PROJECT SUMMARY Transcription of protein-coding genes governs cell identity and fate through cell specific gene expression programs. During transcription, RNA polymerase II must traverse through its native chromatin template. Chromatin and its fundamental unit, the nucleosome, impose a significant hurdle to nuclear processes such as transcription. Importantly, the transcription machinery must not only traverse the genome without disrupting chromatin organization, but transcriptional activity is also epigenetically regulated through post-translational modifications of histone proteins. Often these processes are embedded in elaborate feedback loops where chromatin architecture, transcription, and epigenetics regulate each other. These regulatory mechanisms play a pivotal for gene expression control and dysregulation of gene expression often results in the emergence of cancers. The biochemical and structural foundation for the crosstalk between the transcription machinery, chromatin, and epigenetics, however, remains poorly understood, partly because of the complexity of cellular systems and limitations in experimental approaches. The goal of this proposal is to overcome our lack of understanding of gene expression regulation by using an integrated reconstitution strategy to study chromatin organization, transcription, and epigenetics in parallel. Here, I propose the development of a combinatorial biochemical and structural approach termed visual biochemistry. Visual biochemistry combines a fully reconstituted chromatin transcription system and time-resolved single-particle cryogenic electron microscopy. Our work has revealed that visual biochemistry can be successfully employed to understand how nucleosomes and epigenetic information are retained during transcription. Our proposed research will identify the structural basis of how the transcription machinery transcribes higher-order chromatin in a native-like environement (Aim 1). Coupled with a broad biochemical screen, our experiments will identify novel factors that regulate gene expression in chromatin (Aim 2). Next, we will reveal how transcription affects and is affected by epigenetic crosstalk and feedback loops (Aim 3). Our research will define the molecular rules that govern transcription through chromatin in human biology and explain fundamental mechanisms that drive differential gene expression in health and disease.

Project Narrative Regulatory processes at the intersection of chromatin, transcription, and chromatin are fundamental to cellular development and growth. The mechanistic basis of gene expression regulation, however, is not well understood. Our proposal will develop an integrated strategy to reconstitute transcription and epigenetic processes in their native chromatin environment and employ a novel cryogenic electron microscopy approach to improve our understanding of gene expression regulation in health and disease and to facilitate the development of novel therapeutics against cancers.