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之谜是相同的DNA基因组如何在生物体中指定不同的细胞类型。转录，即在DNA模板上合成RNA，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。关键的未回答的问题是如何在整个基因组中调节转录以产生稳定的、细胞类型特异性的基因表达模式。通过将经典的生物化学方法与全球测序相结合，该项目将确定如何通过靶向负责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，该项目将解决如何将多层信息包装到同一DNA序列中并从中提取的一般问题。该项目涉及启动子附近RNA聚合酶II（Pol II）暂停的现象，它发生在许多后生动物基因的前100个核苷酸内，并受大多数（如果不是全部）Pol II-转录启动子。暂停是由负延伸因子（NELF）建立的，并且除了众所周知的生产性延伸之外，可以通过提前终止而在没有mRNA合成的情况下交替地释放。本计画将探讨人类间充质干细胞在脂肪细胞分化过程中，暂停建立与释放的因子所扮演的角色。目标1将集中在建立暂停，以确定如何NELF定义基因暂停。这个问题将通过在不同的时间尺度上扰动NELF的水平并使用我们公布的以及新的方法（包括5 '-加帽RNA测序和多重高锰酸盐足迹法）监测Pol II暂停分布和动态来解决。目的2将集中在暂停释放，以确定如何决定mRNA的生产与终止调节基因的表达，在响应模型热休克刺激。这个问题将通过将Pol II ChIP测序和全球运行分析与来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暂停作为一种突出但仍然知之甚少的现象，可能会发现一种将基因组织成网络的新原理。参与正在进行的研究的学生将做出更明智的决定是否追求科学事业，这将提高基础科学在公众特别是农村社区中的声誉。该奖项反映了NSF的法定使命，并通过使用基金会的智力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.