Our research program combines detailed biochemical reconstitution experiments with powerful cell-based assays, with a goal of gaining fundamental mechanistic insights about RNA polymerase II (pol II) function and its regulation. The 12-subunit human pol II enzyme transcribes all protein-coding and many non-coding RNAs in the human genome. Pol II transcription initiation is regulated by the 4.0 MDa Pre-Initiation Complex (PIC), which contains TFIIA, IIB, IID, IIE, IIF, IIH, pol II, and Mediator. Together with sequence-specific, DNA-binding transcription factors (TFs), the PIC helps direct the timing, location, and direction of pol II transcription, genome- wide. How TFs and the PIC work together during different stages of pol II transcription (e.g. initiation, pausing, elongation) remain incompletely understood; moreover, new insights over the past 5+ years have transformed our understanding of transcription. For instance, enhancer RNA (eRNA) transcription and enhancer-promoter communication appear to drive lineage- or signal-specific (or oncogenic) gene expression programs, and liquid phase separated molecular condensates correlate with pol II activity in cells. Although new mechanistic models have emerged, such models cannot be reliably tested using only cell-based methods, in part because of the enormous complexity of cellular systems. For instance, the identity and concentration of the proteins, nucleic acids, and biochemicals that are present at any given gene in a population of cells cannot possibly be defined. In the next 5 years, we propose to leverage our unique expertise in biochemical reconstitution with cutting- edge cellular methods to address the following high-impact areas: 1) Liquid phase-separated molecular condensates and pol II function. We seek to define how (or whether) molecular condensates regulate transcription, including whether distinct compositions help control different stages of pol II transcription (e.g. initiation vs. elongation). 2) Regulation of pol II initiation, pausing, and elongation by the transcriptional kinases CDK7 (TFIIH subunit), CDK8 (Mediator-associated kinase), and CDK9 (P-TEFb kinase). We will assess what each kinase, alone and in combination with the others, contributes to the regulation of pol II activity. This will include potential ?downstream? impacts on elongation rates or RNA processing. 3) Enhancer RNA (eRNA) transcription and super-enhancer function. We will dissect the mechanistic requirements for bidirectional eRNA transcription, to determine whether they are distinct from typical protein-coding genes. Furthermore, we seek to reconstitute super-enhancer function in vitro, which would serve as a framework for understanding the ?rules? by which super-enhancers drive high-level transcription in human cells. (Although this aspect is ambitious, we note our recent success with reconstitution of pol II promoter-proximal pausing, which the field long considered difficult if not impossible.) Finally, we emphasize that an equally important aspect of our research plan is to rigorously test the models that emerge from our detailed and systematic in vitro assays through targeted, follow-up cell- based assays, which will implement genome-editing, chemical biology, transcriptomics, and other approaches.
Transcription represents a means by which the human genome is decoded in each of our cells. Regulation of transcription is fundamentally important to maintain normal growth and development, whereas breakdown in transcription regulation underlies many diseases including cancer, heart disease, and neurological disorders. In this study, we seek to apply diverse and complementary approaches to uncover molecular mechanisms that drive developmental or disease-related transcriptional programs in human cells; we anticipate that the mechanistic insights gained will enable development of novel strategies to therapeutically target aberrant transcription in human cells.