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

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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.