The SRC gene encodes pp60c-Src, a 60 kDa non-receptor tyrosine kinase that is frequently activated and/or overexpressed in many cancers including colon cancer. In a subset of colon cancer cell lines, it has been shown, that the overexpression of c-Src can be explained, in part, by the transcriptional activation of the SRC gene. As a result, the general goal of this thesis was to further characterize how SRC is transcriptionally regulated in human cancer cell lines. Two highly dissimilar promoters, the housekeeping-like SRC1A promoter, as well as the HIF-1Ñ regulated tissue-specific SRC1Ñ promoter, regulate SRC expression. hnRNP K and the Sp family of factors regulate the SRC1A promoter; however, the true impact of Sp3 on SRC1A activity was not understood. In this thesis, a comprehensive analysis of the effect of Sp3 on SRC1A activity was performed. Physiologically, Sp3 exists as four translational isoforms that, in part, dictate the activation potential of Sp3. In general, the longer forms of Sp3 were modest transcriptional activators of the SRC1A promoter whereas the shorter forms were unable to activate the SRC1A promoter. An analysis of all Sp3 isoforms identified that the shorter Sp3 isoforms could be converted into transcriptional activators of SRC1A if the SUMOylation of a critical lysine residue within the inhibitory domain was prevented. Conversely, SUMOylation of the same isoform had little effect on the activation potential of the longer Sp3 isoforms at the SRC1A promoter. These results suggest that transcriptional activation by Sp3 is promoter context-, isoform- and modification-dependent.<p>SRC is transcriptionally repressed by histone deacetylase inhibitors (HDIs) and despite unsuccessful studies attempting to identify HDI-responsive elements within the SRC promoter regions none could be identified. This finding also suggests that histone deacetylases (HDACs) may be required for SRC expression. Historically, it was believed that HDIs act at the histone level to alter chromatin dynamics through the inactivation of HDACs to result in histone hyperacetylation and increased transcriptional activation. As such, a systematic investigation of the changes in histone H3 and H4 acetylation status at the transcriptionally repressed SRC promoter regions and the transcriptionally activated p21WAF1 promoter region was performed. The p21WAF1 promoter was used as control in this study as p21WAF1 is a classical example of a gene transcriptionally activated by HDIs. Interestingly, similar changes in histone acetylation at the p21WAF1 promoter and both SRC promoter regions were observed. Upon closer examination of acetylation changes at discreet histone residues, it was observed that in the rare case that a particular residue was differentially acetylated upon treatment at the promoter regions analyzed, the SRC1Ñ and p21WAF1 promoter regions demonstrated more similar changes in acetylation as compared to SRC1A. Taken together, these results suggest that histone acetylation status is not an accurate indicator of transcriptional activity following HDI treatment. To further investigate HDI-mediated SRC repression, RNA Pol. II occupancy at the promoter and regions downstream of the promoter were assessed. Despite the continued occupancy of RNA Pol. II at the promoter regions, RNA Pol. II was lost from the 3¡¦ UTR upon treatment with HDIs. These findings suggest that RNA Pol. II . may be sequestered at the promoter regions upon treatment with HDIs possibly as a result of impeded transcription initiation and/or elongation. Further analysis of the phosphorylation status of RNA Pol. II identified that transcriptional initiation was indeed occurring despite HDI treatment; however, productive transcriptional elongation could not be confirmed thus suggesting a role for abrogated elongation in HDI mediated SRC repression. Complimentary analysis of the effects of HDACs on SRC expression suggested that while class I HDACs abrogated SRC expression, class II HDACs were required for the maintenance of SRC transcript levels in a promoter-independent fashion. Together, these results provide the basis for a model whereby HDIs repress SRC transcriptional expression through the inhibition of class II HDAC activity to eventually result in curtailed SRC transcriptional elongation.

Alternative splicing functions to generate proteomic diversity and to regulate gene expression in higher eukaryotes. Genome-wide analyses suggest that alternative splicing and transcription typically regulate different gene sets to achieve cell- and tissue-type specificity. However, within individual cell-types, most alternative splicing events occur co-transcriptionally and are impacted by the transcriptional machinery. Despite many focused studies on co-transcriptional regulation of alternative splicing, its mechanisms and functions in regulation of gene expression are still poorly understood. To investigate relationships between transcription and alternative splicing, I performed microarray profiling of alternative splicing and transcript levels during activation of a T cell line. This experiment revealed that different sets of genes and associated functional categories are regulated by alternative splicing and transcription during T cell activation. I next employed inhibitors of RNA polymerase II (Pol II) elongation and microarray profiling to identify genes with coupled changes in splicing and transcript levels when transcription is impeded in activated T cell. Genes that were affected at both levels were significantly enriched in RNA binding and processing functions, and generally displayed increased alternative exon inclusion and decreased transcript levels when transcription elongation was disrupted. Similar effects were observed when transcription was driven by mutant polymerases with reduced elongation activity, and when cells were subjected to stress treatments. Many of the elongation inhibition-sensitive exons from the affected genes introduce premature termination codons into the mRNA, resulting in spliced mRNAs that are substrates of the nonsense-mediated decay pathway and further reduction in mRNA levels. ChIP-Seq experiment demonstrated that Pol II occupancy specifically increased in introns flanking the affected exons. These results provide evidence that a physiological function of transcription elongation-coupled alternative splicing regulation is to regulate the levels of RNA processing factors under conditions that reduce elongation activity, including cell stress. In summary, my thesis research has provided new insights into the integration of transcription and splicing control. While these two regulatory levels can control different gene sets during the activation of T cells, within a given cell type, they are closely coupled to control specific alternative splicing events that appear to coordinate mRNA and RNA processing factors levels.

Alternative splicing functions to generate proteomic diversity and to regulate gene expression in higher eukaryotes. Genome-wide analyses suggest that alternative splicing and transcription typically regulate different gene sets to achieve cell- and tissue-type specificity. However, within individual cell-types, most alternative splicing events occur co-transcriptionally and are impacted by the transcriptional machinery. Despite many focused studies on co-transcriptional regulation of alternative splicing, its mechanisms and functions in regulation of gene expression are still poorly understood. To investigate relationships between transcription and alternative splicing, I performed microarray profiling of alternative splicing and transcript levels during activation of a T cell line. This experiment revealed that different sets of genes and associated functional categories are regulated by alternative splicing and transcription during T cell activation. I next employed inhibitors of RNA polymerase II (Pol II) elongation and microarray profiling to identify genes with coupled changes in splicing and transcript levels when transcription is impeded in activated T cell. Genes that were affected at both levels were significantly enriched in RNA binding and processing functions, and generally displayed increased alternative exon inclusion and decreased transcript levels when transcription elongation was disrupted. Similar effects were observed when transcription was driven by mutant polymerases with reduced elongation activity, and when cells were subjected to stress treatments. Many of the elongation inhibition-sensitive exons from the affected genes introduce premature termination codons into the mRNA, resulting in spliced mRNAs that are substrates of the nonsense-mediated decay pathway and further reduction in mRNA levels. ChIP-Seq experiment demonstrated that Pol II occupancy specifically increased in introns flanking the affected exons. These results provide evidence that a physiological function of transcription elongation-coupled alternative splicing regulation is to regulate the levels of RNA processing factors under conditions that reduce elongation activity, including cell stress. In summary, my thesis research has provided new insights into the integration of transcription and splicing control. While these two regulatory levels can control different gene sets during the activation of T cells, within a given cell type, they are closely coupled to control specific alternative splicing events that appear to coordinate mRNA and RNA processing factors levels.

The SRC gene encodes pp60c-Src, a 60 kDa non-receptor tyrosine kinase that is frequently activated and/or overexpressed in many cancers including colon cancer. In a subset of colon cancer cell lines, it has been shown, that the overexpression of c-Src can be explained, in part, by the transcriptional activation of the SRC gene. As a result, the general goal of this thesis was to further characterize how SRC is transcriptionally regulated in human cancer cell lines. Two highly dissimilar promoters, the housekeeping-like SRC1A promoter, as well as the HIF-1Ñ regulated tissue-specific SRC1Ñ promoter, regulate SRC expression. hnRNP K and the Sp family of factors regulate the SRC1A promoter; however, the true impact of Sp3 on SRC1A activity was not understood. In this thesis, a comprehensive analysis of the effect of Sp3 on SRC1A activity was performed. Physiologically, Sp3 exists as four translational isoforms that, in part, dictate the activation potential of Sp3. In general, the longer forms of Sp3 were modest transcriptional activators of the SRC1A promoter whereas the shorter forms were unable to activate the SRC1A promoter. An analysis of all Sp3 isoforms identified that the shorter Sp3 isoforms could be converted into transcriptional activators of SRC1A if the SUMOylation of a critical lysine residue within the inhibitory domain was prevented. Conversely, SUMOylation of the same isoform had little effect on the activation potential of the longer Sp3 isoforms at the SRC1A promoter. These results suggest that transcriptional activation by Sp3 is promoter context-, isoform- and modification-dependent.<p>SRC is transcriptionally repressed by histone deacetylase inhibitors (HDIs) and despite unsuccessful studies attempting to identify HDI-responsive elements within the SRC promoter regions none could be identified. This finding also suggests that histone deacetylases (HDACs) may be required for SRC expression. Historically, it was believed that HDIs act at the histone level to alter chromatin dynamics through the inactivation of HDACs to result in histone hyperacetylation and increased transcriptional activation. As such, a systematic investigation of the changes in histone H3 and H4 acetylation status at the transcriptionally repressed SRC promoter regions and the transcriptionally activated p21WAF1 promoter region was performed. The p21WAF1 promoter was used as control in this study as p21WAF1 is a classical example of a gene transcriptionally activated by HDIs. Interestingly, similar changes in histone acetylation at the p21WAF1 promoter and both SRC promoter regions were observed. Upon closer examination of acetylation changes at discreet histone residues, it was observed that in the rare case that a particular residue was differentially acetylated upon treatment at the promoter regions analyzed, the SRC1Ñ and p21WAF1 promoter regions demonstrated more similar changes in acetylation as compared to SRC1A. Taken together, these results suggest that histone acetylation status is not an accurate indicator of transcriptional activity following HDI treatment. To further investigate HDI-mediated SRC repression, RNA Pol. II occupancy at the promoter and regions downstream of the promoter were assessed. Despite the continued occupancy of RNA Pol. II at the promoter regions, RNA Pol. II was lost from the 3¡¦ UTR upon treatment with HDIs. These findings suggest that RNA Pol. II . may be sequestered at the promoter regions upon treatment with HDIs possibly as a result of impeded transcription initiation and/or elongation. Further analysis of the phosphorylation status of RNA Pol. II identified that transcriptional initiation was indeed occurring despite HDI treatment; however, productive transcriptional elongation could not be confirmed thus suggesting a role for abrogated elongation in HDI mediated SRC repression. Complimentary analysis of the effects of HDACs on SRC expression suggested that while class I HDACs abrogated SRC expression, class II HDACs were required for the maintenance of SRC transcript levels in a promoter-independent fashion. Together, these results provide the basis for a model whereby HDIs repress SRC transcriptional expression through the inhibition of class II HDAC activity to eventually result in curtailed SRC transcriptional elongation.

The process of eukaryotic transcription and its regulation has been an interesting area of research for decades. With more insights into the process of transcriptional regulation of genes, studies have revealed a transcriptional regulation at the level of RNA polymerase II in response to nutritional stress. Further studies in our laboratory and others’, using Saccharomyces cerevisiae as a model system, had shown that two subunits of core RNA polymerase II, RPB4 and RPB7 play a crucial role in response to nutritional starvation. Similarly, these proteins are also known to play important roles in stress response in higher eukaryotes. Additionally, altering levels of Rpb4 and Rpb7 can differentially affect starvation response in S. cerevisiae (Singh et al., 2007). Multiple tissue blot analyses had shown that both these subunits are differentially expressed in different human tissues more significantly in heart, kidney and brain (Khazak et al., 1995; Khazak et al., 1998; Schoen et al., 1997). These findings have led us to investigate in Dictyostelium discoideum, a cellular slime mold, the possible role of these subunits during starvation-induced development. D. discoideum cells exist as unicellular amoebae in soil. In this organism, growth and differentiation phases are distinctly separated, which is an advantage for investigating the functions of these subunits during growth and development. Cells respond to nutritional starvation by undergoing a series of morphological changes coordinated with transcriptional changes giving rise to a terminally differentiated structure referred to as fruiting body which has live spores suspended on top of stalk of dead cells. Though starvation-induced development is accompanied by differential expression of genes, few studies related to the transcription machinery, RNA polymerase II have been reported so far. Purification and presence of all three RNA polymerases from D. discoideum had been reported earlier but the details of their structures and regulation have not been explored in detail (Pong and Loomis, 1973; Renart et al., 1985). One interesting observation reported by Lam and colleagues (Lam et al., 1992) was that CTD of the largest subunit of RNA polymerase II, Rpb1, is highly conserved with 24 heptapeptide repeats and expression of RPB1 transcript was regulated during development. Thus, we carried out experiments to characterize Rpb4 and Rpb7, two subunits of D. discoideum RNA polymerase II to understand any role of these subunits during development. Identification of Rpb4 and Rpb7, two subunits of D. discoideum RNA polymerase II To identify the homologs of S. cerevisiae Rpb4 and Rpb7 in D. discoideum, we employed bioinformatics and genetic approaches. Firstly, we searched D. discoideum database for all protein sequences of S. cerevisiae RNA polymerase II subunits. We could obtain sequences homologous to all twelve subunits in D. discoideum. Among the 12 subunits of D. discoideum RNA polymerase II, we chose to characterize two subunits, DdRpb4 and DdRpb7. We cloned the open reading frames of these two genes from D. discoideum Ax2 cells and cloned them in yeast expression vectors for complementation studies. In S. cerevisiae, Rpb4 is a non-essential protein but rpb4∆ cells show abnormal phenotypes. Few phenotypes of rpb4∆ cells, such as temperature sensitivity, defective in response to nutritional starvation and defective in activated transcription, were employed to identify the D. discoideum homolog of ScRpb4 (Woychik and Young, 1989; Pillai et al., 2001: Pillai et al., 2003). We observed that DdRPB4 can rescue temperature sensitivity corroborated with its ability to activate transcription from HSE containing promoters and sporulation defects of Scrpb4Δ mutant to the wild type. However, DdRPB4 can rescue neither the defect in activated transcription of GAL10 and INO1 promoters nor the elongated morphology exhibited by Scrpb4Δ mutant. On the other hand, we observed that DdRPB7 can complement the lethality associated with ScRPB7 deletion and can partially rescue the phenotypes associated with Scrpb4∆ strain similar to ScRPB7 (Sharma and Sadhale, 1999; Singh et al., 2004). Taken together, we have identified D. discoideum Rpb4 and Rpb7 as bona fide homologs of S. cerevisiae Rpb4 and Rpb7, respectively. Analysis of Rpb4 and Rpb7 in D. discoideum Since yeast RNA polymerase II subunits, Rpb4 and Rpb7, play an important role in the regulation of genes responsive to starvation stress, we carried out experiments to characterize Rpb4 and Rpb7 during growth and starvation-induced development in D. discoideum. Temporal and spatial expression profiles show avaried but similar pattern of RPB4 and RPB7 transcripts during D. discoideum development. We observed similarity between ScRpb4 and DdRpb4 in its ability to interact with DdRpb7 and to localise in both nuclear and cytoplasmic compartments. Attempts to knock out or reduce the levels of DdRpb4 and DdRpb7 by homologous recombination and antisense approaches, respectively, failed. However, since altering levels of Rpb4 and Rpb7 in S. cerevisiae can affect different stress response pathways, we had used overexpression to alter the level of Rpb4 and analysed its effect on D. discoideum development. We overexpressed DdRpb4 as GFP fusion protein in Ax2 cells and observed that D. discoideum cells overexpressing DdRpb4 showed normal growth and development similar to the wild type protein. Interestingly, we observed that Ax2 cells overexpressing DdRpb4 have drastically reduced levels of the endogenous protein. Thus, we have identified a post-transcriptional control on the level of Rpb4 in D. discoideum. Role of S. cerevisiae Rpb4/Rpb7 subcomplex in stress In S. cerevisiae, Rpb4 and Rpb7 interact with each other and carry out important functions (Choder, 2003; Sampath and Sadhale, 2004). Employing the functional conservation of Rpb4 and Rpb7 across various model systems, we further investigated the role of the subcomplex in S. cerevisiae. Since Rpb7 is an essential gene, we have generated rpb7Δstrain in the presence of plasmids expressing Rpb7 or its homologs. We have generated a S. cerevisiae strain lacking both RPB4 and RPB7 and introduced Rpb4 and Rpb7 homologs from either D. discoideum or C. albicans. We analysed these strains under stresses such as high temperature and nutrient starvation. The results of these experiments have provided how the differences in Rpb4 and Rpb7 proteins and their ability to form a subcomplex could be reflected in differential stress responses. Besides the high functional conservation of these proteins, their interaction with other regulatory proteins might also be critical for a proper response to nutritional stress.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 72-93).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 112-124).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Thesis (Ph.D. in Biophysics & Genetics) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 165-185). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [68]-77).