The genomes of higher organisms and their expression are highly regulated in response to a variety of developmental, environmental, and nutritional cues. The failure to execute proper gene regulation can lead to developmental defects and disease states. The overarching goal of this research is to understand basic regulatory mechanisms of genes at the level of transcription, the stage where RNA polymerase II (Pol II) transcribes the genes into mRNA. This regulation is conducted by the concerted effort of DNA elements (promoters and enhancers) and many protein machineries such as Pol II, TFs, and chromatin/histone modifying complexes. Recent studies in Drosophila and mammalian cells have revealed that transcription is primarily regulated at two rate-limiting steps; 1) Recruitment of Pol II to promoter and Pol II pausing at 20-50bp downstream of transcription initiation site, and 2) Release of paused Pol II to productive elongation. A battery of complementary approaches (some novel) are designed to identify human TFs and chromatin remodelers that act at these steps and then rigorously address the mechanism by which they exert transcriptional regulation. by TFs and chromatin modifiers with the highest possible spatiotemporal resolution genome-wide in distinct cell lines, including K562, HCT116, HEK293, and undifferentiated and differentiated WTC-11. The wealth of existing information in these cell lines will be complemented with Run-On assays (RO-seq), and others when necessary, to identify TFs that act primarily on one or both of the rate-limiting regulated steps in transcription. These observational assays include multiple highly-sensitive RO-seq assays for mapping the position and amount of transcription across genes and enhancers genome-wide, ChIP-exo for detecting TF binding and occupancy, STARR-seq assays to measure transcription regulatory activity of enhancers and promoters, and a degron system to rapidly degrade TFs to assess their primary effects by these ?-seq? assays. Together, these systems will test our regulatory models and assess their generality.
In Aim 1, using a novel aptamer-based pulldown and mass spectrometry approach, we will identify pioneering TFs that work through remodeling complexes such as PBAP (SWI/SNF) to open chromatin for subsequent TF and Pol II recruitment.
In Aim 2, we seek to identify cell- type-specific TFs that act at major regulated steps of transcription, Steps 1, 2, or both. This will be achieved by a comparative analysis of cell line specific nascent transcription profiles.
Aim 3 will test the role of TF candidates, which are associated with one or both of the regulatory steps in the transcription cycle, in living cells using a Degron approach and multiple ?-seq? assays.
In Aim 4, we will assess the roles of TFs in the interplay of enhancers and promoters interactions using high-throughput episomal and chromosomal enhancer assays. The proposal will identify TFs that act at specific steps in transcription regulation and provide insights to the mechanics of their action.

Public Health Relevance

The proper development of a human from a single cell embryo and the maintenance of the well-being of a human through encounters with dramatic changes in nutrition and environment require that our 20 thousand genes be exquisitely regulated. This regulation depends on both promoters, which reside close to the genes they regulate, and the interplay of these promoters with enhancers, which can reside up to a million base pairs from the gene promoter, making it nearly impossible to predict the target genes and the effects on the regulated genes. In this study, we propose to characterize regulatory effects of such enhancer regions, to dissect out their molecular building blocks, and to identify enhancer target genes; all of which will lead to a better mechanistic and functional understanding of enhancers and ultimately enable novel therapeutic venues for diseases like cancer where gene regulation is dysfunctional.

National Institute of Health (NIH)
National Human Genome Research Institute (NHGRI)
Research Project with Complex Structure Cooperative Agreement (UM1)
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Gilchrist, Daniel A
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Cornell University
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United States
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Chen, Siwei; Fragoza, Robert; Klei, Lambertus et al. (2018) An interactome perturbation framework prioritizes damaging missense mutations for developmental disorders. Nat Genet 50:1032-1040
Meyer, Michael J; Beltrán, Juan Felipe; Liang, Siqi et al. (2018) Interactome INSIDER: a structural interactome browser for genomic studies. Nat Methods 15:107-114
Tippens, Nathaniel D; Vihervaara, Anniina; Lis, John T (2018) Enhancer transcription: what, where, when, and why? Genes Dev 32:1-3
Tome, Jacob M; Tippens, Nathaniel D; Lis, John T (2018) Single-molecule nascent RNA sequencing identifies regulatory domain architecture at promoters and enhancers. Nat Genet 50:1533-1541