My newly established laboratory at the NIEHS investigates the changes in chromatin architecture and gene activity that results from stimuli from the external environment, focusing on the stress response in Drosophila melanogaster. This model system is ideal for probing the direct, immediate effects of a stress signal on gene activation and repression; within seconds of experiencing a thermal or oxidative stress, Drosophila stress-responsive genes are strongly up-regulated (100 to 1000-fold increase in mRNA levels within 20 minutes). Simultaneously, the chromatin surrounding these genes is dramatically decondensed, and housekeeping genes are shut off. The rapid, robust activation of the major stress-responsive genes and concomitant repression of other cellular transcription allows for a detailed kinetic examination of the links between chromatin structure and gene expression in vivo. We have made use of this system to address several key questions concerning the molecular mechanisms of transcription regulation, as outlined below.? One remarkable property of the stress-repsonse is the speed of gene activation. One proposed reason for this is the presence of RNA polymerase II (Pol II) at the promoter of stress-responsive genes prior to activation. This Pol II has initiated transcription, synthesized a short RNA, and then stalled within the promoter-proximal region. Gene induction by stress rapidly releases this stalled Pol II into the gene, allowing the first wave of Pol II to be observed within the coding regions in seconds. Understanding the fundamental properties of the stalled Pol II, and the mechanisms for maintenance vs. release of Pol II into productive elongation are specific aims of my research. 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 promoters of c-myc, c-fos, junB and the HIV promoter.? To characterize the factors involved in regulating transcription elongation by the stalled Pol II, we have established an efficient genetic assay using RNA interference to deplete specific proteins in Drosophila S2 cells. We have screened a large number of putative transcription elongation and chromatin modifying factors for their effect on RNA production from a key Drosophila stress-responsive gene, Hsp70 (Heat shock protein 70). The Hsp genes in Drosophila represent a well-studied, highly inducible set of genes that are responsive to thermal, oxidative and ionic stress, as well as a number of carcinogens and mutagenic agents. Our genetic screen has identified a number of candidates for further investigation. Among them, the Negative ELongation Factor, or NELF complex, is of particular interest. We have shown by Microarray analysis that depletion of NELF increases basal transcription of a number of Hsp genes as well as affecting a number of other inducible genes, including those responsive to oxidative damage, bacterial pathogens, and cell cycle kinases. Moreover, Chromatin Immunoprecipitation (ChIP) assays have revealed that the majority of NELF-dependent genes possess engaged Pol II near their promoters and that NELF controls the efficiency of transcription through the promoter-proximal region of these genes. This important work established that there is a global link between NELF and stalled Pol II in vivo, and that NELF activity is pivotal for regulating early elongation at a myriad of inducible genes. We propose that NELF functions as a molecular switch to repress gene transcription in the absence of induction, yet allowing for extremely rapid de-repression upon gene activation. These data are particularly interesting in light of recent evidence that NELF plays a critical role in transcription of the junB oncogene in mammalian cells.? The large number of NELF-dependent genes identified by our Microarray analysis suggests that manipulating the efficiency of early elongation is much more prevalent method of gene regulation than previously appreciated. To evaluate this possibility, we are pursuing a genome-wide search for stalled Pol II in Drosophila, employing ChIP-on-chip location analysis (in collaboration with Rick Young?s laboratory at MIT and the NIEHS microarray facility). This technique, which involves probing an array with DNA material derived from a traditional ChIP assay, permits the mapping of Pol II binding sites at high resolution throughout the Drosophila genome. Comparison of Pol II occupancy with RNA expression levels has allowed us to identify a distinct class of genes that posses bound Pol II at their promoters but do not produce significant levels of mRNA. The lack of correlation between Pol II occupancy and gene expression at a subset of genes in Drosophila, in conjunction with recent data from ChIP-on-chip using human fibroblasts, indicates that a significant percentage of genes in higher eukaryotes are regulated at a step after the recruitment of Pol II to the gene promoter. Strikingly, in both Drosophila and mammalian cells, a large number of the promoters that possess a seemingly inactive Pol II are inducible by environmental (e.g steroid hormones, stress) and cell cycle cues. Elucidating this novel paradigm for controlling gene expression will surely provide fresh insights into the activation of not only stress-responsive genes in Drosophila, but also pivotal oncogenes involved in human disease.? By leveraging the power of Drosophila genetics in both embryonic cells and developing organisms, we are continuing to identify the proteins that coordinate the establishment and release of stalled Pol II at these various genes, and to determine the defining features of genes that utilize this novel form of gene regulation.
Showing the most recent 10 out of 24 publications
