Thesis (M. Med. Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 46-54).

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 (M.Med.Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 46-54). Also available in print.

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.

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.

Respiratory syncytial virus (RSV) is the leading cause of respiratory illness in children worldwide. RSV has a negative sense RNA genome, which is the template for viral mRNA transcription and genome replication, and encodes a polymerase to carry out viral RNA synthesis. The promoters for RSV transcription and genome replication are found in a 44-nucleotide (nt), 3´-extragenic region called the leader (Le). Replication is initiated opposite the first nt of the Le, and transcription of the first gene begins at position +45, at a gene start (GS) sequence. However, transcription is also dependent on sequence within Le1-12. Interestingly, Le nucleotides 3-12 bear strong similarity to a GS signal. We hypothesized that this GS-like sequence is the recruitment site for transcribing polymerase. To test this hypothesis, we examined RNA synthesis events at the Le promoter. We identified a previously undescribed RNA initiation site at Le position +3 (Le+3) that was used frequently during RSV infection. Initiation at Le+3 led to the production of a small ~25 nt RNA. Le+3 initiation was shown to occur independently of replication initiation at +1, indicating it is a bona fide initiation site. Mutation of Le1-12 to increase similarity to a GS resulted in elongation of Le+3 RNA and a decrease in transcription initiation at the GS, demonstrating that the Le initiation sequence alters polymerase processivity and impacts downstream transcription events. Preliminary experiments to determine the function of the small RNA showed that it increased levels of viral RNA replication, suggesting it may be involved in influencing a switch from transcription to replication. These studies suggest a model for RSV transcription initiation, whereby the transcribing polymerase enters at the 3´–end of the genome, initiates RNA synthesis from Le+3 and generates a small RNA, and is then positioned to initiate transcription at the first GS. The small RNA that is generated may act as a feedback molecule to promote RNA replication. These findings provide a greater understanding of polymerase behavior at the promoter and may inform rational drug and vaccine design.

Indiana University-Purdue University Indianapolis (IUPUI) / Ssu72, an RNA Pol II CTD phosphatase that is conserved across eukaryotes, has been reported to have a wide array of genetic and physical associations with transcription factors and complexes in RNA transcription. Catalytic mutants of Ssu72 are lethal across many eukaryotes, and mutations to non-catalytic sites in SSU72 phosphatase have been shown to lower function. One spontaneous mutation of the SSU72 gene in Saccharomyces cerevisiae (A to C nucleotide mutation resulting in an L84F mutation in the coded protein) was shown to have transcription termination deficiency (termination override or TOV). This SSU72 mutation was suggested by Loya et al. to cause a lowering of the phosphatase activity of the protein and consequently affect proper termination. In research reported herein, an investigation was completed through in-vitro and ex-vivo approaches with the goal of understanding the impact of the SSU72 TOV mutation on the observed phenotype in S. cerevisiae. It can be concluded from work presented in this report that the SSU72 TOV mutation does not cause a decrease in in-vitro phosphatase activity as compared to wild type. Evidence presented even suggests an increase in phosphatase activity as compared to wild type Ssu72. One model for the observed responses in transcription termination is that the phenylalanine substitution in Ssu72 leads to cooperative interactions with proline residues in the CTD. It is proposed that the corresponding increase in Ssu72 phosphatase activity limits RNA Pol II CTD association with termination factors, such as Nrd1, thus causing deficient transcription termination.

京都大学 / 0048 / 新制・論文博士 / 博士(医科学) / 乙第13169号 / 論医科博第4号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 萩原 正敏, 教授 近藤 玄, 教授 高田 穣 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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