The E2A locus is involved in chromosomal translocations associated with acute lymphoblastic leukemia. The most common of these involves a translocation between chromosomes 1 and 19 (t1;19), resulting in expression of the chimeric oncoprotein E2A-PBX1. A direct interaction between transcriptional activation domain 1 (AD1) of E2A and KIX domain of the histone acetyltransferase (HAT) /co-activator CBP is required for E2A-PBX1-mediated leukemia induction in mice. This thesis examines the functional consequences of the direct, physical interaction between E2A and CBP, for both proteins. We demonstrate that the interaction between E2A and CBP/p300, as well as another HAT/co-activator, p/CAF, results in acetylation of E2A. Mutagenesis-based mapping studies identify several lysine residues as substrates for acetylation. Of particular interest, a conserved lysine (K34) located within AD1 is acetylated in vitro and in vivo. Substitution of this residue to arginine impairs transcriptional activation of a luciferase reporter while substitution to glutamine, mimicking the acetylation, restores E2A-mediated transcriptional activation. Recent studies have shown that several transcription factors can modulate the intrinsic HAT activity of CBP/p300. We were surprised to find that E2A proteins enhance acetylation of histones by CBP, in vitro and in vivo, in a KIX domain-independent manner. Acetylation of E2A is also not required for stimulation of CBP/p300 histone acetylation. It appears that E2A interacts with the other CBP domains to mediate this effect, presumably through allosteric effects. In summary, we demonstrate that acetylation of E2A plays a role in mediating the transcriptional activation activity of E2A. Furthermore, acetylation of E2A enhances its interaction with CBP/p300, at least in the presence of additional nuclear factors. We show evidence that p/CAF may mediate this effect. Enhancement of CBP/p300 HAT activity by oncogenic E2A-PBX1 proteins in vivo, suggests that some of its leukemia-promoting effects may be due to E2A-induced gain of function effects on CBP/p300. The enhanced interaction between acetylated E2A and CBP/p300, as well as the E2A-mediated stimulation of histone acetyltransferase activity might play a role in the DNA-binding-independent induction of proliferation. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2007-09-26 13:37:21.905

Mitochondrial content, an important determinant of muscle metabolic capacity, changes in individuals during development, and in response to physiological and environmental challenges. This phenotypic plasticity is attributed to the coactivator PPARγ coactivator-1α (PGC-1α) but it remains unclear if this transcriptional regulator accounts for evolutionary variation in mitochondrial content. In an attempt to explain why some species have higher muscle mitochondrial enzyme levels than other species, I examined if the transcriptional mechanisms that control mitochondrial content of a tissue in an individual are also responsible for differences between species. If PGC-1α creates differences between the mitochondrial content of species based on variation in promoter binding motifs, then cis-factor evolution may be the guiding force in scaling trends. In this thesis I explored the basis of size-dependent patterns by looking at layers of regulation, from catalytic activities to promoter evolution and regulation. A representative family, Rodentia, was used to collect muscle samples from a size range of approximately 20g up to 17 kg. As expected, in rodent lower limb muscles, mitochondrial and glycolytic enzyme activity exhibited reciprocal scaling patterns, though the scope differed between muscles. Very little of the variation was accounted for when the activity was related to DNA content. However, when COX activities were expressed relative to DNA, the scaling patterns were similar among the 3 muscles. To determine if interspecies differences were linked to transcriptional regulation, ~800bp of the PGC-1α promoter from 56 terrestrial mammals (5g-5000kg) was examined. The basal placental mammalian promoter possesses putative elements for Sp1, HNF3, myogenic factors and metabolic effectors, which have been retained in mammals with little change in order or spacing. To investigate the ability of these promoters to control PGC-1α expression, rodent promoters were cloned into luciferase reporter gene constructs and transfected into a common mouse myoblast background (Sol8 cells). Unlike mitochondrial content, promoter activity did not vary with body size across the rodent family. Likewise, PGC-1α transcript levels did not vary in rodent muscles in a way that would explain differences in COX activity. This suggests that though PGC-1α may be crucial for within species variation, transcriptional regulation of PGC-1α is not responsible for interspecies variation in mitochondrial content. / Thesis (Master, Biology) -- Queen's University, 2010-06-09 10:50:02.133

Deformed epidermal autoregulatory factor -1 (DEAF-1) is a transcription factor mapping to the chromosomal region 11p15.5, a region associated with loss of heterozygosity (LOH) in human cancers. Potential DEAF-1 binding motifs were identified in the PTEN promoter and the ability of DEAF-1 to regulate PTEN gene expression was investigated. DEAF-1 increased transcription 10-14 fold with PTEN sequences between -429 and -221, while mutations in the DNA binding domain (DEAF-ADWA) and nuclear localization signal of DEAF-1 abolished this increase. DEAF-1 was shown to bind sequences between -339 and -233. Rabdomyosarcoma (RD) stable cell lines with inducible expression of DEAF-1 and DEAF-1-ADWA were produced. Increased DEAF-1 expression had no significant effects on PTEN RNA expression or cell proliferation when compared to controls, but did increase susceptibility to UV-induced apoptosis. These studies suggest that DEAF-1 may contribute to the regulation of PTEN gene expression, but other factors may play a more significant role.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18477号 / 農博第2077号 / 新制||農||1025(附属図書館) / 学位論文||H26||N4861(農学部図書室) / 31355 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 左子 芳彦, 教授 澤山 茂樹, 准教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

It is increasingly becoming clear in the post-genomic era that proteins in a cell do not work in isolation but rather work in the context of other proteins and cellular entities during their life time. This has lead to the notion that cellular components can be visualized as wiring diagrams composed of different molecules like proteins, DNA, RNA and metabolites. These systems-approaches for quantitatively and qualitatively studying the dynamic biological systems have provided us unprecedented insights at varying levels of detail into the cellular organization and the interplay between different processes. The work in this thesis attempts to use these systems or network-based approaches to understand the design principles governing different cellular processes and to elucidate the functional and evolutionary consequences of the observed principles. Chapter 1 is an introduction to the concepts of networks and graph theory summarizing the various properties which are frequently studied in biological networks along with an overview of different kinds of cellular networks that are amenable for graph-theoretical analysis, emphasizing in particular on transcriptional, post-transcriptional and functional networks. In Chapter 2, I address the questions, how and why are genes organized on a particular fashion on bacterial genomes and what are the constraints bacterial transcriptional regulatory networks impose on their genomic organization. I then extend this one step further to unravel the constraints imposed on the network of TF-TF interactions and relate it to the numerous phenotypes they can impart to growing bacterial populations. Chapter 3 presents an overview of our current understanding of eukaryotic gene regulation at different levels and then shows evidence for the existence of a higher-order organization of genes across and within chromosomes that is constrained by transcriptional regulation. The results emphasize that specific organization of genes across and within chromosomes that allowed for efficient control of transcription within the nuclear space has been selected during evolution. Chapter 4 first summarizes different computational approaches for inferring the function of uncharacterized genes and then discusses network-based approaches currently employed for predicting function. I then present an overview of a recent high-throughput study performed to provide a 'systems-wide' functional blueprint of the bacterial model, Escherichia coli K-12, with insights into the biological and evolutionary significance of previously uncharacterized proteins. In Chapter 5, I focus on post-transcriptional regulatory networks formed by RBPs. I discuss the sequence attributes and functional processes associated with RBPs, methods used for the construction of the networks formed by them and finally examine the structure and dynamics of these networks based on recent publicly available data. The results obtained here show that RBPs exhibit distinct gene expression dynamics compared to other class of proteins in a eukaryotic cell. Chapter 6 provides a summary of the important aspects of the findings presented in this thesis and their practical implications. Overall, this dissertation presents a framework which can be exploited for the investigation of interactions between different cellular entities to understand biological processes at different levels of resolution.

No description available.

The GADD45 family of proteins consists of three small nuclear proteins, GADD45A, GADD45B, and GADD45G, which are implicated in modulating the cellular response to various types of genotoxic/physiological stress. This family of proteins has been shown to interact with and modulate the function of cell-cycle control proteins, such as p21 and cdc2/cyclin B1, the DNA repair protein, PCNA, key stress response MAP kinases, including MEKK4 (an upstream regulator of JNK kinase), and p38 kinase. Despite similarities in amino acid sequence, structure and function, each gadd45 gene is induced differentially, depending on the type of stress stimuli. For example, the alkylating agent, methylmethane sulfonate (MMS), rapidly induces all three genes, whereas hydrogen peroxide and sorbitol preferentially induce gadd45a and gadd45b, respectively. Studies of the mechanisms of the stress-mediated induction of the gadd45 genes have predominantly focused on gadd45a, with knowledge of gadd45b and gadd45g regulation lacking. Thus, in order to generate a more complete understanding of the collective regulation of the gadd45 genes, a comprehensive analysis of the stress-mediated induction of gadd45b has been carried out. Towards this end, a gadd45b promoter-reporter construct was generated, consisting of 3897bp sequence upstream of the transcription start site of gadd45b, fused to a luciferase reporter. In a human colorectal carcinoma cell line (RKO), in which gadd45b mRNA levels profoundly increase by various stress stimuli, we observe similar, high levels of induction of the gadd45b-luciferase construct with MMS or UVC treatments, but surprisingly not with sorbitol or anisomycin. Linker-scanning mutagenesis of the gadd45b promoter reveals several important MMS and UVC cis-acting responsive elements contained within the proximal promoter, including a GC-rich region and the CCAAT box. Furthermore, we have identified three constitutively bound transcription factors, Sp1, MZF1, and NFY, and one inducible factor, Egr1, which bind to these regions and which contribute to MMS-responsiveness. In contrast, a post-transcriptional mechanism appears to regulate gadd45b induction upon sorbitol treatment, as this treatment increases the gadd45b mRNA half-life, compared to MMS treatment. Interestingly, with the exception of a common cis-element, the stress-mediated induction of gadd45b appears to be mechanistically distinct from gadd45a. In conclusion, this study provides novel evidence that gadd45b induction by distinct stress agents, MMS and sorbitol, is regulated differentially at the level of mRNA transcription or mRNA stability, respectively. / Molecular Biology and Genetics

Despite their name, a number of the cold shock proteins are expressed during normal growth, and not just during cold shock, in several species. The function of these constitutively expressed CspA paralogues is unclear. In Salmonella Typhimurium (a major worldwide cause of gastrointestinal disease) they have been linked to various stress responses and the establishment of virulence. Study of the cold shock proteins as gene regulators is therefore of great interest, and they also have potential as targets for antimicrobial development. CspE in Salmonella Typhimurium is constitutively expressed during normal growth. In order to determine its function, attempts were made to identify the interactions it forms with other cellular proteins. Initially, a proteomic investigation attempted to identify proteins which complex with CspE by in vivo cross-linking and affinity purification followed by mass spectrometry. Although no defined complex was consistently identified, the results suggested a handful of proteins which might interact with CspE in a weak or transient manner. These proteins included many from the nucleoid and ribosomal entry site, hinting at CspE’s cellular localisation. In order to investigate these transient interactions, a bacterial two-hybrid system was employed. Interactions between CspE and HupA, a nucleoid protein identified in the proteomic analysis, were probed, as were interactions between CspE and CsdA, an RNA helicase thought to function co-operatively with CspE. The twohybrid system also allowed investigation of CspE dimerisation, which has been reported in vitro but not investigated in vivo until this study. CspE appears not to interact significantly with either HupA, CsdA, or itself at 37oC. Finally in a further attempt to identify interactions of CspE, a genomic library was created to test CspE interactions by two-hybrid assay with random peptides derived from the whole Salmonella genome. The library was successfully created and screened for evidence of interaction, and revealed an association between CspE and a transcriptional repressor, DeoT. DeoT is a repressor of several genes for catabolic processes, suggesting a role for CspE in the regulation of central metabolism. The findings of this work present a number of novel discoveries and several interesting opportunities for further studies.

No description available.