CAREER: Organization of global transcriptomes by stepwise control of Pol II activity at gene promoters

一个长期存在的生物学谜团是相同的DNA基因组如何在生物体中指定不同的细胞类型。转录，即RNA在DNA模板上的合成，是基因表达的第一步，它在很大程度上决定了哪些基因将被激活。关键的未解决的问题是转录是如何在整个基因组中被调节，以产生稳定的、细胞类型特异性的基因表达模式。通过将经典生化方法与全局测序相结合，该项目将确定如何通过针对负责RNA合成的酶的通用机制来稳定特定的转录模式。通过研究转录过程，该项目将解决一个普遍的问题，即多层信息如何被打包到同一个DNA序列中，并从同一个DNA序列中提取出来。该项目研究启动子-近端RNA聚合酶II （Pol II）暂停现象，该现象发生在许多后生动物基因的前100个核苷酸中，由大多数（如果不是全部的话）Pol II转录启动子所涉及的因素控制。暂停是由负伸长因子（NELF）建立的，除了众所周知的生产性伸长，可以通过过早终止在没有mRNA合成的情况下交替释放。本项目将研究在脂肪细胞分化的人间充质干细胞中暂停建立和释放的因素所起的作用。目的1将侧重于建立暂停，以确定NELF如何定义暂停的基因。这个问题将通过在不同的时间尺度上干扰NELF的水平和监测Pol II的暂停分布和动态来解决，这些方法包括5'帽RNA测序和多重高锰酸盐足迹。目的2将关注暂停释放，以确定mRNA产生与终止之间的决定如何调节基因在模型热休克刺激下的表达。该问题将通过Pol II chip测序和全球运行分析与源自暂停复合物的终止特异性小rna的新分析相结合来解决。通过探索Pol II暂停作为一个突出但仍知之甚少的现象，可能会发现一个将基因组织成网络的新原理。从事正在进行的研究的学生将在是否从事科学事业方面做出更明智的决定，这将提高基础科学在公众中的声誉，特别是在农村社区。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

A long-standing biological mystery is how the same DNA genome can specify distinct cell types in an organism. Transcription, the synthesis of RNA on the DNA template, is the first step in gene expression that largely defines which genes will be active. The key unanswered question is how transcription is regulated across the genome to generate stable, cell-type-specific patterns of gene expression. By combining classic biochemical approaches with global sequencing, this project will determine how specific transcription patterns may be stabilized by universal mechanisms targeting the enzymes responsible for RNA synthesis. By studying the process of transcription, the project will tackle a general problem of how multiple layers of information could be packed into, and extracted from, the same DNA sequence.This project deals with the phenomenon of promoter-proximal RNA polymerase II (Pol II) pausing, which occurs within the first 100 nucleotides of many metazoan genes and is controlled by factors involved in most if not all Pol II-transcribed promoters. Pausing is established by the Negative ELongation Factor (NELF) and, apart from the well-known productive elongation, can be alternatively released without mRNA synthesis via premature termination. This project will investigate the roles of factors responsible for pause establishment and release in human mesenchymal stem cells undergoing adipocyte differentiation. Objective 1 will focus on establishing pausing to determine how NELF defines genes to be paused. The question will be addressed by perturbing the levels of NELF at various time scales and monitoring Pol II pausing distribution and dynamics using our published as well as new methods including 5'-capped RNA- sequencing and multiplex permanganate footprinting. Objective 2 will focus on pause release to determine how the decision between mRNA production versus termination regulates expression of genes in response to a model heat shock stimulus. The question will be addressed by combining Pol II ChIP-sequencing and global run-on assays with novel analysis of termination-specific small RNAs derived from paused complexes. By exploring Pol II pausing as a prominent yet still poorly understood phenomenon, a new principle organizing genes into networks may be uncovered. Students engaged into ongoing research will make more informed decisions on whether or not to pursue careers in science, which will elevate the reputation of basic science among general public especially in rural communities.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.