysis; Site; Testing; Transcript; Transcription Elongation; Transcription Process; Transcriptional Regulation; Work; Yeasts; cell growth; cell type; chromatin modification; histone modification; human disease; improved; innovation; insight; mutant; novel; novel strategies; promoter; research study; tool; transcription factor; yeast genetics

DESCRIPTION (provided by applicant): The functions of genomic features and how they regulate gene expression are poorly understood. A recently developed approach, native elongating transcript sequencing (NET-seq), directly monitors transcriptional activity of elongating RNA polymerase II (RNAPII) with single-nucleotide resolution across a genome. Initial application of NET-seq to S. cerevisiae revealed previously unknown features of transcription. We created and tested models of how transcription is influenced by genomic features utilizing comparison of S. cerevisiae NET-seq data with published datasets. To determine the transcriptional consequences of features of the human genome, including the molecular mechanisms of global transcription regulation, there is a critical need to adapt the NET-seq approach to observe transcription in human cells. The long-term goal is to determine how human transcription and co-transcriptional processes are regulated by DNA sequence, chromatin modifications, and transcription factors. Through expanding the NET-seq protocol, the objective of this proposal is to form a fundamental understanding of transcriptional control through NET-seq analysis of yeast mutants and a range of human cell lines covering distinct areas of human biology. The rationale of this proposal is that through a deep understanding of transcriptional activity, it will be possible to dissect the mechanisms by which genomic features and cellular factors control gene expression activity. The following three specific aims have been formulated to accomplish these objectives: 1) Streamline, optimize, and expand the NET-seq approach; 2) determine how transcriptional activity is modulated by key factors in S. cerevisiae; and 3) adapt native elongating transcript sequencing (NET-seq) for human cells. Under the first aim, upgrades to NET-seq will allow it to be used more broadly, both in yeast and in other organisms. Under the second aim, by obtaining NET-seq profiles for a comprehensive set of 55 S. cerevisiae mutants, fundamental insight into mechanisms of transcriptional control will be revealed, which will help to guide future NET-seq experiments in human cells. Under the third aim, establishing NET-seq as a straightforward and high-resolution approach for the study of human transcription will make critical connections between features of the human genome and transcriptional activity. The proposed research is innovative, because it employs and expands NET-seq, a methodology that substantially surpasses the established methodologies in both resolution and in simplicity of the approach. Furthermore, broad application of NET-seq is highly likely to uncover novel aspects of transcription regulation and reveal new mechanisms that control transcription in biological processes. This contribution is significant because it wil supply the field, and beyond, with a foundational understanding of how aspects of transcription are controlled and provide a tool that can be broadly used in all types of mechanistic studies involving transcription. Ultimately, such knowledge has the potential to impact all human diseases as transcription regulation is a critical component of most human biological processes.

描述（由申请人提供）：对基因组特征的功能以及它们如何调节基因表达知之甚少。最近开发的方法是天然延伸转录本测序 (NET-seq)，它可以在整个基因组中以单核苷酸分辨率直接监测延伸 RNA 聚合酶 II (RNAPII) 的转录活性。 NET-seq 在酿酒酵母中的首次应用揭示了以前未知的转录特征。我们利用酿酒酵母 NET-seq 数据与已发布的数据集进行比较，创建并测试了基因组特征如何影响转录的模型。为了确定人类基因组特征的转录后果，包括全局转录调控的分子机制，迫切需要采用 NET-seq 方法来观察人类细胞中的转录。长期目标是确定 DNA 序列、染色质修饰和转录因子如何调控人类转录和共转录​​过程。通过扩展 NET-seq 协议，该提案的目标是通过对酵母突变体和涵盖人类生物学不同领域的一系列人类细胞系的 NET-seq 分析，形成对转录控制的基本理解。该提案的基本原理是，通过深入了解转录活性，将有可能剖析基因组特征和细胞因素控制基因表达活性的机制。为了实现这些目标，制定了以下三个具体目标：1）简化、优化和扩展 NET-seq 方法； 2) 确定酿酒酵母中关键因子如何调节转录活性； 3）针对人类细胞采用天然延伸转录本测序（NET-seq）。根据第一个目标，NET-seq 的升级将使其能够在酵母和其他生物体中得到更广泛的使用。第二个目标是，通过获得 55 个酿酒酵母突变体的 NET-seq 图谱，将揭示转录控制机制的基本见解，这将有助于指导未来在人类细胞中的 NET-seq 实验。在第三个目标下，将 NET-seq 建立为人类转录研究的简单且高分辨率的方法，将在人类基因组特征和转录活动之间建立关键联系。拟议的研究具有创新性，因为它采用并扩展了 NET-seq，这种方法在分辨率和方法简单性方面都大大超越了现有的方法。此外，NET-seq 的广泛应用很可能揭示转录调控的新方面，并揭示生物过程中控制转录的新机制。这一贡献意义重大，因为它将为该领域及其他领域提供对转录各方面如何控制的基础理解，并提供可广泛用于涉及转录的所有类型的机制研究的工具。最终，这些知识有可能影响所有人类疾病，因为转录调控是大多数人类生物过程的关键组成部分。