R35GM139550

Mechanisms of RNA Polymerase II Transcription Regulation - Summary/Abstract (for R35 GM139550)

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.

The Regents Of The University Of Colorado

THE NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES (NIGMS) SUPPORTS BASIC RESEARCH THAT INCREASES OUR UNDERSTANDING OF BIOLOGICAL PROCESSES AND LAYS THE FOUNDATION FOR ADVANCES IN DISEASE DIAGNOSIS, TREATMENT, AND PREVENTION. NIGMS ALSO SUPPORTS RESEARCH IN SPECIFIC CLINICAL AREAS THAT AFFECT MULTIPLE ORGAN SYSTEMS: ANESTHESIOLOGY AND PERI-OPERATIVE PAIN, CLINICAL PHARMACOLOGY ?COMMON TO MULTIPLE DRUGS AND TREATMENTS, AND INJURY, CRITICAL ILLNESS, SEPSIS, AND WOUND HEALING.? NIGMS-FUNDED SCIENTISTS INVESTIGATE HOW LIVING SYSTEMS WORK AT A RANGE OF LEVELSFROM MOLECULES AND CELLS TO TISSUES AND ORGANSIN RESEARCH ORGANISMS, HUMANS, AND POPULATIONS. ADDITIONALLY, TO ENSURE THE VITALITY AND CONTINUED PRODUCTIVITY OF THE RESEARCH ENTERPRISE, NIGMS PROVIDES LEADERSHIP IN SUPPORTING THE TRAINING OF THE NEXT GENERATION OF SCIENTISTS, ENHANCING THE DIVERSITY OF THE SCIENTIFIC WORKFORCE, AND DEVELOPING RESEARCH CAPACITY THROUGHOUT THE COUNTRY.

93.859 - Biomedical Research and Research Training

National Institute of General Medical Sciences (NIGMS) [HHS - NIH]

Colorado United States

State-Wide

Maximizing Investigators' Research Award (R35 - Clinical Trial Optional) (PAR-19-367)

Amendment Since initial award the total obligations have increased 408% from $633,029 to $3,214,349.