No description available.

Several mechanisms have been proposed for the pol I transcript termination in Schizosaccharomyces pombe. Two well known models are “Pause and Release” and “Torpedo”. Each mechanism explains the role of some of the cis- and trans-factors in transcript termination and the eventual maturation of the ribosomal RNA, but neither mechanism can explain all the experimental observations. A recent study has suggested that each of the two mechanisms can terminate the pol I transcription independently but with significantly less efficiency than the presence of both mechanisms. To help clarify the reasons for the discrepancies in these data, in this study the suggested mechanisms were examined further in three areas by using alternative techniques. First, the effect of uracil concentration or selection times on the transformation frequency of alternative 3’external transcribed spacer (3’ETS) constructs were assessed. Consistent with the previous results a construct containing the full 3’ETS showed the higher transformation frequencies compared with a construct containing only the hairpin or only the termination sites. However, results showed neither the uracil concentration nor selection times have a significant effect on the transformation frequency. Second, to further confirm the “pause and release” mechanism, the termination sites identified by S1 nuclease studies were analyzed using ligation-mediated RT-PCR. The 3’ terminus of the mature 25S rRNA was demonstrated readily but, unexpectedly, the 3’termini of the 3’ETS precursor molecules were not detected, possibly because of their specific structure. Finally, the 3’ extended rRNA precursors were studied by semi-quantitative RT-PCR. These appeared not to correspond with past nuclease protection analyses nor did they demonstrate downstream exonuclease function, observations which question our current understanding of Pol I transcript termination.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.

Transcription of the ribosomal genes accounts for the majority of transcription in the cell due to the constant high demand for ribosomes. The number of proteins synthesized correlates with an effective ribosomal biogenesis, which is regulated by cell growth and proliferation. In the work presented in this thesis, we have investigated the ribosomal RNA genes 45S and 5S rRNA, which are transcribed by RNA Pol I and RNA Pol III, respectively. The focus of this work is the chromatin remodelling complex B-WICH, which is composed of WSTF, the ATPase SNF2h and NM1. We have studied in particular its role in ribosomal gene transcription. We showed in Study I that B-WICH is required to set the stage at rRNA gene promoters by remodelling the chromatin into an open, transcriptionally active configuration. This results in the binding of histone acetyl transferases to the genes and subsequent histone acetylation, which is needed for ribosomal gene activation. Study II investigated the role of B-WICH in transcription mediated by RNA polymerase III. We showed that B-WICH is essential to create an accessible chromatin atmosphere at 5S rRNA genes, which is compatible with the results obtained in Study 1. In this case, however, B-WICH operates as a licensing factor for c-Myc and the Myc/Max/Mxd network. Study III confirmed the importance and the function of the B-WICH complex as an activator of ribosomal genes. We demonstrated that B-WICH is important for the remodelling of the rDNA chromatin into an active, competent state in response to extracellular stimuli, and that the association of the B-WICH complex to the rRNA gene promoter is regulated by proliferative and metabolic changes in cells. The work presented in this thesis has confirmed that the B-WICH complex is an important regulator and activator of Pol I and Pol III transcription. We conclude that B-WICH is essential for remodelling the rDNA chromatin into a transcriptionally active state, as required for efficient ribosomal gene transcription. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.</p><p> </p>

Dans les cellules eucaryotes, il existe 3 ARN Polymérases nucléaires (Pol I, II et III), chacune ayant une fonction de synthèse d'ARN qui lui est propre. L'ARN polymérase I (Pol I) est responsable de la synthèse du précurseur des grands ARN ribosomiques (ARNr), ce qui correspond à une activité de transcription massive dans la cellule. Des données structurales sur cette enzyme de 14 sous-unités sont disponibles. Ceci permet une meilleure compréhension de son mode de fonctionnement, et a confirmé que l'ARN Pol I possède 3 sous-unités spécifiques, aussi appelées "Built-in Transcription Factors", responsables d'activités régulatrices. Deux d'entres elles, Rpa49 / Rpa34, forment un hétérodimère structuralement proche des facteurs de transcription TFIIF et TFIIE de la Pol II, impliqués tant dans l'initiation que dans l'élongation de la transcription. La dernière, Rpa12, est connue pour avoir un rôle dans la stabilité de l'ARN Pol I et l'activité de clivage du transcrit de la polymérase en cours de pause (via son extrémité C-terminale), comme son homologue TFIIS, facteur de transcription de l'ARN Pol II. Nous avons effectué des études génétiques sur des mutants de l'ARN Pol I dépourvus de la sous-unité Rpa49 (rpa49Δ). Cette délétion est viable mais entraîne des problèmes d'initiation et élongation. Nous étudions dans ce travail le clonage et la caractérisation de "suppresseurs" extragéniques correspondant à des mutations ponctuelles de trois sous-unité de la Pol I qui rétablissent une synthèse d'ARNr en l'absence de la sous-unité Rpa49. Toutes ces mutations suppressives identifiées ont été localisées premièrement dans les deux grandes sous-unités Rpa190 et Rpa135, structuralement très proches de Rpa12, mais également au sein même de Rpa12, indiquant une possible interaction entre les sous-unités Rpa49 et Rpa12 ou la région autour. Notamment, un élément spécifique de l'ARN Pol I dans Rpa190, le "DNA Mimicking Loop", est structuralement très proche de la région où l'on trouve ces mutations suppresseur. Les caractérisations génétiques, structurales, mais aussi biochimiques et fonctionnelles de ces suppresseurs nous permettent de proposer des hypothèses sur les rôles de Rpa49 et de Rpa12, mais aussi de cette petite région de Rpa190, en l'initiation de la transcription, ce qui n'a jamais été mis en évidence auparavant. / In eukaryotes cells, there are 3 nuclear RNA Polymerases (Pol I, II and III), each having a particular RNA synthesis function. The RNA Polymerase I (Pol I) produces a single transcript: the precursor of the large ribosomal RNA (rRNA), which correspond to a massive transcription activity in the cell. Structural data of this 14-subunits enzyme is now available. This allows a better understanding of its operating mode, and confirmed that the Pol I has 3 specific subunits, also called "Built-in transcription factors", capable of regulatory activities. Two of them, Rpa49/Rpa34, are forming a heterodimer structurally related to the Pol II transcription factors TFIIF and TFIIE, implicated in both initiation and elongation of the transcription. The last one, Rpa12, is known to have a role in Pol I stability and the cleavage activity of the paused Pol I (via its C-terminus part), like its homologous TFIIS in the Pol II. We performed extensive genetic studies of Pol I mutants lacking one of these subunits: Rpa49 (rpa49Δ). This depletion is viable, but results in initiation and elongation problems. Here, we report the cloning and characterization of extragenic suppressors mapping point mutations in three subunits of Pol I restoring efficient Pol I activities in absence of Rpa49. All suppressor mutations identified were structurally mapped firstly in the two largest subunits Rpa190 and Rpa135 very closed to Rpa12, and then in Rpa12 itself, indicating a possible interplay between the Rpa49 and Rpa12 subunits, and the area around. Notably, a RNA pol I specific element in Rpa190, called "DNA Mimicking Loop", is structurally very closed to the region in which we find all of our suppressor mutations. The genetic, structural but also biochemical and functional characterizations of these suppressors allow us to propose roles of these Rpa49 and Rpa12 subunits, but also of the small area of Rpa190, which has never been highlighted before.

RNR Polymerase I (RNA Pol I) is a “factory” that orchestrate the transcription of ribosomal rRNA for constructing ribosomes as a primary workshop for protein translation to sustain cell growth. Misregulation of RNA Pol I can cause uncontrolled cell proliferation, which leads to the development of cancer. Yeast (Saccharomyces cerevisiae) is a valuable model system to study RNA Pol I. Recently, the X-ray crystal structure of the yeast homologue of RNA Pol I was elucidated, offering the structural basis to selectively target this transcriptional machinery. The approach to selective RNA Pol I targeting was to disrupt the interaction between a specific transcription factor, RRN3 that bind distinct regions of RNA Pol I. For this purpose, a recombined plasmid was designed to carry human rDNA plus its promoter as target together with a selection marker gene. Therefore, this plasmid could not only introduce the target gene into the yeast (host), but also facilitate the passage of this target gene into a stable yeast strain. In this project, one uracil deficient yeast strain of YBR140C was transformed with the recombined yeast integrative plasmid of pHmrDNA-YIPlac211-TG1. This is a recombined plasmid containing not only the human rDNA but also the URA3 gene as a selection marker. PCR amplification of the human ribosomal DNA was indicative of successful integration of the human ribosomal DNA into the genome of the two yeast strains. Virtual screening using a library of 700 FDA-approved compounds was docked into the RRN3-RNA Pol I complex to identify small molecule disruptors of the RRN3-RNA Pol I as a selective strategy. Using growth assays, gel electrophoresis and transcriptional assays, we identified cerivastatin sodium as a lead virtual hit. The result implicates cerivastatin sodium as a selective RNA Pol I inhibitor worthy of further development with potential as targeted anticancer therapeutic.

The 35S rDNA gene clusters on the X and Y chromosomes of Drosophila melanogaster are repeats of approximately 150 to 225 copies. Each are transcribed as a single unit by RNA Polymerase I and modified into the 18S, 5.8S, 2S and 28S ribosomal rRNAs. Reduction in the array copy number results in a bobbed phenotype, characterized by truncated bristles and herniations of abdominal cuticle, due to a decrease in protein production. In some copies within the arrays, R1 and R2 retrotransposable elements are inserted in a conserved region of the 28S gene which represses the transcription of a functional rRNA. Inserted arrays are transcribed at very low levels, but it is not clear how they are identified for repression. Similarly, a subset of uninserted arrays are silenced, and the epigenetic mechanism controlling how this decision is made it is also unknown. The CCCTC binding factor (CTCF) is a boundary element binding protein and a transcriptional regulator found in the nucleolus of differentiated mammalian cells, whose localization requires poly (ADP-ribosyl)ation. We investigated whether CTCF might be involved in the regulation of rDNA expression in Drosophila. Our data show that CTCF is found at the nucleolus of both polytene and diploid nuclei, and we have identified binding sites in the 28S gene, R1 and R2 elements by a bioinformatic approach. ChIP data indicate that CTCF binds only to the site in the R1 retrotransposon. Reduction of CTCF or members of the poly(ADP-ribosyl)ation pathway by RNAi in S2 cells causes an increase in the amount of 35S rDNA gene, R1, and R2 transcripts. In flies, CTCF and PARG mutant alleles show disrupted nucleoli and increased rRNA transcripts. Mutant alleles of CTCF suppress variegation of a P-element inserted in a 35S rDNA array, but not of elements inserted elsewhere in the genome. Consistent with a role for CTCF in rRNA regulation, we found that during oogenesis CTCF is recruited to the nucleolus of nurse cells at early stages when the demand of ribosomes is low and it leaves this compartment in later stages when the cell increases rRNA production. We conclude from these studies that CTCF acts as a regulation of rDNA transcription by RNA polymerase I.

Um conjunto de quadros clínicos conhecidos genericamente por Leishmaniose, que representa um sério problema de Saúde Pública, tem como agentes etiológicos protozoários do gênero Leishmania. Em seu ciclo de vida, o parasita apresenta dois hospedeiros, um vertebrado e um invertebrado. O gênero Leishmania é dividido em dois subgêneros: Leishmania (Leishmania) e LeishmaniaViannia), de acordo com a posição ocupada pela forma promastigota no tubo digestivo do hospedeiro invertebrado. Genes que codificam RNA ribossômico são exclusivamente transcritos pela RNA polimerase I. A região promotora dessa polimerase é conhecida como espécie-específica. Estudos anteriores, baseados em ensaios de expressão transiente com construções nas quais a expressão do gene repórter CAT era dirigido por diferentes regiões do promotor mostraram que em Leishmania há um reconhecimento espécie-específico, mas que espécies filogeneticamente relacionadas reconhecem melhor o promotor do que a espécie homóloga. A caracterização estrutural da região promotora de RNA pol I de L. (V.) braziliensis possibilitou mapear o sítio de início de transcrição e definir a provável região promotora de RNA pol I desse organismo. Quando comparamos o 5´ ETS da região estudada, com a região correspondente das demais espécies do subgênero L. (Leishmania), notamos uma alta similaridade tanto no tamanho, quanto na seqüência de nucleotídeos. Já na região IGS os blocos de repetição apresentam tamanho similar, porém seqüências distintas, assim como a seqüência à montante do ETS. Não foi determinado o número de blocos de repetição presente em cada cístron de L.(V.) braziliensis. Os estudos de ligação de fatores nucleares à região central do promotor mostraram que houve um reconhecimento diferencial dessa região entre a espécie homóloga L. (V.) braziliensis e as espécies heterólogas L. (V.) guyanensis, pertencente ao mesmo subgênero e L. (L.) amazonenis. Os complexos protéicos associados a essa região central apresentaram maior afinidade com a espécie heteróloga L. (V.) guyanensis. O fato de ser o ensaio de CAT trabalhoso e demorado levou à busca de outro repórter que evidenciasse a funcionalidade da região promotora ao mesmo tempo em que permitisse a quantificação dessa expressão, de modo a medir a força do promotor. Neste trabalho iniciamos a padronização para utilizar GFP como gene repórter no estudo da funcionalidade de promotores. Foi possível mostrar que a GFP é detectável em microscopia confocal e que as células que expressam GFP podem ser quantificadas em FACS, no entanto, essas detecções só foram possíveis em parasitas selecionados por uma marca de seleção, ou seja, numa transfecção estável. Assim, substituir o gene repórter CAT pelo gene GFP foi inadequado para os ensaios de transfecção transiente, impedindo que fosse medida a atividade da região promotora de L. (V.) braziliensis em diferentes espécies. / Leishmaniasis is the generic name of a serious public health problem that is caused by protozoa of the genus Leishmania. In its lifecycle, the parasite has two hosts, a vertebrate and an invertebrate. The genus Leishmania is divided into two subgenera: Leishmania (Leishmania) and Leishmania (Vianna), according to the position occupied by the promastigotes form at the gut of the invertebrate host. Genes that encode ribosomal RNA are exclusively transcribed by RNA polymerase I. The promoter region of the RNA polymerase I is known to present a species-specific recognition. Previous studies, based on transient expression assays with constructions in which the expression of the reporter gene CAT was directed by different regions of the promoter showed that RNA pol I promoter in Leishmania is species-specific, but in phylogenetically related species the expression of the reporter gene is higher than in the homologous species. The structural characterization of the promoter region of RNA pol I of L. (V.) braziliensis enabled to map the site of initiation of transcription and to define the promoter region of RNA pol I. The comparison of the determined 5\' region of the ETS region, with the corresponding region of other species of the subgenus L. (Leishmania), showed a high similarity both in size, as in the sequence of nucleotides. However, the IGS region and the repetitive blocks of nucleotides have similar size but different sequences. The studies of binding of nuclear factors to the central region of the promoter showed that there was a recognition between the species counterpart L. (V.) braziliensis and the heterologous species L. (V.) guyanensis, belonging to the same subgenus and L. (L.) amazonensis, that belongs to another subgenus. The complex protein associated with this central region showed greater affinity with the heterologous species L. (V.) guyanensis. The fact that to quantify the CAT expression represents a laborious and time consuming test lead us to search for another reporter that could identified the functionality of the promoter region and at the same time allowed the quantification of expression in order to measure the strength of the promoter. In the present work, we began the standardization for the use of GFP gene as reporter in the study of the functionality of promoters. It was possible to show that GFP is detectable in confocal microscope and the cells that express GFP can be quantified in FACS, however, these findings were made possible only in parasites selected by the mark, ie in a stable transfection. So, the replacement of CAT by GFP reporter showed to be inadequate for testing promoters in transient transfection. This prevented the measure of the activity of the promoter region of L. (V.) braziliensis in different species.

Um conjunto de quadros clínicos conhecidos genericamente por Leishmaniose, que representa um sério problema de Saúde Pública, tem como agentes etiológicos protozoários do gênero Leishmania. Em seu ciclo de vida, o parasita apresenta dois hospedeiros, um vertebrado e um invertebrado. O gênero Leishmania é dividido em dois subgêneros: Leishmania (Leishmania) e LeishmaniaViannia), de acordo com a posição ocupada pela forma promastigota no tubo digestivo do hospedeiro invertebrado. Genes que codificam RNA ribossômico são exclusivamente transcritos pela RNA polimerase I. A região promotora dessa polimerase é conhecida como espécie-específica. Estudos anteriores, baseados em ensaios de expressão transiente com construções nas quais a expressão do gene repórter CAT era dirigido por diferentes regiões do promotor mostraram que em Leishmania há um reconhecimento espécie-específico, mas que espécies filogeneticamente relacionadas reconhecem melhor o promotor do que a espécie homóloga. A caracterização estrutural da região promotora de RNA pol I de L. (V.) braziliensis possibilitou mapear o sítio de início de transcrição e definir a provável região promotora de RNA pol I desse organismo. Quando comparamos o 5´ ETS da região estudada, com a região correspondente das demais espécies do subgênero L. (Leishmania), notamos uma alta similaridade tanto no tamanho, quanto na seqüência de nucleotídeos. Já na região IGS os blocos de repetição apresentam tamanho similar, porém seqüências distintas, assim como a seqüência à montante do ETS. Não foi determinado o número de blocos de repetição presente em cada cístron de L.(V.) braziliensis. Os estudos de ligação de fatores nucleares à região central do promotor mostraram que houve um reconhecimento diferencial dessa região entre a espécie homóloga L. (V.) braziliensis e as espécies heterólogas L. (V.) guyanensis, pertencente ao mesmo subgênero e L. (L.) amazonenis. Os complexos protéicos associados a essa região central apresentaram maior afinidade com a espécie heteróloga L. (V.) guyanensis. O fato de ser o ensaio de CAT trabalhoso e demorado levou à busca de outro repórter que evidenciasse a funcionalidade da região promotora ao mesmo tempo em que permitisse a quantificação dessa expressão, de modo a medir a força do promotor. Neste trabalho iniciamos a padronização para utilizar GFP como gene repórter no estudo da funcionalidade de promotores. Foi possível mostrar que a GFP é detectável em microscopia confocal e que as células que expressam GFP podem ser quantificadas em FACS, no entanto, essas detecções só foram possíveis em parasitas selecionados por uma marca de seleção, ou seja, numa transfecção estável. Assim, substituir o gene repórter CAT pelo gene GFP foi inadequado para os ensaios de transfecção transiente, impedindo que fosse medida a atividade da região promotora de L. (V.) braziliensis em diferentes espécies. / Leishmaniasis is the generic name of a serious public health problem that is caused by protozoa of the genus Leishmania. In its lifecycle, the parasite has two hosts, a vertebrate and an invertebrate. The genus Leishmania is divided into two subgenera: Leishmania (Leishmania) and Leishmania (Vianna), according to the position occupied by the promastigotes form at the gut of the invertebrate host. Genes that encode ribosomal RNA are exclusively transcribed by RNA polymerase I. The promoter region of the RNA polymerase I is known to present a species-specific recognition. Previous studies, based on transient expression assays with constructions in which the expression of the reporter gene CAT was directed by different regions of the promoter showed that RNA pol I promoter in Leishmania is species-specific, but in phylogenetically related species the expression of the reporter gene is higher than in the homologous species. The structural characterization of the promoter region of RNA pol I of L. (V.) braziliensis enabled to map the site of initiation of transcription and to define the promoter region of RNA pol I. The comparison of the determined 5\' region of the ETS region, with the corresponding region of other species of the subgenus L. (Leishmania), showed a high similarity both in size, as in the sequence of nucleotides. However, the IGS region and the repetitive blocks of nucleotides have similar size but different sequences. The studies of binding of nuclear factors to the central region of the promoter showed that there was a recognition between the species counterpart L. (V.) braziliensis and the heterologous species L. (V.) guyanensis, belonging to the same subgenus and L. (L.) amazonensis, that belongs to another subgenus. The complex protein associated with this central region showed greater affinity with the heterologous species L. (V.) guyanensis. The fact that to quantify the CAT expression represents a laborious and time consuming test lead us to search for another reporter that could identified the functionality of the promoter region and at the same time allowed the quantification of expression in order to measure the strength of the promoter. In the present work, we began the standardization for the use of GFP gene as reporter in the study of the functionality of promoters. It was possible to show that GFP is detectable in confocal microscope and the cells that express GFP can be quantified in FACS, however, these findings were made possible only in parasites selected by the mark, ie in a stable transfection. So, the replacement of CAT by GFP reporter showed to be inadequate for testing promoters in transient transfection. This prevented the measure of the activity of the promoter region of L. (V.) braziliensis in different species.

Nous montrons dans cette étude que dans les cellules déplétées pour la nucléoline, une plus faible accumulation de pré-ARNr est associée à une augmentation de marques d’hétérochromatine (H3K9me2) et une diminution de marques d’euchromatine (H4K12ac et H3K4me3) sur la chromatine des gènes ribosomiques. Des expériences de ChIP-seq montrent que la nucléoline est enrichie dans la région codante et promotrice de l’ADNr et est préférentiellement associée avec les gènes non méthylés des ARNr. La déplétion de la nucléoline entraîne une accumulation de l’ARN Pol I au début de l’ADNr et une diminution de UBF sur la région codante et promotrice. La nucléoline interfère avec la liaison de TTF-1 sur le promoteur-proximal T0, inhibant ainsi le recrutement de TIP5 du complexe NoRC, et établissant un état d’hétérochromatine répressive. Ces résultats révèlent l’importance de la nucléoline dans le maintien d’un état euchromatinien des ADNr et dans l’élongation de la transcription. Nous montrons aussi dans cette thèse que l’acétylation est une nouvelle modification post-traductionnelle de la nucléoline. Des études d’immunofluorescence utilisant l’anticorps anti nucléoline acétylée montrent que la nucléoline acétylée est exclue des nucléoles. De plus, par ChIP-seq nous n’avons jamais pu détecter d’association significative de la nucléoline acétylée sur la chromatine des ADNr. Aussi, nous n’avons détecté aucune activation de la transcription de Pol II sur des matrices de chromatine avec la nucléoline acétylée. Nous trouvons une distribution de la nucléoline acétylée majoritairement dans le nucléoplasme où elle co-localise parfaitement avec le facteur d’épissage SC35, et partiellement avec les structures marquées avec un anticorps dirigé contre Y12, mais ne co-localise pas avec des structures contenant la coïline, ce qui suggère que cette fraction de la nucléoline pourrait être impliquée dans la synthèse ou le métabolisme des pré-ARNm. / Here we have shown that, in nucleolin depleted cells, lower accumulation of pre-rRNA is associated with the increase in heterochromatin marks (H3K9me2) and decrease of the euchromatin histone marks (H4K12Ac and H3K4me3) in rDNA chromatin. ChIP-seq experiments show that nucleolin is enriched in the coding and promoter region of the rDNA and is preferentially associated with the unmethylated rRNA genes. Nucleolin knockdown results in the accumulation of RNAPI at the beginning of the rDNA and a decrease of UBF in the coding and promoter regions. Nucleolin is able to interfere with the binding of TTF-1 on the promoter-proximal terminator T0 thus inhibiting the recruitment of the NoRC subunit TIP5 and HDAC1 and establishing a repressive heterochromatin state. These results reveal the importance of nucleolin in the maintenance of the euchromatin state of rDNA and transcription elongation.In this thesis we have also shown that acetylation is a novel post-translational modification of nucleolin. Immuno-fluorescence studies using anti-acetylated nucleolin antibody illustrated that acetylated nucleolin is excluded from nucleoli and interestingly, neither could we detect any significant binding of ac-nucleolin on rDNA chromatin by doing ChIP-Seq, nor did we detect any activation of Pol II transcription with ac-nucleolin from DNA and chromatin templates. Moreover, we found acetylated nucleolin had a predominant nucleoplasmic distribution where it associates with the splicing factor SC35 and partially with the structures labeled with Y12 antibody, but not with coilin containing structures.