Appropriate control of gene expression is essential for correct cellular function and is disrupted in many human diseases. RNA polymerase II (Pol II) is the engine of gene expression and the target of regulation for almost every facet of its function. In the nucleus of the cell, Pol II is recruited to promoters, locates transcription start sites, and enters into transcription in a chromatin context. This occurs amidst flanking nucleosomes, which are the primary unit of chromatin. It is clear that control of the earliest stages of transcription is a major aspect of gene regulation in eukaryotes from simple yeast to humans. Genomic studies have suggested that the encounter of Pol II with the first nucleosome immediately downstream of the promoter (the +1 nucleosome) is important in this regulatory process, but the results of those studies are correlations, based on averages of distributed populations. The significance of those correlations is rarely tested by direct biochemical assays. This proposal will provide those tests by adopting biochemical approaches developed in the Luse lab to directly address the functional importance of the promoter proximal nucleosome for assembly of the Pol II transcription complex and the initial transition into transcript elongation. We will also determine the consequences for the +1 nucleosome resulting from the advance of the transcriptional machinery. Our in vitro systems have the unique ability to reveal the fundamental nature of Pol II/nucleosome interactions that additional factors can modulate or regulate. We will employ both the human and yeast transcription systems. These are the best understood eukaryotic transcription systems at the biochemical level and they provide complementary strengths for our experiments. We will incorporate the full complexity of eukaryotic promoters (both containing and lacking the TATA motif) in our work, which allows us to address the real possibility that the response of Pol II to the +1 nucleosome depends on promoter architecture. We will build on the extensive earlier studies by both investigators to begin testing proposed models of RNA polymerase II-nucleosome interactions with approaches that lead to mechanistic conclusions.
The correct control of gene expression is essential for human health. A major regulatory point for gene expression occurs when the transcriptional machinery recognizes the start signals for gene expression (promoters) and begins to advance into transcript elongation. It is widely supposed that the chromatin structure near promoters is an important part of promoter-proximal gene regulation, but this idea has not been directly tested. We will provide such a test through the studies in this proposal. Understanding promoter-proximal gene regulation at a mechanistic level will in turn significantly increase our ability to understand and interpret the effects of drugs that target modifications in chromatin structure or the transcriptional machinery itself.
