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 rate-limiting for expression of a large number of 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. Surprisingly, these data showed that Pol II pausing is much more widespread than previously appreciated, occurring at thousands of promoters genome-wide. We and others have recently 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. Notably, this exciting new technique revealed a role for the DNA sequence within the initially transcribed region in specifying the efficiency of early elongation, providing insights into why polymerase pausing is more prominent at some genes than at others. In probing the molecular mechanisms governing Pol II stalling, the Negative ELongation Factor, or NELF complex, is of particular interest to the laboratory. NELF has been shown to establish paused Pol II at several genes to date, including the junB and HIV promoters. To globally identify targets of NELF, we have performed a microarray analysis on Drosophila cells that were depleted of NELF using RNA interference. We found that many NELF target genes are involved in stimulus-responsive pathways, with a particular enrichment in the innate immune response. To evaluate the physiological relevance of this finding, we have recently performed NELF depletion in the Drosophila fat body (the main immune responsive tissue), followed by microarray analysis of RNA levels to identify NELF target genes. This work confirms that NELF plays a key role in vivo in regulating expression of components of the innate immune system. Follow-up studies in both cells and flies revealed that NELF-mediated Pol II pausing is essential for an optimal immune response to bacterial challenge and indicated that polymerase pausing tunes the basal expression level of critical immune regulators such as the NF-kB transcription factor Relish (Rel). In addition to our work in Drosophila, we are studying the role of polymerase pausing in the mammalian inflammatory response (using primary macrophages derived from mouse), and during mammalian development (using pluripotent cells). 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.