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 genome-wide location analysis (using a technique called ChIP-chip) 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 nearly 20% of promoters. 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 several Drosophila promoters know to harbor stalled Pol II. To get a comprehensive picture of NELF targets in vivo, we performed a microarray analysis on Drosophila cells that had been 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. Surprisingly, we found that NELF does not only function as a negative elongation factor, but it also stimulates expression of a number of genes, including those activated by immune challenge. Further analysis has revealed that NELF-mediated stalling of Pol II in the promoter-proximal region of these genes enhances gene expression by blocking the assembly of nucleosomes in the promoter region. Thus, by functioning as a nucleosome exclusion factor, stalled Pol II marks these genes for further or future activation. Our goal in the upcoming year is to probe the dynamics of the interactions between Pol II and nucleosomes, in order to better understand how Pol II dictates nucleosome positioning and prevents the formation of repressive chromatin structure at these immune-responsive genes.? In addition to investigating the effects of Pol II stalling on basal levels of gene expression and chromatin architecture at immunity genes, we are currently using both cell-based and whole organism approaches to investigate the role of Pol II stalling during induction of the Drosophila innate immune response upon septic challenge. We have developed ways in which to achieve tissue-specific depletion of the NELF protein in the Drosophila immune-responsive organ, the fat body, and are actively pursuing the effects of this depletion on the production of anti-microbial peptides and other innate immunity genes. We hope to determine the functional, physiological role of Pol II stalling during the immune response, and to asses its importance in allowing the organism to recover from septic injury.

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National Institute of Environmental Health Sciences (NIEHS)
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Pai, Athma A; Paggi, Joseph M; Yan, Paul et al. (2018) Numerous recursive sites contribute to accuracy of splicing in long introns in flies. PLoS Genet 14:e1007588
Henriques, Telmo; Scruggs, Benjamin S; Inouye, Michiko O et al. (2018) Widespread transcriptional pausing and elongation control at enhancers. Genes Dev 32:26-41
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Pai, Athma A; Henriques, Telmo; McCue, Kayla et al. (2017) The kinetics of pre-mRNA splicing in the Drosophila genome and the influence of gene architecture. Elife 6:
Scruggs, Benjamin S; Gilchrist, Daniel A; Nechaev, Sergei et al. (2015) Bidirectional Transcription Arises from Two Distinct Hubs of Transcription Factor Binding and Active Chromatin. Mol Cell 58:1101-12
Williams, Lucy H; Fromm, George; Gokey, Nolan G et al. (2015) Pausing of RNA polymerase II regulates mammalian developmental potential through control of signaling networks. Mol Cell 58:311-322
Rogatsky, Inez; Adelman, Karen (2014) Preparing the first responders: building the inflammatory transcriptome from the ground up. Mol Cell 54:245-54
Gupte, Rebecca; Muse, Ginger W; Chinenov, Yurii et al. (2013) Glucocorticoid receptor represses proinflammatory genes at distinct steps of the transcription cycle. Proc Natl Acad Sci U S A 110:14616-21
Schröder, Sebastian; Herker, Eva; Itzen, Friederike et al. (2013) Acetylation of RNA polymerase II regulates growth-factor-induced gene transcription in mammalian cells. Mol Cell 52:314-24

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