Systematic analysis of context-specific functions of transcriptional CDKs in human cells

本项目研究DNA分子中储存的遗传信息在细胞中表达的过程。这一过程的第一部分被称为基因转录，涉及将DNA片段（基因）复制成RNA分子。尽管正确控制基因转录在生物学中的重要性是无可争议的，但在这一领域仍然存在一些重大的未解之谜。细胞如何调整其基因转录以应对环境的变化？在多细胞生物的发育过程中，基因转录是如何被控制的？在这些不同的情况下，细胞内调节转录的关键酶和其他蛋白质是什么？在这个框架内，研究小组将研究一组被称为“转录CDK”的酶，它们是从酵母到人类的生物体中公认的基因转录调节因子。这项研究将为转录CDK如何使细胞在不同情况下改变其转录活性提供新的见解。因此，产生的知识将推进分子生物学领域，并扩大我们对DNA内遗传信息如何表达以最终控制细胞行为的集体理解。该研究将由一个多元化的教学研究团队进行，该团队在培养从本科到博士后水平的科学家方面有着良好的记录，包括促进学术界代表性不足的少数民族，以及提高整个社会的科学素养。该项目将使用化学遗传学方法阐明转录CDK的功能特化。利用基因组编辑技术，研究人员将创建每个激酶的等位基因，这些等位基因可以被庞大的ATP底物类似物抑制。然后，这组匹配的等基因人类细胞系将用于：1）通过RNA合成、加工和稳态水平的全基因组测量来破译每个CDK对转录周期的贡献; 2）鉴定每个CDK靶向磷酸化的独特和共享蛋白质;以及c）在人类细胞中使用基于CRISPR的遗传筛选来鉴定每个激酶的遗传相互作用物。总的来说，这些努力将提供一个先进的机制如何转录CDKs调节基因转录的理解，以及进化机制，导致其在脊椎动物细胞的功能专业化。这个奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的智力价值和更广泛的影响审查标准的支持。

This project investigates the process by which the genetic information stored in the DNA molecule is expressed in cells. The first part of this process, known as gene transcription, involves the copying of segments of DNA (genes) into RNA molecules. Despite the undisputed importance of proper control of gene transcription in biology, there are still major unanswered questions in this field. How do cells adjust their gene transcription in response to changes in their environment? How is gene transcription controlled during the development of multicellular organisms? What are the key enzymes and other proteins within the cell that regulate transcription in these different contexts? Within this framework, the research team will investigate a group of enzymes known as "transcriptional CDKs" that are well recognized regulators of gene transcription in organisms from yeast to humans. This research will provide new insights into how transcriptional CDKs enable cells to alter their transcriptional activity in different contexts. Therefore, the knowledge generated will advance the field of molecular biology and expand our collective understanding of how the genetic information within DNA is expressed to ultimately control cell behavior. The research will be carried out by a diverse teaching-research team with a proven track record of training scientists from undergraduate to post-doctoral levels, including promoting underrepresented minorities in academia, and increasing scientific literacy in society at large.This project will elucidate the functional specialization of the transcriptional CDKs using a chemical genetics approach. Using genome editing technology, researchers will create alleles of each kinase that can be inhibited by bulky ATP substrate analogs. This panel of matching isogenic human cell lines will then be used to: 1) decipher the contribution of each CDK to the transcription cycle via genome-wide measurements of RNA synthesis, processing, and steady-state levels; 2) identify unique and shared proteins targeted for phosphorylation by each CDK; and c) identify genetic interactors for each kinase using CRISPR-based genetic screening in human cells. Collectively, these efforts will provide an advanced mechanistic understanding of how transcriptional CDKs regulate gene transcription, as well as the evolutionary mechanisms leading to their functional specialization in vertebrate cells.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.