Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 130-153).

Thesis (Ph. D.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

GLK1 and GLK2 transcription factors have been suggested to be involved in the regulation of chloroplast development, organic nitrogen signaling, disease resistance and circadian rhythmicity (Waters et al. 2009; Gutiérrez et al. 2008; Savitch et al. 2007; Sprott et al. 2010). This implies that multiple factors may play roles in regulation of GLK genes. In the present study, transcriptional regulation of GLK1 and GLK2 in Arabidopsis by various endogenous and environmental stimuli was investigated with the objective of elucidating the primary signalling pathway affecting expression of these two genes. Collectively, results of GLK1 and GLK2 expression in response to the experimental treatments of Arabidopsis point to the regulation of the two genes by changes in photosynthetic metabolism and reactive oxygen species (ROS) levels, and by organic nitrogen signalling. Changes in ROS levels and organic nitrogen signalling may also affect the two genes indirectly by interfering with or altering photosynthetic metabolism.

The aim of this study was to gain more information about the interactions between DNA and the histone octamer during the process of transcription. This work used a pUC8 plasmid derivative that contained the core promoter region of the RNA polymerase I of Acanthamoeba castellanii, placed upstream of four repeats of the 5S rDNA nucleosome positioning sequence from the sea urchin, Lytechinus variegatus. The plasmid was reconstituted into chromatin via addition of chicken erythrocyte histone octamers, using polyglutamic acid as a nucleosome assembly factor. The positioning of nucleosomes on the insert was monitored by restriction enzyme digestion. Proper nucleosome positioning was shown to be dependent on the presence of preassembled transcription complexes on the promoter region. The absence of preformed transcription complexes on the promoter region prior to nucleosome reconstitution perturbed the distribution of histone octamers on the repeats of the 5S rDNA. This "mispositioning" effect was related to the location of the RNA polymerase I promoter region upstream of the four repeats of the 5S rDNA fragment. Band shift assays in polyacrylamide gel electrophoresis were used to determine the relative efficiency of nucleosome formation on the promoter-containing fragment, on 5S rDNA and finally on nucleosome core particle DNA. The results indicate that the promoter fragment forms a nucleoprotein complex at lower concentration of histone than the 5S positioning sequence. This complex may not be a nucleosomal structure. The reconstituted plasmid was then used to investigate the transcriptional elongation by RNA polymerase I using the chromatin-like template containing positioned nucleosomes as compared to transcription on improperly positioned nucleosomes and on free DNA. The efficiency of transcription was related to the proper positioning of nucleosomes with regard to the tandemly repeated 208-bp 5S rDNA. The presence of phased nucleosomes in the path of the transcription complex seemed not to inhibit nor to significantly slow down the elongation as compared to free DNA. Furthermore, nucleosome positioning, as assayed by restriction endonuclease digestion, did not change after passage of the polymerase I transcription complex. / Graduation date: 1993

Although numerous transcription factors (TFs) are expressed by oligodendrocytes (OGs), the role(s) of most of these TFs in oligodendrogenesis remains to be elucidated. One such TF is Hoxa2, which was recently shown to be expressed by O4-positive (+) pro-OGs. Hence, the main objectives of this thesis were to determine the expression profile and function(s) of Hoxa2 during OG development. Immunocytochemical analysis of primary mixed glial cultures demonstrated that Hoxa2 is expressed throughout oligodendrogenesis, diminishing only with the acquisition of a myelinating phenotype. Subsequently, immunohistochemical analysis suggested that Hoxa2 is expressed by migratory oligodendroglial cells in the embryonic spinal cord. However, double immunofluorescent analysis of Hoxa2 transgenic knockout mice showed that OG specification and early maturation proceed normally in the absence of Hoxa2 in the spinal cord. As Hoxa2 is one of 39 murine Hox genes, which exhibit extensive overlapping expression profiles in the spinal cord, we decided to examine the expression of an additional Hox TF, Hoxb4, during OG development. Immunocytochemical analysis of primary mixed glial cultures demonstrated that Hoxb4 is also expressed throughout OG development. Furthermore, comparison of the expression profiles of Hoxb4 and Olig2 suggested that Hoxb4 is expressed by oligodendroglial cells in the spinal cord. Hence, Hoxb4, as well as other Hox TFs could compensate for Hoxa2 in the spinal cord in its absence. As the anterior boundary of most Hox genes has been found to be in the hindbrain or spinal cord, we decided to look at the telencephalon which would be less likely to have compensatory mechanisms. Our results showed that similar to the spinal cord, Hoxa2 is expressed by oligodendroglial cells in the telencephalon. Subsequently, it was found that over-expressing Hoxa2 in CG4 cells, an oligodendroglial cell line derived from the perinatal rat cerebral cortex, impairs their differentiation. In an attempt to determine the mechanism by which it accomplishes this, we examined the expression of polysialylated neural cell adhesion molecule (PSA-NCAM), which has been implicated in this process. Contrary to our expectations, however, it was found that over-expressing Hoxa2 in CG4 cells results in significantly fewer PSA-NCAM+ cells. Hence, the results suggest that Hoxa2s effect on OG differentiation is independent of its effect on PSA-NCAM expression. The expression of Hox genes is enhanced by retinoic acid (RA), which, in turn, both inhibits, as well as promotes OG differentiation. Although the reason for these opposing roles is uncertain, examination of the experimental protocols utilized by different research groups reveals disparities in age, CNS region, as well as RA concentration. As a result, RAs effect on oligodendrogenesis could be stage- and/or concentration-dependent. In order to determine which of these factors could impact RAs effect on OG differentiation we treated CG4 cells with two different concentrations of RA at two distinct time points. The results showed that both factors (concentration and time/stage) can impact RAs effect on CG4 cell differentiation. In an attempt to determine the mechanism by which it accomplishes this, we examined the expression of PSA-NCAM. Contrary to our expectations, the results suggest that RAs effect on CG4 differentiation is independent of its effect on PSA-NCAM expression. The results of this thesis suggest that Hoxa2 and RA could play multiple roles in OG development. Although these roles appear to be similar, further research will be needed to determine whether Hoxa2 acts a downstream effector in the RA signaling pathway in oligodendroglial cells.

Isothiocyanates (ITCs) are phytochemicals highly expressed in cruciferous vegetables and these compounds are associated with the decreased incidence of cancers in populations consuming high levels of cruciferous vegetables. Several individual ITCs including phenethyl isothiocyanate (PEITC) inhibit tumor growth and angiogenesis and their anticancer activity has been linked to inhibition of cancer cell growth, survival and inflammation (NFB). It has also been demonstrated that PEITC induces reactive oxygen species (ROS) and that ROS is largely responsible for PEITC-induced cell death. To confirm PEITC-induced cancer cell death we have investigated the mechanism of action of PEITC in pancreatic cancer cell lines and PEITC induces ROS and inhibits growth and induces apoptosis (PARP cleavage). In addition, PEITC downregulates expression of several gene products including vascular endothelial growth factor (VEGF), cyclin D1 (CD1), Bcl2 and survivin and these have previously been reported in other studies. However, since these gene products are all regulated by specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4, which are overexpressed in cancer cells and tumors, we investigated the effects of PEITC on Sp proteins and observed that PEITC decreased expression of Sp1, Sp3 and Sp4 in pancreatic cancer cells. These results demonstrate for the first time that an important underlying mechanism of action of ITCs likely involves targeting Sp transcription factors through an ROS-mediated mechanism and the pathways required for ITC-induced Sp downregulation were investigated and the results are presented in this paper.

The human genome contains slightly more than 20 000 genes that are expressed in a tissue specific manner. Transcription factors play a key role in gene regulation. By mapping the transcription factor binding sites genome-wide we can understand their role in different biological processes. In this thesis we have mapped transcription factors and histone marks along with nucleosome positions and RNA levels. In papers I and II, we used ChIP-seq to map five liver specific transcription factors that are crucial for liver development and function. We showed that the mapped transcription factors are involved in metabolism and other cellular processes. We showed that ChIP-seq can also be used to detect protein-protein interactions and functional SNPs. Finally, we showed that the epigenetic histone mark studied in paper I is associated with transcriptional activity at promoters. In paper III, we mapped nucleosome positions before and after treatment with transforming growth factor  β (TGFβ) and found that many nucleosomes changed positions when expression changed. After treatment with TGFβ, the transcription factor HNF4α was replaced by a nucleosome in some regions. In paper IV, we mapped USF1 transcription factor and three active chromatin marks in normal liver tissue and in liver tissue of patients diagnosed with alcoholic steatohepatitis. Using gene ontology, we as expected identified many metabolism related genes as active in normal samples whereas genes in cancer pathways were active in steatohepatitis tissue. Cancer is a common complication to the disease and early signs of this were found. We also found many novel and GWAS catalogue SNPs that are candidates to be functional. In conclusion, our results have provided information on location and structure of regulatory elements which will lead to better knowledge on liver function and disease.

No description available.

Notch signaling is important for development in Caenorhabditis elegans and the REF-1 family pro-teins, a set of the bHLH transcription factors, are the first targets of Notch signaling. Little is known about the molecular mechanisms employed by the REF-1 family to regulate development. In this project, I iden-tified novel targets of three REF-1 family proteins, HLH-25/HLH-28/HLH-29, and determined which target genes are activated and which are repressed by the REF-1 proteins. These targets were identified by gene expression microarray and were functionally categorized by Gene Oncology analysis. A systems biology approach was performed to identify networks associated with those targets. In addition to the mo-lecular genetics studies, I identified and better characterized the range of phenotypes induced by muta-tions in ref-1 family genes.