The first wave of the inducible gene network up-regulated by pathogen-stimulated mononuclear cells encodes a variety of effector proteins with pleitropic biological activities. This class of primary immediate early (IE) genes codes for potent pro-inflammatory cytokines and chemokines that play a prominent role during the manifestation of inflammatory response. In an attempt to better understand induction mechanisms for such genes, I have focused on those coding for human interleukin-1β (IL1B) and tumor necrosis factor α (TNF), which exhibit both transient IE induction as well as cell-type restriction. Employing a combined approach using cell lines and primary cells, reporter transient transfection, chromatin conformational capture and immunoprecipitation, evaluation of transcript integrity, ectopic expression in a non-competent cell type, and comparison to mouse orthologs, I have determined that a complex array of mechanisms interplay in order to distinctly regulate the Toll-like receptor (TLR) signaling-dependent induction of these two important pro-inflammatory genes whose deregulation provides the etiology for numerous diseases. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), which are the hallmarks of poised IE genes. In contrast, IL1B is stringently regulated by long-distance chromosome gyrations, multistep activation through a unique doubly-paused Pol II which, in association with the monocyte lineage factor Spi1/PU.1 (Spi1), maintains a low TBP and Pol II occupancy prior to activation. Activation and DNA binding of the transcription factors C/EBPβ and NF-κB resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of the positive elongation factor b (P-TEFb). This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Nucleosome occupancy and chromatin modification analyses of the IL1B and TNF promoters, revealed activation-specific changes in chromatin marks that are supportive for nucleosome clearance and formation of nucleosome free regions (NFR). Furthermore, ectopic expression of Spi1, along with a TLR surrogate (over-expressed TNF receptor associated factor 6, TRAF6), in a cell line incompetent for IL1B transcription, is observed to prime the cell's endogenous genome for IL1B induction by appropriately phasing promoter nucleosomes and recruiting paused Pol II in a manner reminiscent of that observed in competent monocytes. Here I report a novel connection between the metabolic state of cells and HIF-1α in regulating murine Il1b gene expression. With regard to the lipopolysaccharide (LPS) unresponsive state known as endotoxin tolerance, my data revealed that following transient induction, IL1B and TNF remained marked with paused Pol II complexes for up to 24 hours post-stimulation. Upon subsequent LPS exposure, tolerized TNF remained in an unresponsive paused state, while IL1B resumed transcription due to recruitment of positive elongation kinase P-TEFb. Emerging evidence suggests that inflammatory responses of LPS/TLR4 activated macrophages are interconnected with metabolic pathways, resulting in the shift of energy utilization by the cells. Here I report that inhibition of either phosphoinositide 3-kinase (PI3K) or glucose metabolism had a greater affect on the transcriptional response of Il1b than of Tnf. The differences between these two genes, especially for endotoxin tolerance, suggest that il1b may play a distinct role from tnf in chronic inflammation. / Bayer School of Natural and Environmental Sciences; / Biological Sciences / PhD; / Dissertation;

<p>The unique C-terminal domain (CTD) of RNA polymerase II (RNAPII), composed of tandem heptad repeats of the consensus sequence YSPTSPS, is subject to differential phosphorylation throughout the transcription cycle. Several RNA processing factors have been shown to bind the appropriately phosphorylated CTD, and this facilitates their localization to nascent pre-mRNA during transcription. In <italic>Saccharomyces cerevisiae</italic>, the mRNA export protein Yra1 (ALY/REF in metazoa) has been shown to cotranscriptionally associate with mRNA and is thought to deliver it to the nuclear pore complex for export to the cytoplasm. Based on a previous proteomics screen, I hypothesized that Yra1 is a <italic>bona fide</italic> phosphoCTD associated protein (PCAP) and that this interaction is responsible for the pattern of Yra1 cotranscriptional association observed <italic>in vivo</italic>. Using <italic>in vitro</italic> binding assays, I show that Yra1 directly binds the hyperphosphorylated form of the CTD characteristic of elongating RNAPII. Using truncations of Yra1, I determined that its phosphoCTD-interacting domain (PCID) resides in the segment comprising amino acids 18-184, which, interestingly, also contains the RNA Recognition Motif (RRM) (residues 77-184). Using UV crosslinking, I found that the RRM alone can bind RNA, although a larger protein segment, extending to the C-terminus (aa 77-226), displays stronger RNA binding activity. Even though the RRM is implicated in both RNA and CTD binding, certain RRM point mutations separate these two functions: thus, mutations that produce defects in RNA binding do not affect CTD binding. Both functions are important <italic>in vivo</italic>, in that RNA binding-defective or CTD binding-defective versions of Yra1 engender growth and mRNA export defects. I also report the construction and characterization of a useful new temperature sensitive <italic>YRA1</italic> allele (<italic>R107AF126A</italic>). Finally, using chromatin immunoprecipitation, I demonstrate that removing the N-terminal 76 amino acids of Yra1 (all of the PCID up to the RRM) results in a 10-fold decrease in Yra1 recruitment to genes during elongation. These results indicate that the PCTD is likely involved directly in cotranscriptional recruitment of Yra1 to active genes.</p> / Dissertation

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Tübingen, Univ., Diss., 1978.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 74-87).

Thesis (Ph.D. in Molecular Biology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 185-214). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 130-152). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Deformed epidermal autoregulatory factor 1 (DEAF1) is a transcription factor that binds to (T/C)TCG(G/T) half-sites and has been shown to be involved in human diseases of cancer, diabetes, depression and intellectual disorders. We used chromatin immunoprecipitation assays to assess endogenous levels of DEAF1 and RNA polymerase II occupancy on the promoter and 5'UTR of the DEAF1 gene. In exponentially growing HEK293 cells, low levels of DEAF1 bind to sequences between -718 and +232, with +1 marking the start of translation. Within 0.5 hr of treating the cells with 500 µM H2O2, DEAF1 occupancy is increased between 7-18 fold at B (-718/-569), -577/-444, C (-432/-299), D (-205/-112) and E(-97/17). There were no statistically significant changes in either RNA polymerase II phospho-serine 5 (RNA PolII pS5) or RNA polymerase II phospho-serine 2 (RNA PolII pS2) binding with H2O2 treatment compared to control. With media change, there is an increase in RNA PolII pS2 and pS5 occupancy at both a distal site -1462/-1326 and in the coding region at 133/232, while no significant change in DEAF1 occupancy was detected. DEAF1 occupancy at the DEAF1 promoter and 5'UTR are inversely correlated with RNA polymerase II occupancy, however, there were no measurable differences in DEAF1 RNA levels at 0.5 hr and 1 hr time points. In summary, these data indicate that there is increased occupancy of DEAF1 at its own promoter following stress, which inversely affects occupancy of RNA polymerase at proximal promoter and 5'UTR sites of the DEAF1 gene.

An automated detection process for Eukaryotic ribonucleic acid (RNA) Polymerase II Promoter is presented in this dissertation. We employ an artificial neural network (ANN) in conjunction with features that were selected using an information-theoretic approach. Firstly an introduction is given where the problem is described briefly. Some background is given about the biological and genetic principles involved in DNA, RNA and Promoter detection. The automation process is described with each step given in detail. This includes the data information gathering, feature generation, and the full ANN process. The ANN section of the project is split up in a generation process, a training section as well as a testing section. Lastly the final detection program was tested and compared to other promoter detection systems. An improvement of at least 10% in positive prediction value (PPV) in comparison with current state-of-the-art solutions was obtained. Note: A Companion CD should accompany this report that contains all the program code and some of the source data that was used in this project. All the references to “Companion CD”, reference number [18] are references to these programs.acquisition process, how the different samples were split into different sets and statistical. / Dissertation (MEng (Computer Engineering))--University of Pretoria, 2007. / Electrical, Electronic and Computer Engineering / unrestricted

Microsatellite repeats are a phenomenon found in DNA where a short sequence, usually 1-6bps, is repeated dozens to hundreds of times. Microsatellite repeats that are able to be transcribed are termed expanded tandem repeat-containing RNA (xtrRNA) [1]. xtrRNA have been associated with many diseases, such as Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD), which are both caused by a repeat in the C9ORF72 gene. Recent research has been focused on trying to provide treatments for patients with these diseases. This study focuses on creating a drug screening process for therapeutics targeting transcription by stopping or slowing the transcription of C9ORF72 repeat expansions. One project has focused on interrupting the interaction of two transcription factors, SUPT5H and SUPT4H1, to slow transcription. Another project has focused on slowing transcription by using transcriptional inhibitors or nucleoside analogs at low concentrations. Our hypothesis is that if transcription rates are slowed enough, pausing or arrest of RNA polymerase will be induced at complex sequences, including GC-rich regions and repeats. This should reduce synthesis of xtrRNA and provide a starting point for therapeutic development.