Investigating Transcriptional Responses to the Environment

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Whereas traditional models for gene regulation posit that recruitment of Pol II to the promoter is both necessary and sufficient for gene expression, we have recently found that release of stalled Pol II from the promoter-proximal region is rate-limiting at a large number of genes. Our work employed a combination of global location analysis (using technique called ChIP-chip and ChIP-seq) as well as in vivo footprinting assays to probe the prevalence of stalled Pol II in Drosophila. Surprisingly, these data show that Pol II stalling is much more widespread than previously appreciated, occurring at thousands of promoters genome-wide. Moreover, these results reveal that Pol II is pre-loaded in the uninduced state at many genes that respond to environmental or developmental stimuli, suggesting that the presence of Pol II, poised for escape into the gene, facilitates efficient, integrated responses to a changing environment. Understanding the fundamental properties of stalled Pol II, and the mechanisms for 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 the stress-response, this work is anticipated to elucidate gene expression during the development of cancer and AIDS, since similarly stalled Pol II are observed at the mammalian promoters of c-myc, c-fos, junB and the HIV promoter. 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 stalled Pol II at several genes to date, including the junB and HIV promoters, as well as at Drosophila promoters know to harbor stalled Pol II. 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 regulating expression of components of the innate immune system in vivo. Follow-up studies in cells revealed that NELF-mediated Pol II stalling is essential for an optimal immune response and indicated that polymerase stalling is necessary for either the binding or activity of the NF-kB transcription factor Relish (Rel). this result is consistent with our earlier suggestion that Pol II stalling helps establish a nucleosome-deprived, accessible chromatin architecture around gene promoters, facilitating future activation. Further experiments are underway in NELF-depleted flies to assess the importance of NELF in allowing the organism to recover from septic injury. In addition to our work in Drosophila, we have begun to study the role of polymerase stalling in the mammalian immune response, using primary macrophages derived from mouse. These ongoing investigations reveal that many immediate early response genes, like TNF-alpha, TTP, junB and tnfaip3, possess stalled Pol II and NELF in their promoter regions in resting, uninduced cells. In contrast, late primary and secondary response genes generally lack stalled Pol II and NELF prior to induction. We are pursuing the relationship between the presence of stalled Pol II and the kinetics of the immune response, as well as evaluating the role of NELF in this process.