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Principal Investigator: Dylan J Taatjes
Organization: UNIVERSITY OF COLORADO
Fiscal Year: 2024
Award: $633,029
Funding agency: National Institute of General Medical Sciences
SUMMARY/ABSTRACT
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.
Terms: <Active Follow-up><Address><Area><Assay><BTF2 transcription factor><Basal Transcription Factor><Basal transcription factor genes><Bioassay><Biochemical><Biological Assay><Biology><Cancers><Cardiac Diseases><Cardiac Disorders><Cell Body><Cell Communication and Signaling><Cell Signaling><Cells><Chemicals><Code><Coding System><Communication><Complex><DNA Binding><DNA Binding Interaction><DNA Polymerase II><DNA Polymerase epsilon><DNA bound><DNA-Dependent DNA Polymerase II><DNA-Dependent RNA Polymerase II><Development><Disease><Disorder><Enhancers><Enzyme Gene><Enzymes><Functional RNA><Gene Expression><Gene Transcription><General Transcription Factor Gene><General Transcription Factor IIA><General Transcription Factors><Genes><Genetic Transcription><Goals><Growth and Development><Growth and Development function><Heart Diseases><Human><Human Genome><In Vitro><Intracellular Communication and Signaling><Kinases><Liquid substance><Location><Malignant Neoplasms><Malignant Tumor><Mediator><Methods><Modeling><Modern Man><Molecular><Nervous System Diseases><Nervous System Disorder><Neurologic Disorders><Neurological Disorders><Non-Coding><Non-Coding RNA><Non-Polyadenylated RNA><Non-translated RNA><Noncoding RNA><Nontranslated RNA><Nucleic Acids><Oncogenic><P-TEFb><Phase><Phosphotransferase Gene><Phosphotransferases><Physical condensation><Pol II><Polymerase><Population><Positive Transcription Elongation Factor B><Positive Transcriptional Elongation Factor B><Proteins><RNA><RNA Expression><RNA Gene Products><RNA Polymerase B><RNA Polymerase II><RNA Processing><Regulation><Research><Ribonucleic Acid><Signal Transduction><Signal Transduction Systems><Signaling><System><TFIIA><TFIIH><Testing><Transcription><Transcription Elongation><Transcription Factor A RNA Polymerase II><Transcription Factor IIA><Transcription Factor Proto-Oncogene><Transcription Factor TFIIA><Transcription Initiation><Transcription Initiation Factor IIA><Transcription Regulation><Transcription factor genes><Transcriptional Control><Transcriptional Regulation><Transphosphorylases><Untranslated RNA><Work><active followup><basic transcription factor 2><biological signal transduction><condensation><developmental><experiment><experimental research><experimental study><experiments><fluid><follow up><follow-up><followed up><followup><genome editing><genome scale><genome-wide><genomewide><genomic editing><heart disorder><human whole genome><in vitro Assay><insight><liquid><malignancy><neoplasm/cancer><neurological disease><new approaches><noncoding><novel approaches><novel strategies><novel strategy><programs><promoter><promotor><public health relevance><reconstitute><reconstitution><success><tat-Associated Kinase><therapeutic target><transcription factor><transcription factor IIH><transcription factor TFIIH><transcriptomics>