Mechanisms of Transcriptional Control Revealed by Nascent Transcript Sequencing

描述(由申请人提供)：基因组特征的功能及其如何调节基因表达知之甚少。最近发展的一种方法，自然延伸转录测序(NET-SEQ)，直接监测延伸RNA聚合酶II(RNAPII)的转录活性，具有跨基因组的单核苷酸分辨率。Net-Seq在酿酒酵母中的初步应用揭示了以前未知的转录特征。我们利用酿酒酵母净序列数据与已发表的数据集的比较，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结果，包括全球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的转录。长期目标是确定人类转录和共转录过程是如何受到DNA序列、染色质修饰和转录因子的调节的。通过扩展net-seq协议，这项建议的目标是通过对酵母突变和覆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。这一建议的基本原理是，通过对转录活性的深入了解，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，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)确定酿酒酵母中关键因子如何调节转录活性；以及3)使天然延长转录序列(NET-SEQ)适用于人类细胞。在第一个目标下，对net-seq的升级将使其在酵母和其他有机体中得到更广泛的应用。在第二个目标下，通过获得55个酿酒酵母突变株的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，建立net-seq作为研究人类转录的直接和高分辨率方法，将在人类基因组的特征和转录活动之间建立关键的联系。拟议的研究具有创新性，因为它采用并扩展了Net-seq方法，这一方法在分辨率和方法的简单性上都大大超过了已有的方法。此外，Net-Seq的广泛应用极有可能发现转录调控的新方面，并揭示生物过程中控制转录的新机制。这一贡献意义重大，因为它将为该领域乃至更远的领域提供关于转录方面是如何控制的基础性理解，并提供一个可广泛用于所有类型涉及转录的机制研究的工具。归根结底，这种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.