The genomes of higher organisms are highly regulated and their expression can respond to a variety of developmental, environmental, and nutritional cues. The failure to execute proper gene regulation can lead to developmental defects and disease states. A broad 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. Studies of Drosophila heat shock (HS) genes, and more recently mammalian genes, have revealed that regulation by transcription factors (TFs) in vivo can occur at a point after Pol II has initiated transcription and has elongated to sites 20 to 60 bases downstream where it pauses. This proximal-promoter pausing in early transcriptional elongation is a rate-limiting and often regulated step in transcription. In contrast, traditional models of transcription regulation, based mainly in studies of microbes, indicated that transcription regulation occurs primarily at the recruitment of Pol II to promoters. Understanding the regulatory contributions and molecular mechanisms of TFs at three critical steps; 1) Pol II recruitment, 2) promoter-proximal pausing, and 3) the regulated release from the paused Pol II to productive elongation, are foci of this renewal. A battery of complementary approaches (some novel) are designed to disrupt (as rapidly as possible) critical TFs involved in transcription regulation, while high spatial and temporal resolution assays will allow Pol II and TFs to be observed across the genome both before and during a time-course of gene activation. These observational assays include highly-sensitive PRO-seq assay for mapping the position and amount of engaged Pol II across genes and genomes, and ChIP-nexus for mapping the position of specific transcription factors genome-wide. Both provide near base-pair resolution, which is critical in evaluating regulatory mechanisms. We will use three experimental systems Drosophila; mammalian cell lines, and S. pombe, and each has a foundation of available tools and existing data sets that can be uniquely exploited. Together, these systems will assess the generalities of our regulatory models. They are designed to reveal in many cases the primary effects of disruption of particular TFs, catalytic activity and macromolecular interaction domains.
Aim 1 tests the mechanistic role of TFs, like GAGA factor and M1BP, and the NURF remodeler, in recruitment of Pol II to promoters and enhancers, and test candidate mammalian factors for comparable roles.
Aim 2 tests the mechanistic role of pausing factors NELF and DSIF, and general transcription factors in stabilizing Pol II pausing at both promoters and enhancers.
Aim 3 tests the role of TFs in the release of Pol II to productive elongation.
While Aims 1 -3 seek to disrupt a TF, Aim 4 directly recruits TF domains and coactivators to explore their roles, again using genome-wide high-resolution assays. The proposal's multi-targeted perturbation and genome-wide resolution assays carried out at near base-pair resolution should provide new insights to the mechanics of transcription regulation.
The failure of humans to execute properly programs of gene regulation leads to developmental defects and disease states; moreover, infectious agents and cancer can usurp these regulatory mechanisms for their benefit at the expense of the host. The broad goal of this research is to understand basic gene regulatory mechanisms at the level when genes are transcribed into RNA in both normal and disease states. Such an understanding is critical for precise diagnoses and for developing highly specific therapies.
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