BACKGROUND:14-3-3 proteins are a family of highly conserved proteins that are involved in a wide range of cellular processes. Recent evidence indicates that some of these proteins have oncogenic activity and that they may promote tumorigenesis. We previously showed that one of the 14-3-3 family members, 14-3-3gamma, is over expressed in human lung cancers and that it can induce transformation of rodent cells in vitro.METHODS:qRTPCR and Western blot analysis were performed to examine 14-3-3gamma expression in non-small cell lung cancers (NSCLC). Gene copy number was analyzed by qPCR. P53 mutations were detected by direct sequencing and also by western blot. CHIP and yeast one hybrid assays were used to detect p53 binding to 14-3-3gamma promoter.RESULTS:Quantitative rtPCR results showed that the expression level of 14-3-3gamma was elevated in the majority of NSCLC that we examined which was also consistent with protein expression. Further analysis of the expression pattern of 14-3-3gamma in lung tumors showed a correlation with p53 mutations suggesting that p53 might suppress 14-3-3 gamma expression. Analysis of the gamma promoter sequence revealed the presence of a p53 consensus binding motif and in vitro assays demonstrated that wild-type p53 bound to this motif when activated by ionizing radiation. Deletion of the p53 binding motif eliminated p53's ability to suppress 14-3-3gamma expression.CONCLUSION:Increased expression of 14-3-3gamma in lung cancer coincides with loss of functional p53. Hence, we propose that 14-3-3gamma's oncogenic activities cooperate with loss of p53 to promote lung tumorigenesis.

published_or_final_version / abstract / toc / Molecular Biology / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

La régulation transcriptionnelle des eucaryotes supérieurs est un processus hautement contrôlé du point de vue spatial et temporel lors du développement, ou en réaction à l’environnement. La transcription ciblée des gènes codant requiert l’assemblage d’un complexe de pré-initiation (PIC) aux promoteurs comprenant l’ARN Polymérase (Pol) II et les facteurs généraux de transcription (GTFs) et dépend de la médiation d’un signal par les facteurs activateurs de transcription (TFs). Les années récentes ont montré que la transition de l’initiation vers l’élongation productive de la transcription représente une étape clé de la régulation de l’expression des gènes. Ce processus est également contrôlé par la structure de la chromatine, les modifications d’histones et par la présence d’éléments cis-régulateurs tels que les ‘enhancers’ ou les ‘silencers’. Au cours de ma thèse, nous avons entrepris de décrypter les mécanismes de régulation transcriptionnelle impliqués dans les étapes du développement lymphocytaire. Nous avons essentiellement travaillé sur des thymocytes primaires murins isolés au stade de différenciation double positif (DP, CD4+/CD8+) pour lequel de nombreuses séquences de type ‘enhancers’ ont été caractérisées dans la littérature scientifique. Nous avons également utilisé des lymphocytes B humains (Raji) immortalisés pour certaines des expériences impliquant des manipulation génétiques complexes permettant l’étude de mutants du domaine carboxy-terminal (CTD) de Pol II. En couplant des approches d’analyse à l’échelle du génome au séquençage à haut-débit, nous avons établi des cartographies fines de la localisation de Pol II, des GTFs, des TFs,de modifications d’histones (ChIP-Seq) et de nucléosomes (MNase-seq) ainsi que la caractérisation de populations variées d’ARN par RNA-seq. Nos principaux résultats ont révélé (i) l’assemblage du PIC et la transcription des enhancers tissus-spécifiques, (ii) l’existence de plateforme d’initiation de la transcription (TIPs) aux enhancers et aux promoteurs tissus-spécifique, (ii) que le contenu en GC représente l’un des principaux éléments promoteurs mammifères en permettant une ouverture transcription-indépendante de la chromatine, (iv) l’importance d’une nouvelle modification post-traductionnelle du domaine CTD de Pol II pour la progression de l’enzyme en élongation et finalement (v) que la modification de l’histone H3 sur le résidu K36 methylé corrèle avec l’épissage des transcrits Pol II. Globalement, les résultats les plus important de ce manuscrit consistent dans la mise en évidence de la transcription des enhancers comme caractérisant l’expression des gènes tissus-spécifiques et dans l’importance des ilots CpG comme éléments promoteurs mammifères permettant la formation d’une structure ouverte de la chromatine. / Transcriptional regulation in higher eukaryotes resembles a tightly controlled temporal and spatial process, as exemplified during development or an organism’s response to environmental stimuli. Directed transcription requires the assembly of the preinitiation complex (PIC) at the promoter of protein-coding genes, including RNA Polymerase (Pol) II and the general transcription factors (GTFs), mediated by activating transcription factors (TFs). Several rate-limiting steps further control the progression of Pol II initiation to productive elongation of the gene. This process is further controlled by chromatin structure, histone modifications as well as cis-regulatory elements, such as enhancers or silencers. We set out to decipher some of these regulatory mechanisms during the tightly controlled process of lymphocyte development. Our work primarily made use of primary mouse thymocytes in CD4+/CD8+ double positive (DP, CD4+/CD8+) stage during T-cell development. To our advantage, many developmentally important cis-regulatory regions are well characterized in this cell population. For genetic manipulations, we made use of the Raji B-cell lymphoma cell-line. Using high throughput genome-wide approaches based on next generation sequencing (NGS), we performed both localization studies of Pol II, GTFs, TFs, histone modifying enzymes, histone modifications and nucleosomes as well as deep-sequencing of different RNA transcript populations. In summary, we find that (i) PICs assemble at tissue-specific enhancers leading to local transcription, (ii) large transcription initiation platforms (TIPs) at tissue-specific promoters and enhancers exist, which correlate with high CG-content of the DNA and transcription factor binding sites (TFBS), (iii) GC-content regulates the nucleosomal structure and initiation, including directionality, at promoters, (iv) Pol II is phosphorylated at a new residue of it C-terminal domain (CTD) in the 3’ regions of genes and (v) splicing events can influence the chromatin structure. Altogether, these results show that PIC formation at and transcription of enhancers are important for the regulation of T-cell target genes, that CpG islands represent important if not the major regulatory promoter element in mammals guiding tissue-specific gene expression and nucleosome structure, as well as novel mechanisms of Pol II elongation and the effect on chromatin structure.

Transcriptional regulation of genes is vital to cell success making it an important aspect of research. Transcriptional regulation can occur in many ways; transcription factors bind to the promoter region and block transcription, disrupt an activator protein, or interact with histones to lead to higher order chromatin. Plant HomeoDomain can recognize and bind to different methylation states of histone tails. PHD proteins use other functional regions to carry out functions. Two associated domains having DNA-binding capacity were characterized in this study; the ARID domains of JARID1A and JARID1C and the DDT domains of BAZ1A, BAZ1B and BAZ2A. These genes are important because of their roles in various diseases such as cancer. The consensus sequences for BAZ1A-DDT is GGACGGRnnGG, GnGAGRGCRnnGGnG, RAGGGGGRnG and CRYCGGT. Consensus sequences for BAZ1B-DDT were CGnCCAnCTTnTGGG and YGCCCCTCCCCnR. Consensus sequences for BAZ2A-DDT were TACnnAGCnY and CnnCCRGCnRTGnYY. Consensus sequence for JARID1A-ARID was GnYnGCGYRCYnCnG. Consensus sequences for JARID1C-ARID was RGGRGCCRGGY. / by Emmanuel MacDonald. / Thesis (M.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Transcriptional regulation by the family of SNAG (Snail/Gfi-1) zinc fingers has been shown to play a role in various developmental states and diseases. These transcriptional repressors have function in both DNA- and protein-binding, allowing for multiple interactions by a single family member. This work aims to characterize the SNAG members Slug, Smuc, Snail, Scratch, Gfi-1, Gfi-1B, and IA-1 in terms of both DNA-protein and protein-protein interactions. The specific DNA sequences to which the zinc finger regions bind were determined for each member, and a general consensus of TGCACCTGTCCGA, was developed for four of the members. Via these studies, we also reveal thebinding affinities of E-box (CANNTG) sequences to the members, since this core is found for multiple members' binding sites. Additionally, protein-protein interactions of SNAG members to other biological molecules were investigated. The Slug domain and Scratch domain have unknown function, yet through yeast two-hybrid screening, we were able to determine protein interaction partners for them as well as for other full length SNAG members. These protein-interacting partners have suggested function as corepressors during transcriptional repression. The comprehensive information determined from these studies allow for a better understanding of the functional relationship between SNAG-ZFPs and other genes. The collected data not only creates a new profile for each member investigated, but it also allows for further studies to be initiated from the results. / by Cindy Chiang. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

Transcription factors are macromolecules that are involved in transcriptional regulation by interacting with specific DNA regions, and they can cause activation or silencing of their target genes. Gene regulation by transcriptional control explains different biological processes such as development, function, and disease. Even though transcriptional control has been of great interest for molecular biology, much still remains unknown. This study was designed to generate the most current list of human transcription factor genes. Unique entries of transcription factor genes were collected and entered into Microsoft Office 2007 Access Database along with information about each gene. Microsoft Office 2007 Access tools were used to analyze and group collected entries according to different properties such as activator or repressor record, or presence of certain protein domains. Furthermore, protein sequence alignments of members of different groups were performed, and phylogenetic trees were used to analyze relationship between different members of each group. This work contributes to the existing knowledge of transcriptional regulation in humans. / by Ala Savitskaya. / Thesis (M.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Transcriptional regulation is an important area of research due to the fact that it leads to gene expression. Transcription factors associated with the regulation can either be activators or repressors of target genes, acting directly or with the aid of other factors. A majority of transcriptional repressors are zinc finger proteins (ZFPs) which bind to specific DNA sequences. The Snail/Gfi (SNAG) domain family, with members such as Slug, Smuc, Snail, and Scratch, are transcriptional repressors shown to play a role in various diseases such as cancer. The SNAG transcription factors contain a conserved SNAG repression domain and DNA binding domain zinc fingers. The specific DNA sequences to which each SNAG-ZFP binds, as well as a general consensus -TGCACCTGTCCGA, have been determined. Also, putative protein-protein interactions in which the Slug domain participates has been identified via binding assays. All these results contribute to better understanding of SNAG-ZFP functions. / by Cindy Chung-Yue Chiang. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

An important hallmark of cancer is the inactivation of tumor suppressor genes. The most common genetic alteration in cancer is the mutation of the TP53 gene occurring in about half of all cancers, but very little progress has been made on how to therapeutically target the signaling defects in these cancers. Additionally, the PTEN tumor suppressor is mutated in a wide variety of cancer types, and its expression is often lost in the absence of mutation. PTEN is a haploinsufficient tumor suppressor that exhibits dose-dependent effects in cells. In the context where PTEN is lost or downregulated, PI3K signaling and downstream signaling through AKT is overactive, leading to an increase in cell growth and proliferation, among other effects. Acting as both a protein and lipid phosphatase, loss of PTEN also affects the PI3K-independent signaling of PTEN, and results in an increase of migration and invasion phenotypes. Importantly, PTEN transcript level is the key determinant for PTEN protein expression, and downregulation of PTEN is part of a poor-prognosis gene expression signature in breast cancer. Downregulation of tumor suppressor gene expression represents a reversible change that is often sufficient to drive tumorigenesis. However, our understanding of the broad molecular mechanisms by which the expression of these tumor suppressors is lost remains limited, but is required to develop effective therapeutic strategies to target malignancies driven by tumor suppressor loss. In Chapter 2, we characterize the problem of transcriptional downregulation of PTEN in breast cancer. We investigate the expression of PTEN in various normal and tumor cells at both the transcript and protein level. We identify various model systems that we believe are suitable to model normal PTEN expression and the PTEN downregulation that mimics what is observed in tumors. We employ a sophisticated approach that couples RNA-sequencing with Nanostring nCounter analysis in order to obtain a detailed and thorough transcriptional profile of the PTEN and pseudogene PTENP1 genomic loci, as well as expression of the poor-prognosis gene signature associated with PTEN downregulation. In this study, we obtained an understanding of the changes in the PTEN transcriptional profile that occur in the progression from normal to cancer, and we believe this approach could be applied to other key tumor suppressor genes. In Chapter 3, we discovered that basally expressed p53 maintains expression of thirteen well-validated tumor suppressors. p53 is expressed at low levels under normal, low-stress conditions, and is expressed at much higher levels under enhanced stress, leading to the activation of stress-response genes. We begin the study by highlighting an association between TP53 mutation and downregulation of PTEN expression. Upon performing chromatin immunoprecipitation coupled with next generation sequencing for p53 under normal, low-stress conditions, we found that p53 binds in the vicinity of thirteen tumor suppressor genes, including PTEN. Basally expressed p53 binds to classic consensus binding sites in enhancers and promoters of target tumor suppressors to maintain their expression at baseline. CRISPR/Cas9-mediated knockout of the endogenous basal p53 binding site upstream of PTEN led to a decrease in PTEN expression and an increase in tumorigenic phenotypes. Given that mutation of TP53 leads to tumorigenesis in mice, but loss of p53 stress-response targets or loss of the ability of p53 to activate these stress-response targets does not lead to spontaneous tumorigenesis, it is likely that these tumor suppressor targets of basal p53 contribute to p53-mediated tumor suppression. In Chapter 4, we identified yet another mechanism by which transcriptional repression of PTEN occurs in triple-negative breast cancer (TNBC) through polycomb repressive complex 2 (PRC2)-mediated repression of the PTEN promoter and upstream regulatory region. Previous research has shown that mutated NOTCH1 represses PTEN through the HES-1 transcription factor in acute myeloid leukemia (AML), and that NOTCH translocations are frequent in TNBC and are sufficient for transformation in vitro. We discovered that NOTCH1 and NOTCH2 mutations and translocations correlate with PTEN downregulation by immunohistochemistry in a cohort of TNBC cases. The TNBC cell line exhibiting PRC2-mediated repression of PTEN also harbors a SEC22B-NOTCH2 translocation that creates a gene product resembling the NOTCH2 intracellular domain. The NOTCH target HES-1 co-localizes on the PTEN promoter with EZH2 (the lysine methyltransferase involved in PRC2-mediated transcriptional repression), and knockdown of NOTCH2 in this cell line led to decreased expression of EZH2, and restoration of PTEN expression at the transcript and protein level. We also demonstrated that EZH2 inhibitors, HDAC inhibitors, and DNA hypomethylating agents robustly restore PTEN transcript levels. Taken together, these results elucidate another mechanism by which PTEN is transcriptionally repressed in the highly aggressive and poor-prognosis TNBC subtype of breast cancer that may be applicable to other cancer types. The results also suggest that this repression is reversible by pharmacological approaches, highlighting a promising therapeutic avenue. Taken together, the studies presented in this thesis begin to unravel the complex mechanisms of transcriptional repression of tumor suppressor genes in cancer. As is the case with PTEN and p53, multiple regulatory mechanisms can influence expression in combination or in a context-dependent manner. The loss of expression of tumor suppressor genes is one of the key hallmarks of cancer, yet very few of the therapeutic approaches used in the clinic today aim to restore tumor suppressor expression. Our results demonstrate proof of concept that restoration of tumor suppressor expression is a plausible and promising therapeutic approach for many different types of cancer, but requires a detailed understanding of the underlying molecular mechanisms of transcriptional regulation.