Cancer cells take more glucose to provide energy and phosphoryl intermediates for cancer progression. Meanwhile, energy-provider function of mitochondria in cancer cells is disrupted. This phenomenon is so-called Warburg effect, which is discovered over eighty years ago. The detail mechanisms for Warburg effect are not well defined. How glycolytic enzymes contribute to cancer progression is not well known. PKM2 is a glycolytic enzyme dominantly localized in the cytosol, catalyzing the production of ATP from PEP. In this study, we discovered that there were more nuclear PKM2 expressed in highly proliferative cancer cells. The nuclear PKM2 levels are correlated with cell proliferation rates. According to our microarry analyses, MEK5 gene was upregulated in PKM2 overexpression cells. Our studies showed that PKM2 regulated MEK5 gene transcription to promote cell proliferation. Moreover, nuclear PKM2 phosphorylated Stat3 at Y705 site using PEP as a phosphoryl group donor to regulate MEK5 gene transcription. Our study also showed that double phosphorylated p68 RNA helicase at Y593/595 interacted with PKM2 at its FBP binding site. Under the stimulation of growth factors, p68 interacted with PKM2 to promote the conversion from tetrameraic to dimeric form so as to regulate its protein kinase activity. Overexpression PKM2 in less aggressive cancer cells induced the formation of multinuclei by regulating Cdc14A gene transcription. Overall, this study presents a step forward in understanding the Warburg effect.

is a functionally diverse large group of proteins sharing a jelly roll β-barrel fold. An enzymatic member 3-hydroxyanthranilate-3,4-dioxygenase (HAO) and a non-enzymatic member pirin, which is a human nuclear metalloprotein of unknown function present in all human tissues, were selected for structural and functional studies in this dissertation work. HAO is an important enzyme for tryptophan catabolism and for 2-nitrobenzoic acid biodegradation. In this work, seven catalytic intermediate were captured in HAO single crystals, enabling for the first time a nearly complete structural snapshot viewing of the entire molecular oxygen activation and insertion mechanism in an iron- and O2-depedent enzyme. The rapid catalytic turnover rate was found achieved in large part by protein dynamics that facilitates O2 binding to the catalytic iron, which is bound to the enzyme by a facile 2-His-1-carboxylate ligand motif. An iron storage and chaperon mechanism was also discovered in the bacterial source of this enzyme, which led to a proposed novel biological function of a mononuclear iron-sulfur center. Although human pirin protein shares the same structural fold with HAO, its iron ion is coordinated by a 3-His-1-carboxylate ligand motif. Pirin belongs to a subset of proteins whose members are playing regulatory functions in the superfamily. In this work, pirin is shown to act as a redox sensor for the NF-κB transcription factor, a critical mediator of intracellular signaling that has been linked to cellular responses to pro-inflammatory signals which controls the expression of a vast array of genes involved in immune and stress responses.

The success of Mycobacterium tuberculosis as a pathogen is due to its remarkable ability to: (i). adapt to and survive inside activated macrophages under nonproliferating condition, (ii). put up drug resistance and (iii). enter into hypoxia-induced dormancy and remain in nonproliferating condition, be resistant to drugs, and get reactivated into proliferation when favourable conditions arise. Thus, regulation of cell division (arrest and resumption) is an obligatory event that is critical to the pathogen for the establishment of successful infection, latency and reactivation process in human host. Therefore, in order to understand and combat the successful survival strategy of the bacterium inside the host macrophages or in granuloma, a basic knowledge of the regulation of cell division in tubercle bacillus is essential. Bacterial cytokinetic protein FtsZ (a tubulin homologue) is the key regulatory molecule for cell division and its intracellular level is critical for initiation of cell division in bacteria. Therefore, in order to understand the regulation cell division by expression and maintenance of ftsZ mRNA and protein, we initiated studies on the transcriptional regulation of ftsZ gene in the slow growing pathogen, M. tuberculosis, and in the fast-growing saprophyte M. smegmatis. Identification of regions containing ftsZMt promoter activity In order to identify promoter activity-containing regions of ftsZ gene of M. tuberculosis H37Rv (ftsZMt) in vivo, different regions upstream of ftsZMt namely, the ftsQ-ftsZ intergenic region, the ftsQ open reading frame (ORF), and different regions of ftsQ ORF, were cloned in a gfp reporter plasmid and analyzed for gfp expression in M. smegmatis mc2155 cells. Flow cytometric analysis of exponentially grown M. smegmatis mc2155 cells containing these transcription fusion constructs revealed GFP expression in the cells harbouring ftsQ-ftsZ intergenic region (172 bp), the entire ftsQ ORF (945 bp), and 5’ 467 bp and 3’ 217 bp regions of ftsQ ORF. RT-PCR analyses on RNA from M. smegmatis mc2155 cell transformants carrying the entire ftsQ ORF-ftsQ-ftsZ intergenic region containing construct, as well as on total RNA from M. tuberculosis confirmed that the regions identified indeed elicit promoter activity. RT-PCR analysis on M. tuberculosis RNA as well as semi-quantitative RT-PCR analyses of gfp transcripts driven by cloned MtftsZ promoter regions in M. smegmatis cells showed that about 70% of the total promoter activity comes from ftsQ ORF and there is co-transcription of ftsQ-ftsZ genes. Multiple transcripts code for ftsZMt Primer extension analysis, using primers annealing at different positions in the ftsQ-ftsZ chromosomal region, on RNA from M. tuberculosis as well as from M. smegmatis transformants containing 1.117 kb ftsZMtpromoter region in a promoter probe vector, identified origin of six different transcripts (T1-T6) for the gene. Among them, five transcripts (T1, T2, T3, T4, and T6) were detected in M. tuberculosis cells at exponential phase of growth. T5 could be detected only in M. smegmatis transformants containing 1.117 kb ftsZMt promoter upstream of mycgfp2+ reporter gene. Transcript T1 and T2 originate in the ftsQ-ftsZ intergenic region, while T3, T4, and T6 start in the ftsQ ORF. Analysis of sequence in the –10 and –35 regions of the corresponding promoters for the individual transcripts identified high GC content of the regions, which is characteristic of promoters of M. tuberculosis. All of the individual promoter sequences were independently cloned in a promoter probe vector and confirmed that they are true promoters, active in M. smegmatis cells, and that the T1-T6 transcripts were not products of RNA processing. Differential expression from the multiple ftsZMt promoters In order to study the activity and regulation of ftsZMt promoters in M. tuberculosis cells, which is a slow grower and also asymptomatically survives as dormant bacteria for decades in human granuloma, a stably genome-integrated plasmid was required where activity of the promoters can be studied by means of stable and enhanced gfp expression. For that purpose, an L5-mycobacteriophage attP (attachment site)-specific integration proficient promoter probe vector, which contains a stable gfp gene (mycgfp2+) whose codon has been optimized for mycobacterial expression, was generated. Using the vector, all the six promoter regions (P1-P6) were studied in M. smegmatis and M. tuberculosis cells. Flow cytometric and semi-quantitative RT-PCR analyses showed that promoter P5 is unable to elicit activity in M. tuberculosis cells, unlike in M. smegmatis transformants. Semi-quantitative RT-PCR analyses showed that expression of P3 is only 10-20% of the total promoter activity. Promoters P1, P2, P4 and P6 showed 50-80% activity of the total promoter activity and their activity were comparable in M. smegmatis and M. tuberculosis. The presence of multiple promoters reflects the requirement to maintain high basal level of, or to differentially regulate a critical level of, FtsZ expression during different pathogenic stages of tubercle bacilli. In order to investigate the role of multiple promoters, we verified the levels of expression of the five transcripts from the five ftsZ promoters in M. tuberculosis cells under conditions of growth inside mouse macrophage cell line and also under various stress conditions mimicking those that exist in the granuloma environment, like conditions of nonreplicating persistence, gradual nutrient depletion stress, oxidative stress, surface tension stress, acidic stress, heat shock, DNA damaging conditions and osmotic stress. For this purpose, individual promoter regions were cloned into a stably inheritable gfp reporter plasmid vector, and into an L5 mycobacteriophage attP (attachment site)-specific integration-proficient variant of the same vector, for the expression of the promoters from the chromosomal locus in M. smegmatis and M. tuberculosis cells. Quantitative primer extension analyses, semiquantitative RT-PCR analyses on RNA from M. tuberculosis cells grown under these different conditions, and quantitative GFP fluorescence analyses in these cells showed differential activation of the five promoters under different conditions of growth. Under hypoxic and nutrient-depleted stationary phase of growth, two new promoters, Tdor and Ts, in the ftsQ ORF were identified, and these promoters showed maximal activity only under those specific conditions of growth. None of the ftsZ promoters were found to be responsive to stringent response mediated by overexpression of M. tuberculosis RelA. None of the promoters were also found to be responsive of overexpression of heat-shock sigma factor SigH in M. tuberculosis, implicating new pathway of regulation of ftsZ promoters. Multiple promoters driving expression of ftsZ gene of M. smegmatis Similar studies, which were carried out on the identification, structural and functional characterization, regulation of the promoters of cell division gene ftsZ in the fast growing saprophyte M. smegmatis cells, showed the presence of four ftsZ promoters, three of which originates from the 249 bp ftsQ-ftsZ intergenic region and one from the ftsQ ORF. RT-PCR analysis showed that both ftsQ and ftsZ are co-transcribed. Cloning and expression analysis of the individual promoters mapped by primer extension in a GFP based reporter plasmid showed that all the four putative regions are true promoters. Quantitative primer extension on RNA from a synchronously grown culture identified P2 promoter to be responsive to either initiation of cell division or stress, although expression of P1, P3, and P4 did not vary with respect to synchronous division. Quantitative primer extension analysis and semi-quantitative RT-PCR analysis on the RNA from M. smegmatis cells showed that under various stress conditions, P2 activity goes down significantly. Under nutrient depleted stationary phase and hypoxic nonreplicating persistence stage-2, the levels of P2 and P3 activity could hardly be detected, whereas, expression from P1 and P4 goes down only in hypoxia. Level of total ftsZ mRNA remains almost the same under various stress conditions, although upon hypoxia and stationary phase the level goes down almost two fold. Thus, in fast growing M. smegmatis too, multiple ftsZ promoters are differentially regulated under various stress conditions and a critical level of ftsZ mRNA is maintained. Taken together, the study of ftsZ promoters of a slow-growing pathogenic mycobacterium and a fast growing non-pathogenic mycobacterium indicate that differential expression of the multiple promoters, along with conditional activation of stage specific promoters like Pdor or Ps, is one of the mechanisms through which the bacilli probably maintain required levels of FtsZ protein that are crucial for the cell survival, probably through cytoskeletal maintenance, and cell division.

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.

is a functionally diverse large group of proteins sharing a jelly roll β-barrel fold. An enzymatic member 3-hydroxyanthranilate-3,4-dioxygenase (HAO) and a non-enzymatic member pirin, which is a human nuclear metalloprotein of unknown function present in all human tissues, were selected for structural and functional studies in this dissertation work. HAO is an important enzyme for tryptophan catabolism and for 2-nitrobenzoic acid biodegradation. In this work, seven catalytic intermediate were captured in HAO single crystals, enabling for the first time a nearly complete structural snapshot viewing of the entire molecular oxygen activation and insertion mechanism in an iron- and O2-depedent enzyme. The rapid catalytic turnover rate was found achieved in large part by protein dynamics that facilitates O2 binding to the catalytic iron, which is bound to the enzyme by a facile 2-His-1-carboxylate ligand motif. An iron storage and chaperon mechanism was also discovered in the bacterial source of this enzyme, which led to a proposed novel biological function of a mononuclear iron-sulfur center. Although human pirin protein shares the same structural fold with HAO, its iron ion is coordinated by a 3-His-1-carboxylate ligand motif. Pirin belongs to a subset of proteins whose members are playing regulatory functions in the superfamily. In this work, pirin is shown to act as a redox sensor for the NF-κB transcription factor, a critical mediator of intracellular signaling that has been linked to cellular responses to pro-inflammatory signals which controls the expression of a vast array of genes involved in immune and stress responses.

Initiation of transcription is a major step in the regulation of gene expression. A dominant theme in regulation of gene expression lies in understanding the mechanism involved in selective expression of the genes in response to external or internal stimuli. Gene regulatory proteins bind DNA at specific sites either cognate to the promoters they act upon or at a distance, thereby exerting their effect by turning on (activation) or turning off (repression) the genes. Response of these factors to the environmental signals is further achieved by the DNA binding affinity of the transcription factors that can be modulated by small ligands, concentrations of which may fluctuate in response to nutrient availability and stress. Bacteriophages achieve a high degree of efficiency in gene expression by evolving elegant strategies of transcriptional control. mom gene of enterobacteriophage Mu serves as an excellent model to understand this elaborate regulation of gene expression. The gene encodes a unique DNA modification function that confers an anti-restriction phenotype to the phage genome. Though dispensable for phage growth, it is fascinating in two respects (i) a novel modification; (ii) regulation follows a complex scheme without precedence in prokaryotes. mom is the last gene to be expressed during the phage lytic life cycle. Premature expression of the gene is deleterious to both host and phage and hence it is under a complex regulatory network. Dam methylase, a host encoded protein acts as a positive regulator of gene expression, an example where methylation has been shown to play a positive role in regulating tranascription. OxyR, another host encoded protein negatively regulates mom gene expression. Dam methylation prevents the binding OxyR to its site located in the mom regulatory region. The regulatory interplay also involves two phage encoded proteins. C, a middle gene product is essential for transcriptional switch from middle to late genes and Com, a late gene product, for enhancing translation of mom mRNA. Thus, C and Com serve as transcriptional and translational activators of mom gene expression. Pmom is a weak promoter with both -10 and -35 elements away from consensus and a sub-optimal 19 bp spacer element encompassing a stretch of 6T residues that act as negative elements. ‘T stretch’ is known to induce a kink in the DNA. The sub-optimal spacer region makes the promoter elements out of phase and RNAP by itself cannot bind at mom promoter. C protein exerts its effect in activation in a multistep mechanism. The protein binds DNA as a dimer overlapping the promoter and unwinds the DNA, realigning the promoter elements, thus recruiting the RNAP. In the next step, it enhances the promoter clearance by the enzyme, thus enhancing the rate of productive transcription. With this prevailing knowledge on C mediated mom gene expression, the present thesis work describes the experiments carried out to further understand the molecular mechanism of second step activation at Pmom. Genetic and biochemical analysis were carried out to identify the interacting surface of C protein on RNAP. Subsequently, studies have been extended to understand the C mediated transactivation at other late promoters- lys, I, P, which encode for the lysis and morphogenetic functions of the phage. Finally, Mg2+ coordinating residues in C protein were identified to decipher the ligand induced conformational changes in the activator protein required for its transactivator function. Chapter I, a general introduction to the thesis, deals with the detailed discussion on gene expression and its regulatory mechanisms. RNA polymerase (RNAP) being the central molecule of gene expression (transcription) its organization and assembly are discussed. With the availability of the high resolution crystal structures of bacterial RNAP, an in-depth review on RNAP structure in terms of its potential regulatory targets, conformational changes associated with the formation of a functional holoenzyme, and during its transition from initiation to elongation processes have been described. Regulation of transcription with an emphasis on activation mechanism, ligand mediated allosteric transitions in regulatory proteins and the polymerase-activator interactions are discussed citing a few examples. The chapter concludes by introducing bacteriophage Mu and mom gene and its regulation by C. The objectives of the thesis form the concluding section of the chapter. Activators are capable of resurrecting defective promoters in response to cellular demands. The unusual, multistep activation of mom promoter (Pmom) by C protein involves activator mediated promoter unwinding to recruit RNA Polymerase (RNAP) and subsequent enhanced promoter clearance of the enzyme. The first step of transactivation is an interaction independent step, while the later might involve a transient interaction between C and one of the subunits of RNAP. Previous studies pointed out β′ subunit to be the most probable interaction partner. Chapter II comprises the genetic and biochemical studies carried out to confirm this observation. Employing a genetic screen mutations in rpoC gene (encoding the β′ subunit of RNAP), were isolated which result in the defective RNAP. The mutant RNAPs were assayed for their C specific activity by in vivo transactivation assays. Such mutants have been purified and characterized to understand their effect at different steps of C mediated mom gene expression during transcription initiation. The mutant RNAP had normal transcription activity with typical σ70 promoters but exhibited reduced productive transcription and enhanced abortive initiation on C-dependent Pmom. Experiments carried out to probe the interaction between C and mutant RNAP revealed that the physical interaction per se is not disrupted between the two proteins. Post C-mediated recruitment of RNAP to the promoter, transient interactions between the two proteins appears to induce subtle conformational changes in RNAP leading to an enhanced promoter clearance. Transactiavtor protein C is essential for the expression of other late genes lys, I, P apart from mom during the phage life cycle. Although the mechanism of multistep activation at Pmom has been elucidated, little is known on the transactivation from lys, I and P promoters. Chapter III includes studies carried out to understand the process of activation at these promoters. Owing to the differences in their C-binding site and promoter architecture it was important to investigate the differential effect of C, if any at lys, I , P promoters compared to that at Pmom. Activators in prokaryotes are shown to stimulate different steps of transcription initiation pathway ranging from the polymerase binding to the promoters to the post recruitment steps of isomerization and promoter clearance. Effect of C at different steps of transcription initiation pathway was analysed. The results indicate that C is absolutely essential for transcription from lys, I and P promoters similar to mom. However, at these promoters C exerts its effect at the step of Isomerisation from closed complex to open complex formation. Thus, C acts at a single step here and the mode of activation is different from that observed at Pmom. C dimer binds DNA with high affinity and sequence specificity, to an interrupted palindromic sequence overlapping the -35 element of mom promoter. Mg2+ mediated conformational transitions in C protein are essential for its DNA binding and transactivation functions. Chapter IV deals with the identification of the Mg2+ coordinating residues in C protein. Primary sequence analyses lead to the identification of a putative metal coordinating motif (EXDXD) towards the N-terminus of the protein. These residues were subjected to site directed mutagenesis to infer their role in Mg2+ coordination, its associated allosteric transition required for specific interaction with DNA. Mutants showed an altered Mg2+ induced conformation, compromised DNA binding and reduced levels of transcription activation when compared to C protein. Though Mg2+ is widely used in various DNA transaction reactions, this study provides the first insights on the importance of metal-ion induced allosteric transitions in regulating transcription factor function.

No description available.

Chromatin structure and spatial interactions between proximal and distal gene regulatory elements, including gene core promoters and enhancers, are important in the control of gene transcription. In this issue, Oh et al. characterized an AP-1-dependent enhancer at the epidermal differentiation complex locus that establishes spatial interactions with numerous gene promoter regions at that locus.

Mammalian genomes are characterised by global and pervasive DNA methylation and this modification is generally thought to be inhibitory to transcription. An exception to this widespread DNA modification are genomic elements called CpG islands (CGI), contiguous regions of non-methylated DNA which encompass the transcription start site of two thirds of mammalian genes. Although CGIs represent the most prominent feature of mammalian promoters, the contribution of these elements to promoter function remains unclear. Work in this study shows that the histone lysine demethylase KDM2B (FBXL10/ JHDM1B) is a nuclear protein which binds specifically to non-methylated CpG dinucleotides and associates with CGI elements genome-wide through its zinc-finger CxxC (ZF-CxxC) DNA binding domain. Furthermore, in mouse embryonic stem cells, biochemical investigation revealed that KDM2B associates with Polycomb group E3 ubiquitin ligase RING1B to form a variant Polycomb repressive complex 1 (PRC1) characterized by the PCGF1 subunit. Considering that KDM2B has clear DNA-binding activity and that CGIs were reported to function as nucleation sites for polycomb repressive complexes, a potential role for KDM2B in mediating PRC1 recruitment to target genes was investigated. Stable depletion studies indicated that KDM2B is required for the normal targeting of RING1B to CGIs and the regulation of expression of a subset of Polycomb-occupied genes. By taking advantage of a genetic ablation system in which the DNA binding domain of KDM2B can be conditionally deleted, results in this thesis reveal that the ability of KDM2B to recognize non-methylated DNA is essential for polycomb domain formation and normal embryonic development. Finally, through the use of a de novo targeting assay, an unexpected PRC2 recruitment pathway was discovered which is dependent on PRC1-mediated H2AK119ub1 deposition. Together this work uncovers a novel mechanism linking KDM2B-dependent recognition of non-methylated DNA with recruitment of Polycomb proteins and provides the framework on which to further investigate the contribution of CGIs to formation of polycomb domains.

A função e a arquitetura dos genes está começando a ser elucidada a partir do estudo de genomas completos tanto de procariotos como de eucariotos. Diversos estudos foram ultimamente realizados a respeito de exon shuffling do ponto de vista evolutivo, fenômeno relacionado à origem de novos genes através de recombinações de DNA mediadas por introns. Apesar de eventos de exon shuffling serem responsáveis pelo aumento da modularidade gênica, outros processos foram desenvolvidos ao longo da evolução para que houvesse o aumento da diversidade do proteoma sem a conseqüente expansão dos genomas, sendo splicing alternativo um dos mais freqüentes. Apresentamos nesta dissertação duas extensivas análises: 1) a análise de uma base de dados de genes eucarióticos contendo pelo menos um intron que apresentou excesso de introns de fase 0 e exons simétricos, dados que suportam exon shuffling como um importante mecanismo de evolução gênica. Avaliamos também a confiabilidade de introns preditos por programas de computador através de alinhamento de ESTs; e 2) a análise do uso alternativo de exons (UAE), um tipo de splicing alternativo, em transcritos humanos detectando que cerca de 51% dos genes humanos possuem mais de uma variante de splicing e que este tipo de processamento pós-transcricional parece ser mais freqüentemente encontrado em tecidos tumorais. / Abstract not available.