CAREER: Exploring the Role of Kin28/Cdk7 in Triggering Sequential Histone and Polymerase Modifications

在过去的几十年里，转录调控领域出现了两大范式。组蛋白密码假说认为，组蛋白修饰的不同组合定义了一个密码，该密码被不同组蛋白结合蛋白组破译。这些蛋白的顺序结合导致组蛋白的进一步修饰，这些事件在基因转录调控中起关键作用。同样，基于RNA聚合酶II c端结构域（CTD）的序列修饰，提出了CTD编码假说。这一假设将CTD修饰与不同蛋白复合物的关联联系起来，这些蛋白复合物参与启动子逃逸、转录延伸、mrna前体加工和转录终止。两个编码之间的功能性串扰可能是由Kin28介导的，Kin28是一种细胞周期蛋白依赖性激酶，可使CTD磷酸化。在这个项目中，化学基因组学方法将用于研究Kin28 （Cdk7在后生动物中的作用）。高特异性小分子对Kin28的体内抑制表明，该激酶不是转录起始所必需的，但可能在mrna前加工和染色质重塑中发挥更深远的作用。该激酶的这些新作用将通过以下目的进行研究：1:Kin28是否启动CTD代码？2 . Kin28是否触发组蛋白序列修饰？安萨里实验室整合了化学，基因组学，生物物理学和分子遗传学方法来探索真核基因转录中的棘手问题。我们的化学抑制Kin28的结果导致了许多模型的重新评估，这些模型是基于该激酶的温度敏感等位基因的结果得出的。Kin28ts等位基因破坏多酶TFIIH复合物的稳定性，因此不能直接解决激酶在各种过程中的作用。这一认识对那些使用其他转录机制组件的温度敏感等位基因的人产生了巨大的影响。这些研究将加强对控制转录周期各个阶段的力量的理解。此外，通过与纳米工程小组以及有机合成和计算基因组学实验室合作，安萨里实验室正在开发新的工具来解决生物学中的挑战性问题。这些合作让工程师和统计学家接触到了生物学中的问题。学科之间丰富的界面向学生介绍了新的科学概念，并培养了解决科学问题的多学科方法。本科生和高中生从事实验室研究，以确保他们开发的项目提供有意义的结果，并有助于科学地理解基因表达。该项目的综合影响将是探索新的生物学问题，扩大与我们合作的学生和同事的视野，并为更广泛的科学界提供强大的新工具。

Over the past decades, two major paradigms have emerged in the field of transcription regulation. The histone code hypothesis posits that different combinations of histone modifications define a code that is deciphered by different sets of histone-binding proteins. The sequential association of these proteins leads to further modifications of histones and these events play a key role in the regulation of gene transcription. Similarly, based on sequential modifications of the C-terminal domain (CTD) of RNA polymerase II, a CTD code hypothesis was proposed. This hypothesis links CTD modifications with the association of different protein complexes, which participate in promoter escape, transcription elongation, pre-mRNA processing, and transcriptional termination. The functional crosstalk between the two codes may be mediated by Kin28, a cyclin-dependent kinase that phosphorylates the CTD. In this project, a chemical-genomics approach will be used to investigate the role of Kin28 (Cdk7 in metazoans). In vivo inhibition of Kin28 by highly specific small molecules demonstrates that the kinase is not required for transcriptional initiation but may have more profound roles in pre-mRNA processing and chromatin remodeling. These novel roles of this kinase will be investigated through pursuing the following aims: 1: Does Kin28 prime the CTD code? and 2: Does Kin28 trigger sequential histone modifications?The Ansari lab integrates chemical, genomic, biophysical, and molecular genetic approaches to probe intractable questions in eukaryotic gene transcription. Our results with chemical inhibition of Kin28 have led to re-evaluation of many models that were derived on the results with temperature sensitive alleles of this kinase. The Kin28ts alleles disrupt the stability of the multi-enzyme TFIIH complex and thus do not directly address the role of the kinase in various processes. This realization has had a tremendous impact on those who use temperature sensitive alleles of other components of the transcriptional machinery. These studies will enhance the understanding of the forces that govern various stages of the transcription cycle. Moreover, by collaborating with nanoengineering groups as well as with organic synthesis and computational genomics laboratories the Ansari lab is developing new tools to tackle challenging questions in biology. The collaborations expose engineers and statisticians to problems in biology. The rich interface between disciplines introduces students to new scientific concepts and fosters a multidisciplinary approach to scientific problems. Undergraduates and high school students are engaged in laboratory research to ensure that they develop projects that provide meaningful results and contribute to scientific understanding of gene expression. The composite impact of this project will be to explore new biological problems, expand the horizons of the students and colleagues that we work with, and provide powerful new tools to the broader scientific community.