Investigating Transcriptional Responses to the Environment

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Our laboratory has found that release of paused Pol II from the promoter-proximal region is essential, regulated step in the expression of most metazoan genes. Our initial work investigated the prevalence of paused Pol II in Drosophila, employing a combination of global location analysis (using techniques called ChIP-chip and ChIP-seq) as well as in vivo footprinting assays. These data showed that Pol II pausing is much more widespread than previously appreciated. We and others have extended these findings to mammalian systems (mouse and human), demonstrating that pausing a prevalent gene regulatory strategy in higher organisms. Moreover, our results reveal that Pol II is constitutively present at many genes in environmentally- or developmentally-responsive gene networks, suggesting that the presence of Pol II facilitates efficient, integrated responses to a dynamically changing environment. Understanding the fundamental properties of paused Pol II, and the factors that govern maintenance vs. release of promoter-proximal Pol II into productive elongation are specific aims of research in the Adelman laboratory. In addition to providing crucial insight into stress-responses, this work is anticipated to elucidate gene expression during the development of cancer and AIDS, since similarly paused Pol II are observed at the mammalian promoters of proto-oncogenes like c-myc, c-fos and junB, as well as at the HIV promoter. As part of our efforts to better define the mechanisms underlying pausing, we have recently developed a novel technique for isolating the short RNA transcripts generated by paused Pol II, and analyzed them through massively-parallel sequencing of individual RNA molecules. This strategy allowed us to pinpoint both the locations of transcription initiation and pausing, at single-nucleotide resolution. In probing the molecular mechanisms governing Pol II pausing, the Negative ELongation Factor, or NELF complex, is of particular interest to the laboratory. Mechanistically, we have shown that NELF is broadly important for the stable pausing of Pol II. To define its physiological role, we have developed conditional knockout mice. Studies in both cells and in vivo revealed that NELF-mediated Pol II pausing is essential for an optimal immune response to bacterial challenge and and for early embryonic development. Notably, in both systems we find that disruption of pausing dramatically impacts cellular responsiveness to environmental or extrinsic cues. Dissection of this effect has revealed that pausing plays a key role in determining the expression level of critical hubs of signal transduction machineries. In this way, pausing tunes signaling responses, and the consequences of signaling on gene expression. Taken together, the data suggest that pausing of Pol II allows for coordinated tuning of both basal gene expression and activation, to enable precise, balanced responses to environmental or developmental cues.