Mechanisms of Transcriptional Control Revealed by Nascent Transcript Sequencing

描述（由申请人提供）：基因组特征的功能以及它们如何调节基因表达的了解甚少。最近开发的一种方法，天然延伸转录测序（NET-seq），直接监测延伸RNA聚合酶II（RNAPII）的转录活性与整个基因组的单核苷酸分辨率。NET-seq在S. cerevisiae揭示了以前未知的转录功能。我们利用S. cerevisiae NET-seq数据与已发布的数据集。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，迫切需要调整NET-seq方法来观察人类细胞中的转录。长期目标是确定人类转录和共转录过程如何受DNA序列、染色质修饰和转录因子的调控。通过扩展NET-seq协议，该提案的目标是通过酵母突变体和一系列涵盖人类生物学不同领域的人类细胞系的NET-seq分析，形成对转录控制的基本理解。这一建议的基本原理是，通过对转录活性的深入理解，将有可能剖析基因组特征和细胞因子控制基因表达活性的机制。为了实现这些目标，已经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S.酿酒酵母;和3）使天然延伸转录物测序（NET-seq）适用于人细胞。在第一个目标下，NET-seq的升级将使其在酵母和其他生物中得到更广泛的应用。在第二个目标下，通过获得55个S.酿酒酵母突变体，转录控制机制的基本见解将被揭示，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，建立NET-seq作为一种直接和高分辨率的人类转录研究方法，将在人类基因组特征和转录活性之间建立关键联系。拟议的研究是创新的，因为它采用并扩展了NET-seq，这是一种在分辨率和方法简单性方面都大大超过现有方法的方法。此外，NET-seq的广泛应用极有可能揭示转录调控的新方面，并揭示控制生物过程中转录的新机制。这一贡献是重要的，因为它将提供该领域，以及超越，与转录的各个方面是如何控制的基本理解，并提供了一个工具，可以广泛用于所有类型的机制研究涉及转录。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。

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.