Tesi sul tema "Transcriptional interference"

Eukaryotic cells transcribe a vast array of non-coding RNAs, most of which have not been assigned a functional role. The work presented here reveals a novel mechanism of transcriptional repression that is mediated by the non-coding RNA prt (pho1-repressing transcript). The prt transcript is shown to recruit a histone deacetylase, Clr3, to repress pho1. This gene encodes a secreted acid phosphatase essential for phosphate acquisition in fission yeast. In the presence of phosphate, prt is produced from an upstream promoter and leads to silencing of pho1. Thus far, this has been explained by prt transcription leading to deposition of repressive methylation over the locus. However, this explanation is known to be incomplete since deletion of the only known histone methyltransferase does not lead to pho1 induction comparable to deletion of the prt promoter. This suggests that another mechanism must be involved in mediating transcriptional interference via non-coding transcription. In the present study the putative ncRNA-binding protein Seb1, together with the chromatin modifying complex SHREC, is demonstrated to associate with prt to elicit silencing of pho1 by a mechanism that is independent of H3K9 methylation and instead relies on deacetylase activity provided by the Clr3 component of SHREC. These data reveal a previously uncharacterised layer of ncRNA-mediated gene regulation and provide important conceptual advances in understanding the mechanisms governing the phenomenon known as transcriptional interference.

Gene expression of human immunodeficiency virus type-1 (HIV-1), which causes Acquired Immunodeficiency Syndrome (AIDS), is regulated at the transcriptional level, where negative factors can block elongation that is overcome by HIV Tat protein and P-TEFb. P-TEFb, a positive elongation transcription factor with two subunits, CDK9 and Cyclin T1 (CycT1), catalyzes Tat-dependent phosphorylation of Ser-5 in the Pol II C-terminal domain (CTD), allowing production of longer mRNAs. Ser-5 phosphorylation enables the CTD to recruit mammalian mRNA capping enzyme (Mce1) and stimulate its guanylyltransferase activity. This dissertation demonstrates that stable binding of Mce1 and cap methyltransferase to template-engaged Pol II depends on CTD phosphorylation, but not on nascent RNA. Capping and methylation doesn't occur until nascent pre-mRNA become 19-22 nucleotides long. A second and novel pathway for recruiting and activating Mce1 involved direct physical interaction between the CTD, Tat and Mce1. Tat stimulated the guanylyltransferase and triphosphatase activities of Mce1, thereby enhancing the otherwise low efficiency of cotranscriptional capping of HIV mRNA. These findings imply that multiple mechanisms exist for coupling transcription elongation and mRNA processing at a checkpoint critical to HIV gene expression. To elucidate P-TEFb's function in human (HeLa) cells, RNA interference (RNAi) was used to degrade mRNA for hCycT1 or CDK9. Down-regulation of P-TEFb expression by RNAi can be achieved without causing major toxic or lethal effects and can control Tat transactivation and HIV replication in host cells. High-density oligonucleotide arrays were used to determine the effect of P-TEFb knockdown on global gene expression. Of 44,928 human genes analyzed, 25 were down-regulated and known or likely to be involved in cell proliferation and differentiation. These results provide new insight into P-TEFb function, its potent role in early embryonic development and strong evidence that P-TEFb is a new target for developing AIDS and cancer therapies. To fulfill the promise of RNAi for treating infectious and human genetic diseases, structural and functional mechanisms underlying RNAi in human cells were studied. The status of the 5' hydroxyl terminus of the antisense strand of short interfering RNA (siRNA) duplexes determined RNAi activity, while a 3' terminus block was tolerated in vivo. A perfect A-form helix in siRNA was not required for RNAi, but was required for antisense-target RNA duplexes. Strikingly, crosslinking siRNA duplexes with psoralen did not completely block RNAi, indicating that complete unwinding of the siRNA helix is not necessary for RNAi in vivo. These results suggest that RNA amplification by RNA-dependent RNA polymerase is not essential for RNAi in human cells.

Retroelements are genomic parasites which make up ~42% of the human genome and 38% of the mouse genome. Most are degenerate, but a large number have relatively intact promoter elements, suggesting that they are capable of transcription. Transcriptionally active retroelements can perturb normal transcription units in their vicinity through a variety of mechanisms, leading to phenotypic effects and in some cases disease. This phenomenon of transcriptional interference has been observed in organisms as diverse as maize, Drosophila, and the mouse. We analysed the extent of retroelement transcription in normal and diseased tissues, by searching the mouse and human EST databases for transcripts originating in retroelement promoters, and found a large number of transcripts from LINEs, SINEs and ERVs. Retroelement transcripts were found to be initiated in both sense and antisense orientations, and to be equally as common in normal and diseased tissue. Several of these transcripts were chimeric, appearing to initiate in retroelements and reading through to cellular genes, suggestive of transcriptional interference. We have used transposon display to identify and recover retroelement transcripts in the mouse. Transcripts initiated in LINE, SINE and ERV promoters are numerous, and many are chimeric with cellular genes. Although the numbers of recovered chimeric transcripts are too large to permit rigorous analysis of more than a small proportion, some of those we have studied further appear to be authentic transcripts that may represent interference with the canonical promoters of the genes in question. Our results suggest that transcriptional interference by retroelements may be a relatively common occurrence in mammals.

Acute myeloid leukemia (AML) is a clonal neoplastic disorder organized as a cellular hierarchy, with the self-renewing leukemia stem cell (LSC) at the apex. Recurrent mutations in transcription factors (TF) and epigenetic regulators suggest that AML is driven by aberrant transcriptional circuits, but these circuits have not been fully defined in an LSC model. To study transcriptional mechanisms relevant to leukemogenesis in vivo, we generated a murine serial transplantation model of MLL-AF9-driven, myelomonocytic leukemia with genetically- and phenotypically-defined LSCs. Using this model, we pursued two related lines of investigation. First, we performed an in vivo RNA interference (RNAi) screen to identify transcription factors required for LSC function. This screen highlighted the circadian rhythm TFs, Clock and Bmal1, as genes essential for the survival of murine leukemia cells, and we validated this finding with CRISPR/Cas-based genome editing and knockdown studies in AML cell lines. Utilizing luciferase reporter mice to track expression of the circadian target gene Per2, we demonstrated that both leukemic and normal hematopoietic cells have the capacity for oscillating, circadian-dependent gene expression. Importantly, using murine knockout models, we found that normal hematopoietic stem and progenitor cells (HSPC), in contrast to leukemia cells, do not depend on Bmal1. We further demonstrated that selective depletion of LSCs following circadian perturbation is mediated through enhanced myeloid differentiation. ChIP-Seq studies revealed that the circadian rhythm network is integrally connected to the LSC self-renewal circuitry and highlighted putative Clock/Bmal1 targets in leukemia, providing a mechanistic basis for our findings. Second, we performed a functional and genomic characterization of our MLL-AF9 serial transplantation model to explore mechanisms of disease evolution and clonal selection in AML. Limiting dilution studies demonstrated that serial transplantation results in a reduction in disease latency, dramatic enrichment of leukemia-initiating cells (LIC), and reconfiguration of the LSC hierarchy. While mutations in known AML-associated genes were not linked to disease progression, RNA-sequencing (RNA-Seq) demonstrated that the increase in LIC frequency in serially transplanted leukemias is driven by changes in cell cycle and differentiation. In aggregate, these studies offer insights into the biological mechanisms regulating LSC self-renewal and disease evolution in AML.

A targeted approach to the development of antifolate therapies has been sought for many years. Central to the success of such development is an understanding of the molecular mechanisms dictating the sensitivity of cells to antifolates and the fundamental differences of these processes between normal and neoplastic phenotypes. This dissertation addressed transcriptional mechanisms and cell-signaling events responsible for the efficacy of antifolate therapies. Transcriptional processes and cell signaling pathways are often aberrant in neoplastic tissues, providing a potential point of distinction between a normal and neoplastic cellular state. Folylpolyglutamate synthetase (FPGS) catalyzes the formation of poly-γ-glutamate derivatives of folates and antifolates, which permits intracellular retention and accumulation of these compounds. The mouse fpgs gene uses two distant promoters to produce functionally distinct isozymes in a tissue-specific pattern. We questioned how the two promoters were differentially controlled. An analysis of DNA methylation and histone post-translational modifications across the length of the mouse fpgs gene showed that epigenetic mechanisms contributed to the tissue-specific control of the upstream (P1), but not the downstream (P2) fpgs promoter. RNAPII complexes and general transcription factors were present over P1 only when P1 was transcribed, but these components were present over P2 in most tissues, and promoter-proximal pausing was evident in brain. Clear promoter occlusion was found over P2 in liver. These studies concluded that tissue-specific coordination of dual promoters required multiple interacting controls. The mammalian target of rapamycin (mTOR) controls protein translation initiation, and is central to a cell-signaling pathway rich in tumor suppressor and oncogenic proteins. mTOR dysregulation is a common feature of several human cancers and inhibition of this protein has been sought as an ideal cancer drug target. We have determined that antifolates inhibiting the two folate-dependent steps of purine synthesis (GART or AICART) activate AMP-dependent protein kinase (AMPK) and inhibit mTOR. The mechanism of AMPK stimulation appears to be mediated by either nucleotide depletion (GART inhibitors), or ZMP accumulation (AICART inhibitors). These studies discovered a new mechanism for antifolates that surprisingly defines them as molecularly targeted therapeutics.

L'expression des gènes est finement régulée dans la cellule et soumise à de multiples contrôles-qualité. Cette régulation intervient à différents niveaux, de façon à garantir une synthèse efficace des produits fonctionnels de l'expression génique, et pour assurer une adaptation à un changement environnemental. Notamment, les régulations transcriptionnelles sont cruciales pour contrôler la cinétique et le niveau d'expression des gènes. La transcription pervasive est une transcription généralisée non-codante et instable qui fut révélée chez la levure Saccharomyces cerevisiae. Bien que son potentiel régulateur ait été démontré de façon ponctuelle, la question de sa fonctionnalité globale restait ouverte. Lors de ma thèse, j'ai pu montrer l'existence de phénomènes multiples d'interférence transcriptionnelle liés à la transcription pervasive, pour co-réguler un ensemble de gènes entre la phase exponentielle et la quiescence. En effet, la transcription non-codante en antisens des gènes concernés conduit à leur répression, dans des conditions où ils ne doivent pas être exprimés. Le mécanisme de répression fait intervenir des modifications de la chromatine. La levure bourgeonnante, dépourvue de la machinerie d'ARN interférence, présente donc un système fin de régulation de l'expression génique utilisant la transcription pervasive
In the cell, gene expression is finely tuned and is submitted to different quality-controls. Gene are regulated at different expression levels in order to guarantee a proper synthesis of functional products, and to ensure an optimal adaptation to environmental changes. In particular, transcriptional regulations are critical for gene expression level and kinetics.Pervasive transcription, defined as a generalized non-coding and unstable transcription, was discovered in the yeast Saccharomyces cerevisiae. Although its regulatory potential was punctually shown, the question of its global functionality still remained. During my PhD, I could show the existence of numerous transcriptional interference mechanisms involved in the co-regulation of a group of genes between exponential phase and quiescence. Indeed, non-coding transcription in antisense to genes promoter leads to its repression in conditions where they have to be switched off. The repression mechanism is allowed by chromatin modifications.Hence, budding yeast that lacks RNA interference machinery has developed a fine regulation system using pervasive transcription

In diverse eukaryotic organisms, double-stranded RNA (dsRNA) induces robust silencing of cellular RNA cognate to either strand of the input dsRNA; a phenomenon now known as RNA interference (RNAi). Within the RNAi pathway, small, 21 nucleotide (nt) duplexed RNA, dubbed small interfering RNAs (siRNAs), derived from the longer input dsRNA, guide the RNA induced silencing complex (RISC) to destroy its target RNA. Due to its ability to silence virtually any gene, whether endogenous or exogenous, in a variety of model organisms and systems, RNAi has become a valuable laboratory tool, and is even being heralded as a potential therapy for an array of human diseases. In order to understand this complex and unique pathway, we have undertaken the biochemical characterization of RNAi in the model insect, Drosophila melanogaster. To begin, we investigated the role of ATP in the RNAi pathway. Our data reveal several ATP-dependent steps and suggest that the RNAi reaction comprises as least five sequential stages: ATP-dependent processing of double-stranded RNA into siRNAs, ATP-independent incorporation of siRNAs into an inactive ~360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, ATP-dependent activation of RISC following siRNA unwinding, and ATP-independent recognition and cleavage of the RNA target. In addition, ATP is used to maintain 5´ phosphates on siRNAs, and only siRNAs with these characteristic 5´ phosphates gain entry into the RNAi pathway. Next, we determined that RISC programmed exogenously with an siRNA, like that programmed endogenously with microRNAs (miRNAs), is an enzyme. However, while RISC behaves like a classical Michaelis-Menten enzyme in the presence of ATP, without ATP, multiple rounds of catalysis are limited by release of RISC-produced cleavage products. Kinetic analysis of RISC suggests that different regions of the siRNA play distinct roles in the cycle of target recognition, cleavage and product release. Bases near the siRNA 5´ end disproportionately contribute to target RNA-binding energy, whereas base pairs formed by the central and 3´ region of the siRNA provide helical geometry required for catalysis. Lastly, the position of the scissile phosphate is determined during RISC assembly, before the siRNA encounters its RNA target. In the course of performing the kinetic assessment of RISC, we observed that when siRNAs are designed with regard to 'functional asymmetry' (by unpairing the 5´ terminal nucleotide of the siRNA's guide strand, i.e. the strand anti-sense to the target RNA), not all of the RISC formed was active for target cleavage. We observed, somewhat paradoxically, that increased siRNA unwinding and subsequent accumulation of single-stranded RNA into RISC led to reduced levels of active RISC formation. This inactive RISC did not act as a competitor for the active fraction. In order to characterize this non-cleaving complex, we performed a series of protein-siRNA photo-crosslinking assays. From these assays we found that thermodynamic stability and termini structure plays a role in determining which proteins an siRNA will associate with, and how association occurs. Furthermore, we have found, by means of the photo-crosslinking assays, that siRNAs commingle with components of the miRNA pathway, particularly Ago1, suggesting overlapping functions or crosstalk for factors thought to be involved in separate, distinct pathways.

L'expression des gènes est finement régulée dans la cellule et soumise à de multiples contrôles-qualité. Cette régulation intervient à différents niveaux, de façon à garantir une synthèse efficace des produits fonctionnels de l'expression génique, et pour assurer une adaptation à un changement environnemental. Notamment, les régulations transcriptionnelles sont cruciales pour contrôler la cinétique et le niveau d'expression des gènes. La transcription pervasive est une transcription généralisée non-codante et instable qui fut révélée chez la levure Saccharomyces cerevisiae. Bien que son potentiel régulateur ait été démontré de façon ponctuelle, la question de sa fonctionnalité globale restait ouverte. Lors de ma thèse, j'ai pu montrer l'existence de phénomènes multiples d'interférence transcriptionnelle liés à la transcription pervasive, pour co-réguler un ensemble de gènes entre la phase exponentielle et la quiescence. En effet, la transcription non-codante en antisens des gènes concernés conduit à leur répression, dans des conditions où ils ne doivent pas être exprimés. Le mécanisme de répression fait intervenir des modifications de la chromatine. La levure bourgeonnante, dépourvue de la machinerie d'ARN interférence, présente donc un système fin de régulation de l'expression génique utilisant la transcription pervasive
In the cell, gene expression is finely tuned and is submitted to different quality-controls. Gene are regulated at different expression levels in order to guarantee a proper synthesis of functional products, and to ensure an optimal adaptation to environmental changes. In particular, transcriptional regulations are critical for gene expression level and kinetics.Pervasive transcription, defined as a generalized non-coding and unstable transcription, was discovered in the yeast Saccharomyces cerevisiae. Although its regulatory potential was punctually shown, the question of its global functionality still remained. During my PhD, I could show the existence of numerous transcriptional interference mechanisms involved in the co-regulation of a group of genes between exponential phase and quiescence. Indeed, non-coding transcription in antisense to genes promoter leads to its repression in conditions where they have to be switched off. The repression mechanism is allowed by chromatin modifications.Hence, budding yeast that lacks RNA interference machinery has developed a fine regulation system using pervasive transcription

The endocrine heart synthesises and secretes two polypeptide hormones: the natriuretic peptides (NP) atrial natriuretic factor (ANF) and B-type natriuretic peptide (BNP). The biological actions of these hormones serve both acutely and chronically to reduce systemic blood pressure and hemodynamic load to the heart, thus contributing to the maintenance of cardiorenal homeostasis. Considerable effort has been focused on the elucidation of the mechanistic underlying ANF and BNP gene expression and secretion but much remains to be determined regarding specific molecular events involved in the cardiocyte secretory function. These hormones are produced by the atrial muscle cells (cardiocytes), which display a dual secretory/muscle phenotype. In contrast, ventricular cardiocytes display mainly a muscle phenotype. Comparatively little information is available regarding the genetic background for this important phenotypic difference with particular reference to the endocrine function of the heart. We postulated that comparison of gene expression profiles between atrial and ventricular muscles would help identify transcripts that underlie the phenotypic differences associated with the endocrine function of the heart as well as identify signaling pathways involved in its regulation. The cardiac atrial and ventricular transcriptomes were analyzed using oligonucleotide microarrays under normal or chronically induced aortocaval shunt volume-overload conditions. Transcriptional differences were validated by RT-PCR and transcripts of interest were knocked-down by RNAi. Comparison of gene expression profiles in the rat heart revealed a total of 1415 differentially expressed genes between normal atrial and ventricular tissues. Functional classification and pathway analysis identified numerous transcripts involved in mechanosensing, vesicle trafficking, hormone secretion, and G protein signaling. Volume-overloaded animals exhibited a progressive increase in cardiac mass over the four-week time course, an increase in expression of known hypertrophic genes, as well as the differential expression of 700 genes within the atria. Volume-overload specifically downregulated the accessory protein for heterotrimeric G protein signaling RASD1 in the atria. In vitro, knockdown of RASD1 in the atrial-derived HL-1 cells, significantly increased ANF secretion, demonstrating a previously unknown negative modulator role for RASD1. The data developed in this investigation provides insight into the expression profiles of genes particularly centered on the secretory function of the heart under normal and chronic hemodynamic overload conditions. Genome-wide expression profile analysis identified RASD1 as being differentially expressed between cardiac tissues as well as being modulated by chronic volume overload. RASD1 emerges as a tonic inhibitor of ANF secretion. The novel function identified herein for RASD1 in the atria is of considerable interest given the fact that secretory impairment of the cardiac natriuretic hormones can negatively impact cardiovascular homeostasis.

El establecimiento de un reservorio latente del virus del HIV en células T CD4+ es la mayor barrera contra la erradicación de esta enfermedad. Para lograr su erradicación, se necesitaría combinar la terapia antirretroviral HAART con drogas capaces de reactivar los virus durmientes. El objetivo principal de este estudio es entender cómo se establece la latencia tras la integración del virus en el genoma, con el propósito de identificar factores involucrados que pudieran ser dianas de una nueva terapia. Hemos generado una librería de clones que contienen un minigenoma latente del virus que expresa GFP como reportero cuando se reactiva. Esta librería permite estudiar la posible relación existente entre estado de la cromatina en el lugar de integración y actividad del promotor. También hemos estudiado la implicación de la interferencia transcripcional en el establecimiento de la latencia en los clones cuya integración del HIV ha tenido lugar en genes transcripcionalmente activos. Para investigar el mecanismo de represión durante la latencia, se han llevado a cabo depleciones de factores relacionados con el reensamblaje de la cromatina y proteínas relacionadas con represión transcripcional. Finalmente, hemos buscado drogas que puedan reactivar el virus latente como posible terapia a combinar con la terapia antiretroviral.
The establishment of a latent HIV reservoir in CD4+ T cells is the main barrier to prevent the eradication of the virus and converts its infection in a chronic disease. To achieve its eradication, it would be needed to combine HAART with drugs able to reactivate the dormant viruses. The main objective of this study is to understand how latency is established after proviral integration into the genome, with the aim of identifying factors involved that could be targeted by new therapeutic approaches. We have generated a library of clones containing a latent HIV minigenome that expresses GFP as a reporter only when reactivated. This library allows the study of the relationship between the chromatin state at the site of integration and HIV promoter activity. We have also studied the implication of transcriptional interference in the establishment of latency in those clones where HIV has integrated in transcriptionally active genes. To further investigate the mechanism of transcriptional repression in latency we have performed knockdowns of known chromatin reassembly factors and repression-related proteins by using shRNA expression. Finally we have searched drugs that can reactivate the latent HIV as a possible therapy to combine with HAART.

Banana bunchy top virus (BBTV) causes one of the most devastating diseases of banana. Transgenic virus resistance is now considered one of the most promising strategies to control BBTV. Pathogen-derived resistance (PDR) strategies have been applied successfully to generate plants that are resistant to numerous different viruses, primarily against those viruses with RNA genomes. BBTV is a circular, single-stranded (css) DNA virus of the family Nanoviridae, which is closely related to the family Geminiviridae. Although there are some successful examples of PDR against geminiviruses, PDR against the nanoviruses has not been reported. Therefore, the aim of this thesis was to investigate the potential of BBTV genes to interfere with virus replication when used as transgenes for engineering banana plants resistance to BBTV. The replication initiation protein (Rep) of nanoviruses is the only viral protein essential for viral replication and represents an ideal target for PDR. Therefore, this thesis focused on the effect of wild-type or mutated Rep genes from BBTV satellite DNAs or the BBTV integral genome on the replication of BBTV in banana embryogenic cell suspensions. A new Rep-encoding satellite DNA, designated BBTV DNA-S4, was isolated from a Vietnamese BBTV isolate and characterised. When the effect of DNA-S4 on the replication of BBTV was examined, it was found that DNA-S4 enhanced the replication of BBTV. When the replicative capabilities of DNA-S4 and the previously characterised Rep-encoding BBTV satellite, DNA-S1, were compared, it was found that the amount of DNA-S4 accumulated to higher levels than DNA-S1. The interaction between BBTV and DNA-S1 was also examined. It was found that over-expression of the Rep encoded by DNA-S1 using ubi1 maize polyubiquitin promoter enhanced replication of BBTV. However, when the Rep-encoded by DNA-S1 was expressed by the native S1 promoter (in plasmid pBT1.1-S1), it suppressed the replication of BBTV. Based on this result, the use of DNA-S1 as a possible transgene to generate PDR against BBTV was investigated. The roles of the Rep-encoding and U5 genes of BBTV DNA-R, and the effects of over-expression of these two genes on BBTV replication were also investigated. Three mutants of BBTV DNA-R were constructed; plasmid pUbi-RepOnly-nos contained the ubi1 promoter driving Rep expression from DNA-R, plasmid pUbi-IntOnly-nos contained the ubi1 promoter driving expression of the DNA-R internal gene product (U5), while plasmid pUbi-R.ORF-nos contained the ubi1 promoter driving the expression of both Rep and the internal U5 gene product. The replication of BBTV was found to be significantly suppressed by pUbi-RepOnly-nos, weakly suppressed by pUbi-IntOnly-nos, but strongly enhanced by pUbi-R.ORF-nos. The effect of mutations in three conserved residues within the BBTV Rep on BBTV replication was also assessed. These mutations were all made in the regions in the ATPase motifs and resulted in changes from hydrophilic to hydrophobic residues (i.e. K187→M, D224→I and N268→L). None of these Rep mutants was able to initiate BBTV replication. However, over-expression of Reps containing the K187→M or N268→L mutations significantly suppressed the replication of BBTV. In summary, the Rep constructs that significantly suppressed replication of DNA-R and -C in banana embryogenic cell suspensions have the potential to confer resistance against BBTV by interfering with virus replication. It may be concluded that BBTV satellite DNAs are not ideal for conferring PDR because they did not suppress BBTV replication consistently. Wild-type Rep transcripts and mutated (i.e. K187→M and N248→L) Rep proteins of BBTV DNA-R, however, when over-expressed by a strong promoter, are all promising candidates for generating BBTV-resistant banana plants.

Banana bunchy top virus (BBTV) causes one of the most devastating diseases of banana. Transgenic virus resistance is now considered one of the most promising strategies to control BBTV. Pathogen-derived resistance (PDR) strategies have been applied successfully to generate plants that are resistant to numerous different viruses, primarily against those viruses with RNA genomes. BBTV is a circular, single-stranded (css) DNA virus of the family Nanoviridae, which is closely related to the family Geminiviridae. Although there are some successful examples of PDR against geminiviruses, PDR against the nanoviruses has not been reported. Therefore, the aim of this thesis was to investigate the potential of BBTV genes to interfere with virus replication when used as transgenes for engineering banana plants resistance to BBTV. The replication initiation protein (Rep) of nanoviruses is the only viral protein essential for viral replication and represents an ideal target for PDR. Therefore, this thesis focused on the effect of wild-type or mutated Rep genes from BBTV satellite DNAs or the BBTV integral genome on the replication of BBTV in banana embryogenic cell suspensions. A new Rep-encoding satellite DNA, designated BBTV DNA-S4, was isolated from a Vietnamese BBTV isolate and characterised. When the effect of DNA-S4 on the replication of BBTV was examined, it was found that DNA-S4 enhanced the replication of BBTV. When the replicative capabilities of DNA-S4 and the previously characterised Rep-encoding BBTV satellite, DNA-S1, were compared, it was found that the amount of DNA-S4 accumulated to higher levels than DNA-S1. The interaction between BBTV and DNA-S1 was also examined. It was found that over-expression of the Rep encoded by DNA-S1 using ubi1 maize polyubiquitin promoter enhanced replication of BBTV. However, when the Rep-encoded by DNA-S1 was expressed by the native S1 promoter (in plasmid pBT1.1-S1), it suppressed the replication of BBTV. Based on this result, the use of DNA-S1 as a possible transgene to generate PDR against BBTV was investigated. The roles of the Rep-encoding and U5 genes of BBTV DNA-R, and the effects of over-expression of these two genes on BBTV replication were also investigated. Three mutants of BBTV DNA-R were constructed; plasmid pUbi-RepOnly-nos contained the ubi1 promoter driving Rep expression from DNA-R, plasmid pUbi-IntOnly-nos contained the ubi1 promoter driving expression of the DNA-R internal gene product (U5), while plasmid pUbi-R.ORF-nos contained the ubi1 promoter driving the expression of both Rep and the internal U5 gene product. The replication of BBTV was found to be significantly suppressed by pUbi-RepOnly-nos, weakly suppressed by pUbi-IntOnly-nos, but strongly enhanced by pUbi-R.ORF-nos. The effect of mutations in three conserved residues within the BBTV Rep on BBTV replication was also assessed. These mutations were all made in the regions in the ATPase motifs and resulted in changes from hydrophilic to hydrophobic residues (i.e. K187→M, D224→I and N268→L). None of these Rep mutants was able to initiate BBTV replication. However, over-expression of Reps containing the K187→M or N268→L mutations significantly suppressed the replication of BBTV. In summary, the Rep constructs that significantly suppressed replication of DNA-R and -C in banana embryogenic cell suspensions have the potential to confer resistance against BBTV by interfering with virus replication. It may be concluded that BBTV satellite DNAs are not ideal for conferring PDR because they did not suppress BBTV replication consistently. Wild-type Rep transcripts and mutated (i.e. K187→M and N248→L) Rep proteins of BBTV DNA-R, however, when over-expressed by a strong promoter, are all promising candidates for generating BBTV-resistant banana plants.

The main object of this thesis is the inference of gene regulation from quantitative gene expression data. This goal is of central importance for health care as complex genetic diseases such as cancer are caused by deregulation or aberrant regulation of genes. The thesis is structured into two main parts corresponding to a theoretical and a practical approach to the inference of gene regulation. In the first part of the thesis a Bayesian hierarchical model for the reconstruction of regulatory networks from gene expression microarray data is presented. In the second part of the thesis a novel experimental design to infer gene regulatory modules from real-time PCR data is presented. The aim is to characterize the IFNa-transcriptional response in human endothelial cells, by identifying key modulators and regulatory modules in which they are involved.
L'oggetto principale di questa tesi è l'inferenza di regolazioni geniche a partire da dati quantitativi di espressione genica. Questo obiettivo è di centrale importanza in ambito sanitario poiché malattie genetiche complesse come il cancro sono causate dalla deregolazione o dalla regolazione aberrante di geni. La tesi è strutturata in due parti principali corrispondenti, rispettivamente, ad un approccio teorico e pratico all'inferenza di regolazioni geniche. Nella la prima parte della tesi viene presentato un modello gerarchico bayesiano per la ricostruzione di reti di regolazione da dati di microarray. Nella seconda parte della tesi viene presentato un nuovo design sperimentale per inferire moduli di regolazione genica da dati di real-time PCR. L'obiettivo è quello di caratterizzare la risposta trascrizionale di IFN- in cellule endoteliali umane, attraverso l'individuazione di modulatori chiave e dei moduli regolatori in cui sono coinvolti.

Tumormodelle, in denen die maligne Transformation durch definierte Onkogene experimentell ausgelöst und unterhalten wird, bieten vielfältige Möglichkeiten, die komplexen Mechanismen der Tumorentstehung und Therapieresistenz zu untersuchen und neue Ansätze für Diagnostik und Therapie auszuarbeiten. KRAS-Onkogen-„getriebene“ Transformationsmodelle spiegeln neben anderen tumorspezifischen Veränderungen insbesondere die charakteristischen Änderungen des Transkriptoms wider. In der vorliegenden Arbeit wird ein Modell für Ovarialtumore auf Grundlage von Rose Zellen („Rat ovarian surface epithelium“) verwendet, um die Rolle von Transkriptionsfaktoren, welche durch die KRAS-vermittelte Signaltransduktion hoch reguliert werden, zu untersuchen. Die KRAS-transfomierten Derivate der normalen Rose Zellen zeigen die typischen Merkmale von ankerunabhängigen und invasiven Tumorzellen. Aufgrund der hohen Komplexität sind die Interaktionen zwischen der zytoplasmatischen Signaltransduktion und dem durch sie regulierten Transkriptionsfaktornetzwerk noch weitgehend unverstanden. Die Transkriptionsfaktoren Fosl1, Hmga2, Klf6, JunB, Otx1, Gfi1 und RelA wurden systematisch mittels RNA-Interferenz in KRAS-transformierten Rose Zellen transient ausgeschaltet. Danach wurden Proliferation, Morphologie (epithelial-mesenchymale Transition, EMT) und Ankerunabhängigkeit der Zellen bestimmt. Alle untersuchten Transkriptionsfaktoren beeinflussten die KRAS-induzierten morphologischen Veränderungen teilweise, belegt durch die Abnahme der EMT-Merkmale nach siRNA-vermittelter Ausschaltung. Der Knock-down der Transkriptionsfaktoren Otx1, Gfi1 und RelA hemmte die Proliferation, während Fosl1, Hmga2, Klf6 und JunB die generelle Proliferationsfähigkeit nicht beeinflussten, jedoch spezifisch die ankerunabhängige Proliferation blockierten. Diesen Faktoren kommt daher eine spezifische Funktion in der neoplastischen Transformation zu, da die Ankerunabhängigkeit sehr gut mit der Tumorigenität korreliert ist. Um die Beteiligung der Transkriptionsfaktoren an der Deregulation von Zielgenen zu erfassen, wurden Genexpressionsmuster aller Zellen, in denen jeweils ein Faktor durch siRNA ausgeschaltet war, mittels Microarray-Analyse identifiziert. Auf dieser Grundlage wurde ein Netzwerk-Modell der regulatorischen Interaktionen zwischen den Transkriptionsfaktoren berechnet. Die Existenz der beiden funktionellen Gruppen wurde im Modell bestätigt. Darüber hinaus zeigte sich eine gegenseitige Abhängigkeit des transkriptionellen Netzwerks und der zytoplasmatischen Signaltransduktion, gemessen mittels Proteinanalyse der mitogenabhängigen Signalkinasen (MAPK). Diese wird als kompensatorische Regulation interpretiert, welche trotz Pertubation, experimentell durch siRNA, das effiziente Überleben der transformierten Zellen sicherstellt. Die vorliegende Studie schafft somit die Voraussetzung und Motivation, das reduzierte Netzwerk aus sieben Komponenten auf alle differentiell exprimierten Transkriptionsfaktoren zu erweitern. Möglicherweise behindern solche Regulationskreise in der klinischen Situation die effektive Wirkung zielgerichteter Therapien.
Tumor models, in which malignant transformation was experimentally triggered and maintained through defined oncogenes, offer manifold opportunities to determine the complex mechanisms of tumor progression and resistance to therapies, and to develop new strategies for diagnosis and therapy. Particularly, KRAS oncogene driven models of transformation reflect the characteristic alterations of the transcriptome, among other tumor specific changes. In the present work a model for ovarian cancer based on Rose („Rat ovarian surface epithelium“) cells has been used to evaluate the role of transcription factors, which are up-regulated through KRAS dependent signaling. The KRAS transformed derivates of normal ROSE cells exhibit typical characteristics of anchorage-independent and invasive tumor cells. Due to the high complexity of cellular networks, the interactions between cytoplasmic signalling and their regulated transcription factors are not well understood. The transcription factors Fosl1, Hmga2, Klf6, JunB, Otx1, Gfi1 and RelA were systematically eliminated by transient RNA interference in KRAS transformed ROSE cells. The proliferation, morphology (epithelial-mesenchymal transition, EMT) and anchorage-independence of the cells were determined. All of the selected transcription factors had partial effect on the KRAS induced morphologic changes, documented by reduction of EMT-properties after siRNA treatment. The knock-down of the transcription factors Otx1, Gfi1 and RelA blocked proliferation in general, whereas Fosl1, Hmga2, Klf6 and JunB had no influence on proliferation but specifically blocked the anchorage-independence. Thus, these factors exhibited essential functions in the process of neoplastic transformation, because the anchorage-independence correlates very well with tumorigenicity. In order to elucidate the involvement of the transcription factors in the genetic deregulation of their target genes, microarray based gene expression profiles were determined from all cells in which one factor was eliminated by siRNA. Based on these data, a network model of regulatory interactions among these transcription factors was calculated. The existence of both functional groups was confirmed by the model. Furthermore, an interdependence of the transcriptional networks and cytoplasmatic signaling was observed by protein analysis of the mitogen dependent signal kinases (MAPK). This was interpreted as compensatory regulation, which in spite of experimental perturbation by siRNA, permitted efficient survival of the transformed cells. Thus, the present work provides the basis and motivation to extend the reduced network composed of seven components to all regulated transcription factors. Potentially, such regulatory networks diminish the efficacy of targeted therapies in clinical situations.

For self-renewal, embryonic stem cells (ESCs) require the expression of specific transcription factors accompanied by a particular chromosome organization to maintain a balance between pluripotency and the capacity for rapid differentiation. However, how transcriptional regulation is linked to chromosome organization in ESCs is not well understood. Here we show that the cohesin component RAD21 exhibits a functional role in maintaining ESC identity through association with the pluripotency transcriptional network. ChIP-seq analyses of RAD21 reveal an ESC specific cohesin binding pattern that is characterized by CTCF independent co-localization of cohesin with pluripotency related transcription factors Oct4, Nanog, Sox2, Esrrb and Klf4. Upon ESC differentiation, most of these binding sites disappear and instead new CTCF independent RAD21 binding sites emerge, which are enriched for binding sites of transcription factors implicated in early differentiation. Furthermore, knock-down of RAD21 causes expression changes that are similar to expression changes after Nanog depletion, demonstrating the functional relevance of the RAD21 - pluripotency transcriptional network association. Finally, we show that Nanog physically interacts with the cohesin or cohesin interacting proteins STAG1 and WAPL further substantiating this association. Based on these findings we propose that a dynamic placement of cohesin by pluripotency transcription factors contributes to a chromosome organization supporting the ESC expression program.

For self-renewal, embryonic stem cells (ESCs) require the expression of specific transcription factors accompanied by a particular chromosome organization to maintain a balance between pluripotency and the capacity for rapid differentiation. However, how transcriptional regulation is linked to chromosome organization in ESCs is not well understood. Here we show that the cohesin component RAD21 exhibits a functional role in maintaining ESC identity through association with the pluripotency transcriptional network. ChIP-seq analyses of RAD21 reveal an ESC specific cohesin binding pattern that is characterized by CTCF independent co-localization of cohesin with pluripotency related transcription factors Oct4, Nanog, Sox2, Esrrb and Klf4. Upon ESC differentiation, most of these binding sites disappear and instead new CTCF independent RAD21 binding sites emerge, which are enriched for binding sites of transcription factors implicated in early differentiation. Furthermore, knock-down of RAD21 causes expression changes that are similar to expression changes after Nanog depletion, demonstrating the functional relevance of the RAD21 - pluripotency transcriptional network association. Finally, we show that Nanog physically interacts with the cohesin or cohesin interacting proteins STAG1 and WAPL further substantiating this association. Based on these findings we propose that a dynamic placement of cohesin by pluripotency transcription factors contributes to a chromosome organization supporting the ESC expression program.

The development of RNAi based technologies has given researchers the tools to interrogate processes as diverse as cancer biology, metabolism and organ development. Here I employ genome-wide shRNA screens to discover the genes involved in two different processes in carcinogenesis, oncogene-induced senescence [OIS] and epigenetic silencing of tumor suppressor genes [TSGs]. OIS is a poorly studied yet significant tumor suppressing mechanism in normal cells where they enter cell cycle arrest [senescence] or programmed cell death [apoptosis] in the presence of an activated oncogene. Here I employ a genomewide shRNA screen and identify a secreted protein, IGFBP7, that induces senescence and apoptosis in melanocytes upon introduction of the oncogene BRAFV600E. Expression of BRAFV600E in primary cells leads to synthesis and secretion of IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce senescence and apoptosis. Apoptosis results from IGFBP7-mediated upregulation of BNIP3L, a proapoptotic BCL2 family protein. Recombinant IGFBP7 has potent pro-apoptotic and anti-tumor activity in mouse xenograft models using BRAFV600E-postive melanoma cell lines. Finally, IGFBP7 is epigenetically silenced in human melanoma samples suggesting IGFBP7 expression is a key barrier to melanoma formation. Next I investigated the factors involved in epigenetic silencing in cancer. The TSG p14ARFis inactivated in a wide range of cancers by promoter hypermethylation through unknown mechanisms. To discover p14ARF epigenetic silencing factors, I performed a genome-wide shRNA screen and identified ZNF304, a zinc finger transcription factor that contains a Krüppel-associated box [KRAB] repressor domain. I show that ZNF304 binds to the p14ARF promoter and recruits a KRAB co-repressor complex containing KAP1, SETDB1 and DNMT1 for silencing. We find oncogenic RAS signaling to promote the silencing of p14ARF by USP28-mediated stabilization of ZNF304. In addition I find ZNF304 to be overexpressed in human colorectal cancers and responsible for hypermethylation of over 50 TSGs known as Group 2 CIMP marker genes. My findings establish ZNF304 as a novel oncogene that directs epigenetic silencing and facilitates tumorigenicity in colorectal cancer.

The development of RNAi based technologies has given researchers the tools to interrogate processes as diverse as cancer biology, metabolism and organ development. Here I employ genome-wide shRNA screens to discover the genes involved in two different processes in carcinogenesis, oncogene-induced senescence [OIS] and epigenetic silencing of tumor suppressor genes [TSGs]. OIS is a poorly studied yet significant tumor suppressing mechanism in normal cells where they enter cell cycle arrest [senescence] or programmed cell death [apoptosis] in the presence of an activated oncogene. Here I employ a genomewide shRNA screen and identify a secreted protein, IGFBP7, that induces senescence and apoptosis in melanocytes upon introduction of the oncogene BRAFV600E. Expression of BRAFV600E in primary cells leads to synthesis and secretion of IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce senescence and apoptosis. Apoptosis results from IGFBP7-mediated upregulation of BNIP3L, a proapoptotic BCL2 family protein. Recombinant IGFBP7 has potent pro-apoptotic and anti-tumor activity in mouse xenograft models using BRAFV600E-postive melanoma cell lines. Finally, IGFBP7 is epigenetically silenced in human melanoma samples suggesting IGFBP7 expression is a key barrier to melanoma formation. Next I investigated the factors involved in epigenetic silencing in cancer. The TSG p14ARFis inactivated in a wide range of cancers by promoter hypermethylation through unknown mechanisms. To discover p14ARF epigenetic silencing factors, I performed a genome-wide shRNA screen and identified ZNF304, a zinc finger transcription factor that contains a Krüppel-associated box [KRAB] repressor domain. I show that ZNF304 binds to the p14ARF promoter and recruits a KRAB co-repressor complex containing KAP1, SETDB1 and DNMT1 for silencing. We find oncogenic RAS signaling to promote the silencing of p14ARF by USP28-mediated stabilization of ZNF304. In addition I find ZNF304 to be overexpressed in human colorectal cancers and responsible for hypermethylation of over 50 TSGs known as Group 2 CIMP marker genes. My findings establish ZNF304 as a novel oncogene that directs epigenetic silencing and facilitates tumorigenicity in colorectal cancer.

RNA interference (RNAi) is a conserved pathway that plays key roles in heterochromatin formation, gene regulation and genome surveillance across a wide range of eukaryotes. One of the most utilised model organisms for studying the RNAi pathway is the fission yeast Schizosaccharomyces pombe. However, this species is somewhat atypical, in that it has not retained the ancestral role for RNAi in the silencing of mobile genetic elements. In contrast, the related fission yeast S. japonicus has a large and diverse retrotransposon complement that appears to give rise to abundant siRNAs. For this reason, we believe that S. japonicus may be a more suitable model for studying the role of RNAi in silencing mobile genetic elements, a function that is conserved in many higher eukaryotes. Functional analysis of the S. japonicus RNAi pathway proved more challenging than expected, as it was generally not possible to recover strains bearing deletions of core RNAi components (Ago1/Clr4/Rdp1/Arb1/Arb2). This suggests that a functional RNAi pathway may be required for viability in S. japonicus, unlike in S. pombe. However, disruption mutants were isolated for the sole Dicer ribonuclease Dcr1, at very low frequency. Analysis of these mutants revealed that disruption of Dcr1 impaired the generation of retrotransposon derived siRNAs, and caused de-repression of retroelement transcript accumulation and mobilisation in an element dependent manner. Surprisingly however, Dcr1 appeared dispensable for the maintenance of H3K9me2 at transposons, suggesting that, in contrast to S. pombe, silencing may occur principally at the post-transcriptional level. It is also possible that the isolated Dcr1 mutants represent rare survivors that are viable due to the presence of suppressor mutations elsewhere in the genome. I utilised my genome wide RNA sequencing data to help improve the annotation of the S. japonicus genome, with a specific focus on the retrotransposon complement. From this, I identified 12 new families of LTR retrotransposon, which increased the annotated retrotransposon complement by around 40% in S. japonicus. Finally, I characterised the integrative preference of the S. japonicus retrotransposon Tj1, and found that it shares characteristics associated with the S. cerevisiae retrotransposons Ty1 and Ty3, mostly integrating upstream of RNA PolIII transcribed tRNA genes. The findings of this work highlight some potentially key differences in the way the RNAi pathway functions across the fission yeast clade, both in terms of its importance for viability and its mode of action. The work undertaken here also contributes to the establishment of S. japonicus as a model for the study of RNA interference and genome regulation.

Hintergrund: Der Transkriptionsfaktor GATA-4 ist für die normale Entwicklung des Endoderms essentiell. Mausmutanten mit einer homozygoten Deletion des gata-4 Gens versterben zwischen den embryonalen Tagen 8.5 - 10.5 aufgrund einer Störung der ventralen Morphogenese und der Ausbildung des Herzschlauches. Zielsetzung und experimentelle Strategie: Um die Bedeutung von GATA-4 auch nach der embryonalen Entwicklung untersuchen zu können, wurden doppelt-transgene Mäuse generiert. Diese Mausmutanten exprimieren einen Tetrazyklin-Repressor und eine gegen GATA-4 gerichtete short hairpin RNA (shGATA-4). Die Expression der shGATA-4 steht dabei unter der Kontrolle eines H1-Promotors, welcher durch ein Tetrazyklin-Operator Element modifiziert wurde. Dadurch ist das System durch Doxyzyklin induzierbar. Ergebnisse: Die Integration der Transgene in dem Genom der Maus wurde durch Southern-Blot Analyse nachgewiesen. Die Expression der shGATA-4 wurde durch die Applikation von Doxyzyklin über das Trinkwasser (20 mg/ml) induziert. Langzeitstudien am Herzen haben dabei eine signifikante Suppression von GATA-4 nach 38 Tagen ergeben (80 %). Diese Reduktion konnte durch Western-Blot Analyse bestätigt werden. Obwohl die Expression verschiedener Zielgene von GATA-4 (ANF, BMP-4) ebenfalls herunterreguliert war, fiel bei den transgenen Mäusen kein kardialer Phänotyp auf. Jedoch wurde die GATA-4 Expression in den Hoden und Ovarien transgener Mäuse supprimiert, nachdem shGATA-4 durch die Applikation von Doxyzyklin induziert wurde. Weiterführende Untersuchungen an adulten Mäusen zeigten eine GATA-4 Reduktion von 20 % auch in nicht mit Doxyzyklin-induzierten Mausmutanten. Diese Reduktion könnte durch einen sog. leaky-Effekt des shGATA-4 Transgens hervorgerufen worden sein, wodurch die stark eingeschränkte Fertilität dieser Mauslinie erklärt werden könnte. Interessanterweise haben ca. 10 % der mit Doxyzyklin behandelten transgenen Weibchen Ovarial-Teratome ausgebildet. Histologisch wiesen diese Teratome überwiegend (neuro-) ektodermale, vereinzelt mesodermale und nahezu keine endodermalen Strukturen auf. Schlussfolgerung: In diesem Modell hat die Suppression von GATA-4 keinen Einfluss auf die Funktion des Herzens der adulten Maus. Jedoch scheint GATA-4 für die Fertilität der Maus von großer Bedeutung zu sein. Weiterhin scheint die Suppression von GATA-4 mit der Ausbildung von Ovarial-Teratomen assoziiert zu sein.
Background: The transcription factor GATA-4 is crucial for the normal endodermal development. In mice, homozygous deficiency of GATA-4 causes defects in ventral morphogenesis and heart tube formation, resulting in embryonic death between day e8.5 and e10.5. Aim and experimental strategy: To analyze the implication of GATA-4 beyond embryonic development a double transgenic mouse expressing the tetracycline repressor (TetR) and an inducible small interfering RNA directed against GATA-4 was generated. This expression construct contains a H1 promoter modified with a tetracycline operator upstream of the coding region for the GATA-4 short hairpin RNA (shGATA-4). Results: The integration of the transgenes in FvB mice (H1:G4/TetR) was confirmed by Southern blot. To induce the expression of the shGATA-4 construct, transgenic mice were treated with doxycycline (20 mg/ml drinking water). In longitudinal analysis, most efficient GATA-4 suppression was detected after 38 days. Quantitative PCR revealed a GATA-4 reduction of about 80 % in the heart, if normalized against the wildtype. Reduction of GATA-4 was confirmed by Western Blot. Although GATA-4 target genes (ANP, BMP-4) were down regulated, the animals showed no clinical phenotype. In opposite to wildtype mice, GATA-4 expression was undetectable in the ovaries and testis of transgenic mice with induced shGATA-4. Additional analysis in adult transgenic mice, which were not treated with doxycycline, also showed a reduction of GATA-4 expression of about 20 %, probably caused by a leaky-effect of the transgene. This may explain the significantly reduced fertility of the colony. Importantly, 10 % of transgenic females treated with doxycycline developed ovarian teratomas. Histological examination of teratomas showed predominantly (neuro-) ectodermal and to a lower degree mesodermal, but almost no endodermal compounds. Conclusions: GATA-4 reduction in the adult murine heart is – at least to a certain degree – clinically redundant. GATA-4 seems to be required for normal fertility. In our model GATA-4 deficiency seems to be associated with an increased risk for developing ovarian teratoma.

Proper development of skeletal muscle occurs through a highly complex process where activation and repression of genes are essential. Control of this process is regulated by timely and spatial expression of specific transcription factors (TFs). Six1 and Six4 are homeodomain TFs known to be essential for skeletal muscle development in mice. Using the C2C12 cell line, a model for skeletal muscle differentiation, I used a functional genomics approach, employing siRNA specific to both these TFs, to characterize their role in skeletal myogenesis. To identify the genes that are regulated by both these TFs, gene expression profiling by microarray of cells treated with siRNA against Six1 and/or Six4 was performed. The knock-down of these TFs caused lower expression of markers of terminal differentiation genes in addition to an impairment of myoblast fusion and differentiation. Interestingly, transcript profiling of cells treated with siRNA against myogenin revealed that several of the Six1 and Six4 target genes are also regulated by myogenin. Through a combination of bioinformatic analyses it was also found that specific knock-down of Six4 causes an up-regulation of genes involved in mitosis and the cell cycle. In summary, these results show that Six1 and Six4 can both independently regulate different genes, but can also cooperate together with other TFs where they play an important role in the proper regulation of skeletal myogenesis.

Recent breakthroughs in creating induced pluripotent stem cells (iPS cells) provide alternative means to obtain embryonic stem (ES) cell-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. However, the molecular basis of reprogramming is largely unknown. To address this question, we employed microRNAs, small molecules, and conducted genome-wide RNAi screen, to investigate the regulatory mechanisms of reprogramming. First we showed that depleting miR-21 and miR-29a enhances reprogramming in mouse embryonic fibroblasts (MEFs). We also showed that p53 and ERK1/2 pathways are regulated by miR-21 and miR-29a and function in reprogramming. Second, we showed that computational chemical biology combined with genomic analysis can be used to identify small molecules regulating reprogramming. We discovered that the NSAID Nabumetone and the anti-cancer drug OHTM could replace Sox2 during reprogramming. Nabumetone could also replace c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPS cells. To identify the cell-fate determinants during reprogramming, we integrated a genome-wide RNAi screen with transcriptome analysis to dissect the molecular requirements in reprogramming. We found that extensive interactions of embryonic stem cell core circuitry regulators are established in mature iPS cells, including Utf1, Nr6a1, Tdgf1, Gsc, Fgf10, T, Chrd, Dppa3, Fgf17, Eomes, Foxa2. Remarkably, genes with non-differential change play the most critical roles in the transitions of reprogramming. Functional validation showed that some genes act as essential or barrier roles to reprogramming. We also identified several genes required for maintaining ES cell properties. Altogether, our results demonstrate the significance of miRNA function in regulating multiple signaling networks involved in reprogramming. And our work further advanced the reprogramming field by identifying several new key modulators.

Recent breakthroughs in creating induced pluripotent stem cells (iPS cells) provide alternative means to obtain embryonic stem (ES) cell-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. However, the molecular basis of reprogramming is largely unknown. To address this question, we employed microRNAs, small molecules, and conducted genome-wide RNAi screen, to investigate the regulatory mechanisms of reprogramming. First we showed that depleting miR-21 and miR-29a enhances reprogramming in mouse embryonic fibroblasts (MEFs). We also showed that p53 and ERK1/2 pathways are regulated by miR-21 and miR-29a and function in reprogramming. Second, we showed that computational chemical biology combined with genomic analysis can be used to identify small molecules regulating reprogramming. We discovered that the NSAID Nabumetone and the anti-cancer drug OHTM could replace Sox2 during reprogramming. Nabumetone could also replace c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPS cells. To identify the cell-fate determinants during reprogramming, we integrated a genome-wide RNAi screen with transcriptome analysis to dissect the molecular requirements in reprogramming. We found that extensive interactions of embryonic stem cell core circuitry regulators are established in mature iPS cells, including Utf1, Nr6a1, Tdgf1, Gsc, Fgf10, T, Chrd, Dppa3, Fgf17, Eomes, Foxa2. Remarkably, genes with non-differential change play the most critical roles in the transitions of reprogramming. Functional validation showed that some genes act as essential or barrier roles to reprogramming. We also identified several genes required for maintaining ES cell properties. Altogether, our results demonstrate the significance of miRNA function in regulating multiple signaling networks involved in reprogramming. And our work further advanced the reprogramming field by identifying several new key modulators.

Orientador: Kleber Gomes Franchini
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Doenças do coração são freqüentemente associadas à hipertrofia miocárdica. Estímulos mecânicos induzem o crescimento hipertrófico e contribuem para a degeneração e morte dos miócitos cardíacos. Dentre os fatores de transcrição envolvidos no processo de hipertrofia miocárdica, estão os da família MEF2 (Myocyte Enhancer Factor-2), que é composto por 4 membros, MEF2A, B, C e D. O MEF2C é descrito como o principal transcrito no miocárdio. Tanto a deleção quanto a hiperexpressão de seu gene causam efeitos deletérios na formação e na função do músculo cardíaco. Estudos anteriores do nosso laboratório demonstraram que o MEF2 é ativado por estiramento de cardiomiócitos e influencia a expressão de genes do programa hipertrófico. O presente estudo tem como objetivo avaliar os efeitos do silenciamento gênico do MEF2C nas alterações estruturais e funcionais do ventrículo esquerdo de camundongos submetidos à sobrecarga pressórica. Para isso, utilizamos a técnica de interferência por RNA para o MEF2C. A padronização constituiu de: 1) avaliação do silenciamento do MEF2C em cultura de células C2C12 e no ventrículo esquerdo de camundongos Swiss; 2) determinação da dose necessária de siRNA para o silenciamento da expressão protéica do MEF2C; 3) determinação do curso temporal do silenciamento; 4) avaliação dos efeitos do tratamento com molécula irrelevante de siRNA direcionada para a proteína exógena GFP; 5) avaliação da especificidade do silenciamento (off-targets) pela análise do RNAm para o MEF2A e das proteínas FAK, GAPDH, JNK1/2 e SHP2; 6) avaliação do silenciamento em outros órgãos, como pulmão e rim; 7) avaliação da efetividade do silenciamento de MEF2C em miócitos cardíacos isolados do ventrículo esquerdo de camundongos. O tratamento com siRNA diminuiu a expressão protéica do MEF2 em 70% das células C2C12. Também verificamos que o tratamento com siRNA silenciou 85% da expressão protéica e do RNAm do MEF2C no ventrículo esquerdo de camundongos em até 4 dias de seguimento. Não foi verificada alteração na expressão de RNAm para o MEF2A e das proteínas FAK, GAPDH, JNK1/2 e SHP2. O silenciamento foi efetivo no pulmão e nos cardiomiócitos isolados do ventrículo esquerdo de camundongos tratado com siRNAMEF2C. Após a padronização do silenciamento, procedeu-se à determinação dos efeitos do silenciamento na estrutura e na função do ventrículo esquerdo de camundongos submetidos à sobrecarga pressórica crônica. Para isso, realizaram-se as análises ecocardiográfica, hemodinâmica, gravimétrica e morfométrica do ventrículo esquerdo de camundongos submetidos à coarctação da aorta com seguimento de 15 dias. Demonstramos que o tratamento com siRNAMEF2C atenuou a hipertrofia cardíaca nos animais coarctados. Esta conclusão foi baseada em dados de ecocardiografia que revelaram menor espessura da parede posterior (30% menor) e por gravimetria que revelou atenuação de aproximadamente 45% da massa do ventrículo esquerdo. Apesar de ter havido aumento do gradiente sistólico nos animais coarctados, a pressão arterial sistêmica não apresentou diferença estatisticamente significativa com o tratamento do siRNAMEF2C. Morfologicamente, o siRNA atenuou a fração de colágeno no ventrículo esquerdo de camundongos coarctado com 15 dias de seguimento. Entretanto, o diâmetro dos miócitos e o infiltrado de células inflamatórias foram comparáveis dentre os grupos. Somente os animais coarctados por 24 horas tiveram maior expressão de ß- MHC, e quando tratados com siRNAMEF2C apresentaram menor razão ATP/ADP. Dessa forma, esses dados sugerem que o MEF2C regula múltiplos aspectos da hipertrofia cardíaca induzida por sobrecarga pressórica tais como a expressão de genes sarcoméricos e genes envolvidos na adaptação metabólica do músculo cardíaco.
Abstract: Heart diseases are frequently associated with myocardial hypertrophy. Mechanical stimuli can trigger hypertrophic growth as well as degeneration and death of the cardiac myocytes. The MEF2C family of transcription factors plays a role in the process of myocardial hypertrophy. It is composed by 4 members, MEF2A, B, C and D, and the MEF2C is the main transcript in the heart. Both the deletion and overexpression of mef2c induce deleterious effects in the formation and function of the heart. Previous studies of the our laboratory has shown that the transcription factor MEF2C is activated by mechanical stretch in cardiomyocytes and regulates the expression of genes related to cardiac hypertrophy. This study was performed to address the effects of MEF2C gene silencing in the structural and functional changes of the left ventricle (LV) induced by pressure overload in mice. To silence MEF2C, it was employed the RNA interference technique, specific siRNA target to MEF2C was administered through the mice jugular vein. To optimize the MEF2C knockdown, it was necessary to 1) analyze the MEF2C silencing in C2C12 cells, 2) determine the dose required to induce significant MEF2C silencing in LV of mice, 3) determine the time course of gene silencing, 4) assess the effects of the treatment with irrelevant siRNA target to the protein GFP, 5) evaluate the specificity of gene silencing by siRNAMEF2C through the expression analysis of the transcription factor MEF2A and other non-related proteins, 6) analyze of the MEF2C knockdown in other organs, 7) determine the effectiveness of the MEF2C silencing in cardiac myocytes harverst from the LV of mice treated systemically with siRNAMEF2C. Treatment with 100ng/mL of siRNAMEF2C induced MEF2C silencing (~70%) in C2C12 cells. Intrajugular delivery of 30µg of siRNAMEF2C in mice induced the reduction in the mRNA and protein levels (~85%) until 4 days after the injection. The treatment with siRNAMEF2C did not affect the expression of MEF2A and other non-related proteins. The MEF2C silencing was effective in lung and in cardiac myocytes harverst from LV of mice treated with siRNAMEF2C. After knockdown optimization, echocardiographic, hemodynamic, gravimetric and morphometric analysis was performed to address the effects of MEF2C silencing in the structure and function of the LV from 15 days aorticbanded mice. Myocardial MEF2C silencing attenuated the load-induced hypertrophy in banded mice, indicated by the reductions of the wall thickness and the mass (~45%) of the LV. An increase in transconstriction gradient was observed in banded mice but the systemic blood pressure did not shown a significant statistically difference with the siRNAMEF2C treatment. The siRNAMEF2C injection reduced the collagen fraction in the LV of 15 days banded mice. On the other hand, the myocytes diameter and inflammatory cells level were comparable between the groups. Only the 24 hours banded mice showed an increase in the â-MHC expression and the treatment with siRNAMEF2C reduced ATP/ADP ratio. This study indicate that MEF2C regulates many aspects of the cardiac hypertrophy induced by pressure overload, like the expression of sarcomeric genes and genes involved in metabolic adaptation of the heart muscle.
Mestrado
Medicina Experimental
Mestre em Fisiopatologia Médica

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E2F1 pertence a uma família de fatores de transcrição e possui papel central no controle da expressão de genes relacionados à regulação da proliferação celular, pois ativa genes que participam da síntese de DNA. A atividade de E2F1 é regulada por meio da proteína pRB que, quando fosforilada por quinases associadas à ciclinas (Ciclinas/CDK) libera este fator de transcrição, promovendo assim a proliferação. A disfunção da complexa via de regulação da divisão celular pode acarretar em proliferação exacerbada, sendo a superexpressão de E2F1 bastante comum em diferentes tipos de tumores. Este fenômeno pode ser o principal fator para a alta proliferação de células tumorais. Desta forma, a inibição da atividade de E2F1 através de RNA de interferência (RNAi) pode ser promissora como tratamento para a diminuição da proliferação de células de melanoma. Assim sendo, objetiva-se neste trabalho inativar por RNAi o gene E2f1 em células B16mCAR, derivadas de melanoma de C57BL/6 e que superexpressam o receptor CAR, e averiguar os efeitos de sua ausência na proliferação celular, tanto in vitro como in vivo.
E2F1 belongs to a family of transcription factors and plays a central role in controlling the expression of genes related to regulation of the cell-cycle progression, since it activates genes involved in DNA synthesis. The activity of E2F1 is regulated by pRB protein, that when phosphorylated by cyclin-dependent kinases cyclins (Cyclins/CDK) releases this transcription factor, thereby promoting proliferation. The dysfunction of the complex regulatory pathway of cell division can lead to excessive proliferation, which overexpression of E2F1 is quite common in different types of tumors. This phenomenon may be the main factor for the high proliferation of tumor cells. Thus, inhibition of E2F1 activity by RNA interference (RNAi) may be promising as a treatment for decreased proliferation of melanoma cells. Therefore, the purpose of this work is the inactivation of the E2f1 gene through RNAi in B16mCAR cells, derived from C57BL/6’s melanoma and overexpresses the CAR receptor, and also verifies the effects of its absence on cell proliferation in vitro and in vivo.
TEDE
BV UNIFESP: Teses e dissertações

Aus den frühen Bindungsstudien des globalen Regulators AbrB mit der ausgedehnten phyC-Promotorregion von Bacillus amyloliquefaciens FZB45 konnte ein mehrstufiger kooperativer Bindungsprozess abgeleitet werden. Dabei verlangt die AbrB-vermittelte Repression von phyC nach Integrität zweier großer Bindungsstellen, ABS1 und ABS2, die 162 bp voneinander entfernt liegen. In der vorliegenden Arbeit wurden die ersten Echtzeitkinetiken zur DNA-AbrB-Interaktion mittels der Oberflächenplasmonresonanz (SPR) gemessen und analysiert. AbrB zeigte hohe Affinitäten zu den 40 bp langen Oligonukleotiden, die den beiden Bindungsstellen entstammen. Dabei verursachten alle Oligonukleotide der ABS2 und nur eine kurze Region innerhalb der ABS1 bei der Bindung von AbrB Konformationsänderungen im Protein und in der DNA (CD - Zirkulardichroismusspektroskopie) und wiesen eine Kooperativität von 2
In previous binding studies it could be demonstrated that a global regulator AbrB and the extensive phyC promoter region of Bacillus amyloliquefaciens FZB45 interact in a complex manner. AbrB binding is a multistep cooperative process. The integrity of both binding sites, ABS1 and ABS2, which are separated by 162 bp, is crucial for the AbrB-mediated repression of phyC. This work presents the first real-time binding kinetics of the AbrB-DNA interaction using surface plasmon resonance (SPR). AbrB exhibited high affinities to all analyzed 40-bp oligonucleotides that were derived from the ABSs of phyC. All parts of the ABS2, but only a small region within ABS1, were bound cooperatively to AbrB with a stoichiometry of 2 DNA to 1 AbrB tetramer and with 2

Chez la guêpe parasitoïde Venturia canescens, des particules virales dépourvues d'ADN appelées VLP (pour "Virus-like Particules") sont produites spécifiquement dans les ovaires et tapissent le chorion des oeufs qui sont injectés dans la chenille hôte. Les VLP ont une fonction immunosuppressive pour l'hôte parasité et permettent ainsi la survie des oeufs du parasitoïde. Ces VLP résultent de l’intégration d’un nudivirus dans le génome de l’ancêtre de la guêpe, nudivirus qui a été ensuite domestiqué pour former des liposomes viraux capables de véhiculer dans l’hôte des protéines de virulence d'origine cellulaire. L’étude réalisée au cours de cette thèse a eu pour objet, d’une part, d'étudier les mécanismes de domestication virale qui ont conduit au virus symbiotique endogène actuel nommé VcENV (pour V. canescens endogenous nudivirus) et d’autre part, d'apporter des éléments de réponse sur le processus de morphogénèse et le mode d'action parasitaire des VLP
Viral particles devoid of DNA called VLPs (for Virus-Like Particles) are specifically produced in the ovaries of the parasitoid wasp Venturia canescens and line the chorion of the wasp’s eggs injected into the host caterpillar. VLPs are immunosuppressive and allow parasitoid eggs survival. These VLPs result from the integration of a nudivirus into the wasp ancestor genome, nudivirus which was then domesticated to form viral liposomes capable of carrying, into the host, virulence proteins of cellular origin. The aim of the study carried out during this thesis was, first, to analyze the viral domestication mechanisms that led to the current endogenous symbiotic virus called VcENV (for V. canescens endogenous nudivirus) and secondly to provide some answers on VLPs morphogenesis process and parasitic mode of action

ABSTRACT Cassava is currently the second most important source of carbohydrates on the African continent. In the last two decades, cassava crops have been severely affected by outbreaks of cassava mosaic disease (CMD). South African cassava mosaic virus (SACMV) has been associated with CMD outbreaks in the Mpumalanga province. Advances in post-transcriptional gene silencing (PTGS) technology have provided promising new strategies for the engineering of virus resistance in plants. Inverted repeat (IR) constructs are currently the most potent inducers of PTGS, however, these constructs are inherently unstable. The purpose of this study was to develop IR constructs with an improved stability for the efficient induction of PTGS in plants. Two mismatched inverted repeat constructs, one targeting the SACMV BC1 open reading frame, the other targeting the Maize streak virus (MSV) AC1 open reading frame, were successfully created. Sodium bisulfite was used to deaminate cytosine residues on the sense arm of the constructs. The resulting number of GT mismatches was seemingly sufficient to stabilize the linear conformation of the IR constructs, as they were efficiently propagated by E.coli DH5!, and subsequently behaved like linear DNA molecules. Furthermore, it was found that the number of mismatches on the BC1 construct (17.5%) was ideal, as the subsequent stability of the predicted RNA hairpin was not affected. Due to the higher number of mismatches on the AC1 construct (23.5%), it was found that the loop region of the RNA hairpin was marginally destabilized. Despite this, long stretches of stable dsRNA were still produced from the AC1 IR construct, and is likely to induce PTGS. Interestingly, it was observed that the mismatched IR constructs, although still replicated in E.coli, were marginally destabilized in Agrobacterium. Therefore, it was deduced that the stability of a mismatched IR construct may be influenced by the particular intracellular environment of an organism. Due to the recalcitrance of cassava to transformation, a model plant system, Nicotiana benthamiana, was used to screen constructs for toxicity, stability, and efficiency of PTGS induction. Agrobacteriummediated transformation and regeneration of N. benthamiana was optimized, and 86% transformation efficiency was achieved when using leaf disk explants. It was found that the addition of an ethylene scrubber, potassium permanganate, substantially increased the rate of regeneration by reducing the frequency of hyperhydritic plants. Transgene iv integration was confirmed by PCR amplification of the hptII gene in the T-DNA region. Transgene expression was confirmed by screening for GUS and GFP reporter genes. No toxic responses to the transgene have been observed thus far. Studies are currently underway to confirm the stability of the mismatched IR constructs in N. benthamiana. PAGE Northern blotting is being done, as the detection of siRNAs derived from the transgene will confirm that constructs are functional. In addition, infectivity assays are underway to determine the efficacy of BC1 knockdown by a stably integrated construct. Due to the enhanced stability of mismatched IR constructs, they may be an appealing alternative to currently available intron-spliced, or exact matched hairpin systems.

Recent estimates indicate that globally there are over 33 million people infected with Human Immunodeficiency Virus type 1 (HIV-1). The epidemic is particularly severe in sub-Saharan Africa which accounts for 67% of all infected individuals and 72% of AIDS deaths in 2007. While current therapies, particularly in combination as a cocktail of highly active antiretroviral therapy (HAART), have had an important positive impact on the morbidity and mortality of HIV-related illness, there remain significant limitations. These include toxicities, resistance and the inability to eradicate a latent infection. In addition, most therapeutic agents have been developed to target HIV-1 subtype B, which affects individuals predominantly in Western Europe and North America. These concerns, along with ensuring patient compliance with treatment and the high cost of improved treatment regimens, have prompted the search for innovative and globally-effective therapies to eradicate HIV infection. More recently, gene therapy strategies based on the naturally-occurring RNA interference (RNAi) pathway has provided an exciting new mechanism to inhibit rogue gene expression. RNAi represents a set of highly conserved cellular pathways whereby double-stranded RNA (dsRNA) precursors are processed into shorter dsRNAs by the successive action of ribonucleases Drosha and Dicer. Processed, 21-23 nucleotide, short interfering RNAs (siRNAs) or antisense RNAs (asRNAs), associate with members of the Argonaute family of proteins to regulate gene expression at the transcriptional and post-transcriptional level. By exploiting the biogenesis of the endogenous mammalian RNAi pathway, several exogenous RNAi pathway mimics have been developed to inhibit unique sequences, including viral targets such as HIV. In the context of HIV, a combinatorial system that allows for the simultaneous suppression of multiple targets is important in preventing viral mutational escape of this rapidly evolving pathogen. The studies presented in this thesis add significantly to the newly emerging body of research on combinatorial RNAi strategies by focusing on the two novel technological approaches using mammalian expression systems. Both RNA Pol III-generated long-hairpin RNAs (lhRNAs) and RNA Pol II-generated polycistronic primary microRNAs (pri-miRNAs) were developed as systems for generating combinatorial RNAi precursors from single transcriptional units that induce post-transcriptional silencing of several highly conserved sequences of HIV-1. These included established therapeutic sites targeted to coding and non-coding regions of the HIV-1 long terminal repeat (LTR), Polymerase, Tat and Integrase. Expressed lhRNAs with ~63 bp duplex dsRNA regions and defined 5' and 3' termini were targeted to the transcribed region of the HIV 5' LTR and effectively suppressed two distinct sites within this region across both subtypes B and C HIV sequences. In addition, to assess whether lhRNAs could inhibit basal levels of HIV transcription, the lhRNAs were shown to suppress Tat-mediated (processive) and Tat-independent (non-processive) transcription when targeting episomal and integrated LTR-driven sequences. Portions of the lhRNAs that produced the most active siRNAs were dissected by using tiled LTR targets cloned into a luciferase reporter gene and by using northern blot analyses. Dicer-processing of expressed lhRNAs was shown to be most effective from the base of the duplex and decreased in efficiency towards the loop, suggesting that a gradient of siRNAs production is possible from a single lhRNA but with decreasing efficacy. This work laid the foundation for improved expressed lhRNAs whereby multiple unique anti-HIV siRNAs were produced from a single lhRNA. The second combinatorial RNAi strategy made use of RNA Pol II-expressed pri-miRNA mimics, where each mimic was derived from a different endogenous scaffold. Polycistronic transcripts consisting of four different pri-miRNA scaffolds and targeting four separate sites in HIV were tested in several combinatorial systems. The pri-miRNA backbone chosen was shown to dramatically affect the concentration and inhibitory efficacy of each generated effector strand, and this was largely independent of the sequence used. A strategy to combine four of the most effective pri-miRNA scaffolds into one expression cassette was developed and significant inhibition of an HIV infectious molecular clone as well as a wild type HIV isolate was demonstrated. Finally, in an attempt to uncover additional asRNAs capable of inducing inhibition via transcriptional gene silencing of the HIV LTR promoter, indiscriminate cell-wide gene activation was shown to occur as result of an unintended off-target effect. These observations demonstrated that caution should be exercised when interpreting RNA-induced gene activation results. Overall, this thesis provides a detailed description of the efficacy of two novel combinatorial RNAi approaches based on single promoter expression systems that are aimed at generating multiple RNAi effector sequences targeted to HIV. These approaches pave the way towards a better understanding of the efficacy of combinatorial RNAi and an effective and sustained gene therapy of HIV.

"March 2003"
Bibliography: leaves 133-143.
x, 143 leaves : ill. (some col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Sciences, 2003

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia
Ataxia espinocerebelosa do tipo 3 (SCA3) ou doença de Machado-Joseph (MJD) é uma doença neurodegenerativa e descrita como como a forma mundialmente mais comum de ataxia com hereditariedade autossómica dominante. Pertencendo ao grupo das doenças de poliglutaminas, SCA3 é causada por uma expansão anormal de uma repetição de trinucleótidos CAG (que codificam glutamina) no exão 10 do gene ATXN3, afectando maioritariamente áreas anteriores do cérebro, tais como o cerebelo, o tronco cerebral e a parte superior da espinal medula.A progressão implacável da MJD e a sua falta de tratamento deram origem ao desenvolvimento de uma miríade de estratégias terapêuticas promissoras, incidindo em diferentes alturas da patogénese da doença, muitas delas usando tecnologia de RNAi. Usando uma estratégia específica apenas para o alelo mutante, alguns estudos foram já capazes de silenciar a forma expandida da ataxina-3 e evitar outros patogénicos mais a jusante. Os resultados destes estudos têm sido altamente promissores, tendo sido capazes de abolir tanto neuropatologia como o resgate do fenótipo normal de coordenação motora em diferentes modelos animais da doença. No entanto, estratégias de RNAi possuem desvantagens inerentes, a maior das quais sendo a sua possível toxicidade para as células, devido ao sequestro de proteínas e saturação da maquinaria enzimática de RNAi endógena. Uma estratégia incidindo num gene a um nível pré-transcripcional, permitiria uma estratégia de silenciamento de um gene fundamentalmente mais segura, permitindo evitar tanto a potencial toxicidade destas estratégias de RNAi e do mRNA mutado do gene ATXN3, como evitar processos patogénicos mais a jusante . Este tipo de estratégias podem utilizar técnicas mais recentes de edição génica, tendo por base o uso de nucleases programáveis, como a tecnologia CRISPR.Sendo uma tecnologia de desenho muito simples e de flexibilidade extrema, com um baixo custo associado, a tecnologia CRISPR permite edição génica rápida e precisa. A sua flexibilidade permitiu o aparecimento de diferentes variantes da técnica, cada uma com usos especializados: CRISPR pode ser usada para silenciar genes através de cortes em DNA (com formações de indels), para correção de mutações ou para modulação via complexos CRISPR cataliticamente inactivos e fundidos com repressores ou indutores de transcrição (CRISPRi).Neste trabalho, propomos usar a variante CRISPRi para obter silenciamento pré-transcripcional, tanto in vitro como in vivo, do gene ATXN3 humano. Para tal, propomos o uso de uma Cas9 cataliticamente inactiva fundido com um repressor transcripcional (neste caso a caixa Krüppel-associada, ou KRAB), para permitir silenciamento sem perda de informação genómica, permitindo assim uma estratégia mais segura quando comparada com estratégias clássicas de RNAi, visto que permitirá evitar toxicidade associada com mRNA mutante do gene ATXN3 mutado. Desenhámos sgRNA direccionados para diferentes áreas do gene ATXN3, tendo testado estas sequências em culturas celulares e num modelo animal trangénico da doença. De um modo geral, esta estratégia foi capaz de reduzir os níveis de ataxina-3 expandida nas culturas celulares e resultados preliminares no modelo trangénico sugerem uma melhoria da coordenação motora.
Spinocerebellar ataxia type-3 (SCA3) or Machado-Joseph disease (MJD) is a neurodegenerative disorder, described as the most common form of dominantly inherited ataxia worldwide. Being a polyglutamine disease, SCA3 is caused by an abnormal expansion of CAG trinucleotide repeat (coding for glutamine) in exon 10 of the ATXN3 gene and affects mainly the areas of the hindbrain, such as the cerebellum, brainstem and the upper part of the spinal cord.MJD relentless progression and lack of treatment have given rise to the development of a plethora of different potential and promising therapeutic approaches, many of which use RNAi technology. By using an allele-specific strategy to target the mutant allele alone, some studies have been able to silence expanded ataxin-3 and avoid downstream pathogenic processes. The results have been extremely promising, with the abolishing of neuropathology and rescue of normal gait and motor coordination. Despite this, RNAi strategies have inherent disadvantages, the greatest of which is its possible toxicity for the cell due to the sequestration of proteins or transcription factors and saturation of the endogenous enzymatic machinery of RNAi. An approach targeting a gene on a pre-transcriptional level would allow for a safer gene silencing strategy, when compared to the classical RNAi approaches, since it would avoid both more downstream pathological events and possible toxicity originating from both these RNAi strategies and mutant ATXN3 mRNA. This can be achieved with current gene editing tools, using as a basis the use of programmable nucleases, one of which being CRISPR technology.Being an extremely flexible and easily designable technology, with a low associated cost, CRISPR technology allows for relatively quick and precise gene targeting. Its flexibility has given rise to several variants of the technology, each with a specialized use: CRISPR can be used from gene silencing via DNA cleavage, to correcting mutations and to gene silencing through a chemically inactive CRISPR complex fused with a transcription repressor.In this work, we proposed to use a variant of the CRISPR editing system, termed CRISPR interference, to achieve pre-transcriptional silencing, both in vitro and in vivo, of the human ATXN3 gene. Eliciting this effect will be done via the use of a chemically inactive, or dead, Cas9, fused with a transcriptional repressor (in this case the Krüppel-associated box, or KRAB), to allow for gene silencing without loss of genome information. We have designed sgRNA directed towards areas of the ATXN3 gene and tested these sequences both in cells and in a transgenic animal model of the disease. This approach was capable of reducing the levels of ataxin-3 in cell cultures and preliminary results from the animal model suggest an improvement in motor coordination.

RNA interference (RNAi) is one of the key mechanisms that are involved in many biological processes such as control of plant gene expression, influence on chromatin arrangement or providing protection against invasive DNA or RNA transposons, viruses and transgenes. In plants, RNAi is triggered by double stranded RNA (dsRNA) that is cleaved by DICER LIKE (DCL) proteins to small RNAs (sRNAs). The size of these sRNAs is in range of 21 - 24 nucleotides (nt). Small RNA acts in the place of origin and they are also a mobile signal which in plants can move to a short distance through plasmodesmata and to a long distance trough phloem. sRNA and Argonaute (AGO) protein form RNA-induced silencing complex (RISC). Together, they recognize the target RNA molecule and contribute to an efficient RNAi phase which may be exhibited by gene silencing at posttranscriptional level (PTGS) or transcriptional level (TGS). The purpose of this study was to compare the effects of silencing constructs, witch in a controlled way differently trigger RNAi directed against the expression of the GFP reporter gene in the model organism Arabidopsis thaliana. Silencing constructs were placed under an inducible promoter activated by the presence of 17-β-estradiol (XVE system). They differed in the way of the dsRNA formation and in the...

Indiana University-Purdue University Indianapolis (IUPUI)
My thesis comprises of two individual projects: 1) we have developed a database for operon prediction using high-throughput sequencing datasets for bacterial genomes. 2) Genomics and mechanistic insights of convergent transcription in bacterial genomes. In the first project we developed a database for the prediction of operons for bacterial genomes using RNA-seq datasets, we predicted operons for bacterial genomes. RNA-seq datasets with different condition for each bacterial genome were taken into account and predicted operons using Rockhopper. We took RNA-seq datasets from NCBI with distinct experimental conditions for each bacterial genome into account and analyzed using tool for operon prediction. Currently our database contains 9 bacterial organisms for which we predicted operons. User interface is simple and easy to use, in terms of visualization, downloading and querying of data. In our database user can browse through reference genome, genes present in that genome and operons predicted from different RNA-seq datasets. Further in the second project, we studied the genomic and mechanistic insights of convergent transcription in bacterial genomes. We know that convergent gene pairs with overlapping head-to-head configuration are widely spread across both eukaryotic and prokaryotic genomes. They are believed to contribute to the regulation of genes at both transcriptional and post-transcriptional levels, although factors contributing to their abundance across genomes and mechanistic basis for their prevalence are poorly understood. In this study, we explore the role of various factors contributing to convergent overlapping transcription in bacterial genomes. Our analysis shows that the proportion of convergent overlapping gene pairs (COGPs) in a genome is affected due to endospore formation, bacterial habitat, oxygen requirement, GC content and the temperature range. In particular, we show that bacterial genomes thriving in specialized habitats, such as thermophiles, exhibit a high proportion of COGPs. Our results also conclude that the density distribution of COGPs across the genomes is high for shorter overlaps with increased conservation of distances for decreasing overlaps. Our study further reveals that COGPs frequently contain stop codon overlaps with the middle base position exhibiting mismatches between complementary strands. Further, for the functional analysis using cluster of orthologous groups (COGs) annotations suggested that cell motility, cell metabolism, storage and cell signaling are enriched among COGPs, suggesting their role in processes beyond regulation. Our analysis provides genomic insights into this unappreciated regulatory phenomenon, allowing a refined understanding of their contribution to bacterial phenotypes.

In this study, I utilized the defined (target) sequence for transcription factor NHR-25 and GFP as a marker to visualize where nhr-25 is active during the development in vivo. After obtaining worm strains carrying these constructs, the expression pattern was analyzed and the specificity of the expression was tested by means of RNAi.

The brain is a highly adaptable organ that is capable of converting sensory information into changes in neuronal function. This plasticity allows behavior to be accommodated to the environment, providing an important evolutionary advantage. Neurons convert environmental stimuli into long-lasting changes in their physiology in part through the synaptic activity-regulated transcription of new gene products. Since the neurotransmitter-dependent regulation of Fos transcription was first discovered nearly 25 years ago, a wealth of studies have enriched our understanding of the molecular pathways that mediate activity-regulated changes in gene transcription. These findings show that a broad range of signaling pathways and transcriptional regulators can be engaged by neuronal activity to sculpt complex programs of stimulus-regulated gene transcription. However, the shear scope of the transcriptional pathways engaged by neuronal activity raises the question of how specificity in the nature of the transcriptional response is achieved in order to encode physiologically relevant responses to divergent stimuli. Here we summarize the general paradigms by which neuronal activity regulates transcription while focusing on the molecular mechanisms that confer differential stimulus-, cell-type-, and developmental-specificity upon activity-regulated programs of neuronal gene transcription. In addition, we preview some of the new technologies that will advance our future understanding of the mechanisms and consequences of activity-regulated gene transcription in the brain.
Dissertation

The RNA interference is a mechanism, which allows cells to regulate their genes functions, to establish and maintain heterochromatin and to defend them against invasive nucleic acids. In plants, RNA interference is initiated by double-stranded RNA, which is processed by Dicer into small RNAs, usually 20-24nt long. These small RNAs form a complex with Argonaut protein that participates in different processes based on sequence complementarity. This complex can guide mRNA cleavage, translation blocking and chromatin modifications, resulting either into posttranscriptional silencing (by preventing translation of already existing mRNA, PTGS) or transcriptional silencing (by preventing transcription of mRNA, TGS). The first step of this thesis was to establish different ways of triggering PTGS and to evaluate their functionality and efficiency. The next step was a preparation of a system which would allow to study the transition from posttrancriptional to transcriptional silencing. These so called "indicator lines" should allow to observe the timing and dynamics of this process by utilizing fluorescent proteins. This system is also going to enable to evaluate, how different factors are involved in this process - one of the factors is RNA-dependent RNA polymerase 6 (RDR6) which plays an essential role in...

Gene deletions are a powerful method to uncover the cellular functions of a given gene in living systems. A limitation to this methodology is that it is not applicable to essential genes. Even for non-essential genes, gene knockouts cause complete absence of gene product thereby limiting genetic analysis of the biological pathway. Alternatives to gene deletions are mutants that are conditional, for e.g, temperature sensitive (ts) mutants are robust tools to understand temporal and spatial functions of genes. By definition, products of such mutants have near normal activity at a lower temperature or near-optimal growth temperature which is called as the permissive temperature and reduced activity at a higher, non-optimal temperature called as the non-permissive temperature. Generation of ts alleles in genes of interest is often time consuming as it requires screening a large population of mutants to identify those that are conditional. Often many essential proteins do not yield ts such alleles even after saturation mutagenesis and extensive screening (Harris et al., 1992; Varadarajan et al., 1996). The limited availability of such mutants in many essential genes prompted us to adopt a biophysical approach to design temperature-sensitive missense mutants in an essential gene of fission yeast. Several studies report that mutations in buried or solvent-inaccessible amino acids cause extensive changes in the thermal stability of proteins and specific substitutions create temperature-sensitive mutants (Rennell et al., 1991; Sandberg et al., 1995). We used the above approach to generate conditional mutants in the fission yeast gene spprp18+encoding an essential predicted second splicing factor based on its homology with human and S. cerevisiae proteins. We have used a missense mutant coupled with a conditional expression system to elucidate the cellular functions of spprp18+. Further, we have employed the same biophysical principle to generate a missense mutant in spago1+ RNA silencing factor that is non-essential for viability but has critical functions in the RNAi pathway of fission yeast. Fission yeast pre-mRNA splicing: cellular functions for the protein factor SpPrp18 Pre-mRNA splicing is an evolutionarily conserved process that excises introns from nascent transcripts. Splicing reactions are catalyzed by the large ribonuclear protein machinery called the spliceosome and occur by two invariant trans-esterification reactions (reviewed in Ruby and Abelson, 1991; Moore et al., 1993). The RNA-RNA, RNA–protein and protein-protein interactions in an assembly of such a large protein complex are numerous and highly dynamic in nature. These interactions in in vitro splicing reactions show ordered recruitment of essential small nuclear ribonucleic particles snRNPs and non–snRNP components on pre-mRNA cis-elements. Further these trans acting factors recognize and poise the catalytic sites in proximity to identify and excise introns. The precision of the process is remarkable given the diversity in architecture for exons and introns in eukaryotic genes (reviewed in Burge et al., 1999; Will and Luhrmann, 2006). Many spliceosomal protein components are conserved across various organisms, yet introns have diverse features with large variations in primary sequence. We hypothesize that co-evolution of splicing factor functions occurs with changes in gene and intron architectures and argue for alternative spliceosomal interactions for spliceosomal proteins that thus enabling splicing of the divergent introns. In vitro biochemical and genetic studies in S. cerevisiae and biochemical studies with human cell lines have indicated that ScPRP18 and its human homolog hPRP18 function during the second catalytic reaction. In S. cerevisiae, ScPrp18 is non-essential for viability at growth temperatures <30°C (Vijayraghavan et al., 1989; Vijayraghavan and Abelson, 1990; Horowitz and Abelson, 1993b). The concerted action of ScSlu7 - ScPrp18 heteromeric complex is essential for proper 3’ss definition during the second catalytic reaction (Zhang and Schwer, 1997; James et al., 2002). These in vitro studies also hinted at a possible intron -specific requirement for ScPrp18 and ScSlu7 factors as they were dispensable for splicing of intron variants made in modified ACT1 intron containing transcripts (Brys and Schwer, 1996; Zhang and Schwer, 1997). A short spacing distance between branch point adenosine to 3’splice site rendered the substrate independent of Prp18 and Slu7 for the second step (Brys and Schwer, 1996; Zhang and Schwer, 1997). Extensive mutational analyses of budding yeast ScPrp18 identified two functional domains and suggested separate roles during splicing (Bacikova and Horowitz, 2002; James et al., 2002). Fission yeast with its genome harboring multiple introns and degenerate splice signals has recently emerged as a unique model to study relationships between splicing factors and their role in genomes with short introns. Previously, studies in our lab had initiated genetic and mutational analysis of S. pombe Prp18, the predicted homolog of budding yeast Prp18. Genetic analysis showed its essentiality, but a set of missense mutants based on studies of budding yeast ScPrp18 (Bacikova and Horowitz, 2002) gave either inactive null or entirely wild type phenotype for the fission yeast protein. In this study, we have extended our previous mutational analysis of fission yeast Prp18 by adopting biophysical and computational approaches to generate temperature-sensitive mutants. A missense mutant was used to understand the splicing functions and interactions of SpPrp18 and the findings are summarized below. Fission yeast SpPrp18 is an essential splicing factor with transcript-specific functions and links efficient splicing with cell cycle progression We initiated our analysis of SpPrp18 by adopting a biophysical approach to generate ts mutants. We used the PREDBUR algorithm to predict a set of buried residues, which when mutated could result in a temperature-sensitive phenotype that complements the null allele at permissive temperature. These predictions are based upon two biophysical properties of amino acids: 1) Hydrophobicity, which is calculated in a window of seven amino acids 2) Hydrophobic moment, which is calculated in a sliding window of nine amino acids in a given protein sequence. Several studies correlate these properties to protein stability and function (Varadarajan et al., 1996). One of the buried residue mutants V194R, in helix 1 of SpPrp18 conferred weak temperature- sensitivity and strong cold-sensitivity even when the protein was over expressed from a plasmid. Through semi-quantitative RT-PCR we showed splicing-defects for tfIId+ intron1 in these cells even when grown at permissive temperature. The primary phenotype was the accumulation of pre-mRNA. Further, we showed this splicing arrest is co-related with reduced levels of SpPrp18 protein, linking protein stability and splicing function. Next we examined the effects of this mutation on function by further reduction of protein levels. This was done by integrating the expression cassette nmt81:spprp18+/spprp18V194R at the leu1 chromosomal locus and by metabolic depletion of the integrated allele. Through RT-PCRs we demonstrated that depletion of wild type or missense protein has intron specific splicing defects. These findings showed its non-global and possibly substrate-specific splicing function. In the affected introns, precursor accumulation is the major phenotype, confirming prior data from our lab that hinted at its likely early splicing role. This contrasts with the second step splicing role of the human or budding yeast Prp18 proteins. Previous data from our lab showed loss of physical interaction between SpPrp18 and SpSlu7 by co-immunoprecipitation studies. This again differs from the strong and functionally important ScPrp18 and ScSlu7 interaction seen in budding yeast. We show the absence of charged residues in SpSlu7 interaction region formed by SpPrp18 helix1 and helix2 which can explain the altered associations for SpPrp18 in fission yeast. Importantly, as the V194R mutation in helix 1 shows splicing defects even at permissive temperature, the data indicate a critical role for helix 1 for splicing interactions, possibly one that bridges or stabilizes the proposed weak association of SpPrp18-SpSlu7 with a yet unknown splicing factor. We also investigated the effects of mutations in other helices; surprisingly we recovered only mutations with very subtle growth phenotypes and very mild splicing defects. Not surprisingly, stop codon at L239 residue predicted to form a truncated protein lacking helices 3, 4 and 5 conferred recessive but null phenotype implicating essential functions for other helices. Other amino acid substitutions at L239 position had near wild type phenotype at 30°C and 37°C. Helix 3 buried residue mutant I259A conferred strong cold-sensitivity when over expressed from plasmid, but semi quantitative analysis indicated no splicing defects for intron1 in the constitutively expressed transcript tfIId+. These findings indicate cold sensitivity either arises due to compromised splicing of yet unknown transcripts or that over-expressed protein has near wild type activity. We find mutations in the helix 5 buried residues L324 also conferred near WT phenotype. Earlier studies in the lab found that substitution of surface residues KR that are in helix 5 with alanine lead to null phenotypes (Piyush Khandelia and Usha Vijayraghavan unpublished data). We report stable expression of all of these mutant proteins; L239A, L239P, L239G, I259A, I259V, L324F, L324A as determined by our immunoblot analysis at 30°C and 37°C. The mild phenotypes of many buried residues can be attributed to orientation of their functional groups into a protein cavity between the helices. Lastly, our microscopic cellular and biochemical analysis of cellular phenotypes of spprp18 mutant provided a novel and direct role of this factor in G1-S transition of cell cycle. Our RT-PCR data suggest spprp18+ is required for efficient splicing of several intron containing transcripts involved in G1-S transition and subsequent activation of MBF complex (MluI cell cycle box-binding factor complex) during S-phase and shows a mechanistic link between cell cycle progression and splicing. A tool to study links between RNA interference, centromeric non-coding RNA transcription and heterochromatin formation S.pombe possesses fully functional RNA interference machinery with a single copy for essential RNAi genes ago1+, dcr1+ and rdp1+. Deletion of any of these genes causes loss of heterochromatinzation with abnormal cytokinesis, cell-cycle deregulation and mating defects (Volpe et al., 2002). In S.pombe, exogenous or endogenously generated dsRNA’s from transcription of centromeric repeats are processed by the RNaseIII enzyme dicer to form siRNA. These siRNA’s are loaded in Ago1 to form minimal RNA induced silencing complex (RISC) complex or specialized transcription machinery complex RNA induced transcriptional silencing (RITS) complex and target chromatin or complementary mRNAs for silencing. Thus as in other eukaryotes, fission yeast cells deploy RNAi mediated silencing machinery to regulate gene-expression and influence chromatin status. Several recent studies point to emerging new roles of RNAi and its association with other RNA processes (Woolcock et al., 2011; Bayane et al., 2008; Kallgren et al., 2014). Many recent reports suggest physical interactions of RISC or RITS and RNA dependent RNA polymerase complex (RDRC) with either some factors of the spliceosomal machinery, heterochromatin machinery (CLRC complex) and the exosome mediated RNA degradation machinery (Bayne et al., 2008 and Chinen et al., 2010 ; Hiriart et al., 2012; Buhler et al., 2008; Bayne et al., 2010 ). Thus we presume conditional alleles in spago1+ will facilitate future studies to probe the genetic network between these complexes as most analyses thus far rely on ago1∆ allele or have been based on proteomic pull down analyses of RISC or RITS complexes. In this study, we employed biophysical and modeling approaches described earlier to generate temperature sensitive mutants in spago1+ and spdcr1+. We tested several mutants for their ability to repress two reporter genes in a conditional manner. Our modeling studies on SpAgo1 PAZ domain indicated structural similarities with human Ago1 PAZ domain. We created site-directed missense mutants at predicted buried residues or in catalytic residues. We also analyzed the effects of random amino acid replacements in specific predicted buried or catalytic residues of SpAgoI. These ago1 mutants were screened as pools for their effects on silencing of GFP or of ura4+ reporter genes. These assays assessed post transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) activity of these mutants. We obtained three temperature sensitive SpAgo1 mutants V324G, V324S and L215V while the V324E replacement was a null allele. Based upon our modeling, a likely explanation for the phenotype of these mutants is structural distortion or mis-orientation of the functional groups caused due to these mutations, which affect activity in a temperature dependent manner. This distortion in the PAZ domain may affect binding of siRNA and thereby lead to heterochromatin formation defects that we observed. Our data on the SpAgo1 V324 mutant shows conditional centromeric heterochromatin formation confirmed by semi quantitative RT-PCR for dh transcripts levels that shows temperature dependent increase in these transcripts. We find reduced H3K9Me2 levels at dh locus by chromatin immunoprecipitation (ChIP) assay, linking the association of siRNAs for establishment of heterochromatin at this loci. The data on PTGS of GFP transcripts show SpAgo1 V324G mutation has decreased slicing activity as semi-quantitative RT-PCR for GFP transcripts show increased levels at non permissive temperature. These studies point out the importance of siRNA binding to the PAZ domain and its effect on slicing activity of SpAgo1. The mutations in Y292 showed residue loss of centromeric heterochromatin formation phenotype. Thus, we ascribe critical siRNA binding and 3’ end recognition functions to this residue of SpAgo1. These studies point out functional and structural conservation across hAgo1 and SpAgo1. Adopting the aforementioned biophysical mutational approach, we generated mutants in spdcr1+ and screened for those with conditional activity. Our modeling studies on SpDcr1 helicase domain shows it adopts the conserved helicase domain structure seen for other DEAD Box helicases. Our data on mutational analysis of a conserved buried residue I143 in the walker motif B created inactive protein. The data confirm critical functions for dicer in generation of siRNAs and also in recognition of dsRNA ends. Mutants in buried residues L1130 and I1228 of RNase IIIb domain were inactive and the proximity of these residues to the catalytic core suggest that the critical structural alignment of catalytic residues is indispensable for carrying out dsRNA cleavage to generate siRNAs. We also attribute critical catalytic functions to SpDcr1 D1185 residue for generation of siRNA and heterochromatin formation as measured by our transcriptional gene silencing assay. Our studies employing biophysical and computational approaches to design temperature-sensitive mutants have been successfully applied to an essential splicing factor SpPrp18, which was refractory for ts mutants by other methods. Using a missense mutant, we showed its intron-specific splicing function for subsets of transcripts and deduced that its ubiquitous splicing role is arguable. We have uncovered a link between the splicing substrates of SpPrp18 and direct evidence of splicing based cell cycle regulation, thus providing a mechanistic link to the cell cycle arrest seen in some splicing factor mutants. The same methodology was applied to another important biological pathway, the RNAi machinery, where central factors SpAgoI and SpDcrI were examined We report the first instance of conditional gene silencing tool by designing Ago1 ts mutants which will be useful for future studies of the global interaction network between RNAi and other RNA processing events.

Gene deletions are a powerful method to uncover the cellular functions of a given gene in living systems. A limitation to this methodology is that it is not applicable to essential genes. Even for non-essential genes, gene knockouts cause complete absence of gene product thereby limiting genetic analysis of the biological pathway. Alternatives to gene deletions are mutants that are conditional, for e.g, temperature sensitive (ts) mutants are robust tools to understand temporal and spatial functions of genes. By definition, products of such mutants have near normal activity at a lower temperature or near-optimal growth temperature which is called as the permissive temperature and reduced activity at a higher, non-optimal temperature called as the non-permissive temperature. Generation of ts alleles in genes of interest is often time consuming as it requires screening a large population of mutants to identify those that are conditional. Often many essential proteins do not yield ts such alleles even after saturation mutagenesis and extensive screening (Harris et al., 1992; Varadarajan et al., 1996). The limited availability of such mutants in many essential genes prompted us to adopt a biophysical approach to design temperature-sensitive missense mutants in an essential gene of fission yeast. Several studies report that mutations in buried or solvent-inaccessible amino acids cause extensive changes in the thermal stability of proteins and specific substitutions create temperature-sensitive mutants (Rennell et al., 1991; Sandberg et al., 1995). We used the above approach to generate conditional mutants in the fission yeast gene spprp18+encoding an essential predicted second splicing factor based on its homology with human and S. cerevisiae proteins. We have used a missense mutant coupled with a conditional expression system to elucidate the cellular functions of spprp18+. Further, we have employed the same biophysical principle to generate a missense mutant in spago1+ RNA silencing factor that is non-essential for viability but has critical functions in the RNAi pathway of fission yeast. Fission yeast pre-mRNA splicing: cellular functions for the protein factor SpPrp18 Pre-mRNA splicing is an evolutionarily conserved process that excises introns from nascent transcripts. Splicing reactions are catalyzed by the large ribonuclear protein machinery called the spliceosome and occur by two invariant trans-esterification reactions (reviewed in Ruby and Abelson, 1991; Moore et al., 1993). The RNA-RNA, RNA–protein and protein-protein interactions in an assembly of such a large protein complex are numerous and highly dynamic in nature. These interactions in in vitro splicing reactions show ordered recruitment of essential small nuclear ribonucleic particles snRNPs and non–snRNP components on pre-mRNA cis-elements. Further these trans acting factors recognize and poise the catalytic sites in proximity to identify and excise introns. The precision of the process is remarkable given the diversity in architecture for exons and introns in eukaryotic genes (reviewed in Burge et al., 1999; Will and Luhrmann, 2006). Many spliceosomal protein components are conserved across various organisms, yet introns have diverse features with large variations in primary sequence. We hypothesize that co-evolution of splicing factor functions occurs with changes in gene and intron architectures and argue for alternative spliceosomal interactions for spliceosomal proteins that thus enabling splicing of the divergent introns. In vitro biochemical and genetic studies in S. cerevisiae and biochemical studies with human cell lines have indicated that ScPRP18 and its human homolog hPRP18 function during the second catalytic reaction. In S. cerevisiae, ScPrp18 is non-essential for viability at growth temperatures <30°C (Vijayraghavan et al., 1989; Vijayraghavan and Abelson, 1990; Horowitz and Abelson, 1993b). The concerted action of ScSlu7 - ScPrp18 heteromeric complex is essential for proper 3’ss definition during the second catalytic reaction (Zhang and Schwer, 1997; James et al., 2002). These in vitro studies also hinted at a possible intron -specific requirement for ScPrp18 and ScSlu7 factors as they were dispensable for splicing of intron variants made in modified ACT1 intron containing transcripts (Brys and Schwer, 1996; Zhang and Schwer, 1997). A short spacing distance between branch point adenosine to 3’splice site rendered the substrate independent of Prp18 and Slu7 for the second step (Brys and Schwer, 1996; Zhang and Schwer, 1997). Extensive mutational analyses of budding yeast ScPrp18 identified two functional domains and suggested separate roles during splicing (Bacikova and Horowitz, 2002; James et al., 2002). Fission yeast with its genome harboring multiple introns and degenerate splice signals has recently emerged as a unique model to study relationships between splicing factors and their role in genomes with short introns. Previously, studies in our lab had initiated genetic and mutational analysis of S. pombe Prp18, the predicted homolog of budding yeast Prp18. Genetic analysis showed its essentiality, but a set of missense mutants based on studies of budding yeast ScPrp18 (Bacikova and Horowitz, 2002) gave either inactive null or entirely wild type phenotype for the fission yeast protein. In this study, we have extended our previous mutational analysis of fission yeast Prp18 by adopting biophysical and computational approaches to generate temperature-sensitive mutants. A missense mutant was used to understand the splicing functions and interactions of SpPrp18 and the findings are summarized below. Fission yeast SpPrp18 is an essential splicing factor with transcript-specific functions and links efficient splicing with cell cycle progression We initiated our analysis of SpPrp18 by adopting a biophysical approach to generate ts mutants. We used the PREDBUR algorithm to predict a set of buried residues, which when mutated could result in a temperature-sensitive phenotype that complements the null allele at permissive temperature. These predictions are based upon two biophysical properties of amino acids: 1) Hydrophobicity, which is calculated in a window of seven amino acids 2) Hydrophobic moment, which is calculated in a sliding window of nine amino acids in a given protein sequence. Several studies correlate these properties to protein stability and function (Varadarajan et al., 1996). One of the buried residue mutants V194R, in helix 1 of SpPrp18 conferred weak temperature- sensitivity and strong cold-sensitivity even when the protein was over expressed from a plasmid. Through semi-quantitative RT-PCR we showed splicing-defects for tfIId+ intron1 in these cells even when grown at permissive temperature. The primary phenotype was the accumulation of pre-mRNA. Further, we showed this splicing arrest is co-related with reduced levels of SpPrp18 protein, linking protein stability and splicing function. Next we examined the effects of this mutation on function by further reduction of protein levels. This was done by integrating the expression cassette nmt81:spprp18+/spprp18V194R at the leu1 chromosomal locus and by metabolic depletion of the integrated allele. Through RT-PCRs we demonstrated that depletion of wild type or missense protein has intron specific splicing defects. These findings showed its non-global and possibly substrate-specific splicing function. In the affected introns, precursor accumulation is the major phenotype, confirming prior data from our lab that hinted at its likely early splicing role. This contrasts with the second step splicing role of the human or budding yeast Prp18 proteins. Previous data from our lab showed loss of physical interaction between SpPrp18 and SpSlu7 by co-immunoprecipitation studies. This again differs from the strong and functionally important ScPrp18 and ScSlu7 interaction seen in budding yeast. We show the absence of charged residues in SpSlu7 interaction region formed by SpPrp18 helix1 and helix2 which can explain the altered associations for SpPrp18 in fission yeast. Importantly, as the V194R mutation in helix 1 shows splicing defects even at permissive temperature, the data indicate a critical role for helix 1 for splicing interactions, possibly one that bridges or stabilizes the proposed weak association of SpPrp18-SpSlu7 with a yet unknown splicing factor. We also investigated the effects of mutations in other helices; surprisingly we recovered only mutations with very subtle growth phenotypes and very mild splicing defects. Not surprisingly, stop codon at L239 residue predicted to form a truncated protein lacking helices 3, 4 and 5 conferred recessive but null phenotype implicating essential functions for other helices. Other amino acid substitutions at L239 position had near wild type phenotype at 30°C and 37°C. Helix 3 buried residue mutant I259A conferred strong cold-sensitivity when over expressed from plasmid, but semi quantitative analysis indicated no splicing defects for intron1 in the constitutively expressed transcript tfIId+. These findings indicate cold sensitivity either arises due to compromised splicing of yet unknown transcripts or that over-expressed protein has near wild type activity. We find mutations in the helix 5 buried residues L324 also conferred near WT phenotype. Earlier studies in the lab found that substitution of surface residues KR that are in helix 5 with alanine lead to null phenotypes (Piyush Khandelia and Usha Vijayraghavan unpublished data). We report stable expression of all of these mutant proteins; L239A, L239P, L239G, I259A, I259V, L324F, L324A as determined by our immunoblot analysis at 30°C and 37°C. The mild phenotypes of many buried residues can be attributed to orientation of their functional groups into a protein cavity between the helices. Lastly, our microscopic cellular and biochemical analysis of cellular phenotypes of spprp18 mutant provided a novel and direct role of this factor in G1-S transition of cell cycle. Our RT-PCR data suggest spprp18+ is required for efficient splicing of several intron containing transcripts involved in G1-S transition and subsequent activation of MBF complex (MluI cell cycle box-binding factor complex) during S-phase and shows a mechanistic link between cell cycle progression and splicing. A tool to study links between RNA interference, centromeric non-coding RNA transcription and heterochromatin formation S.pombe possesses fully functional RNA interference machinery with a single copy for essential RNAi genes ago1+, dcr1+ and rdp1+. Deletion of any of these genes causes loss of heterochromatinzation with abnormal cytokinesis, cell-cycle deregulation and mating defects (Volpe et al., 2002). In S.pombe, exogenous or endogenously generated dsRNA’s from transcription of centromeric repeats are processed by the RNaseIII enzyme dicer to form siRNA. These siRNA’s are loaded in Ago1 to form minimal RNA induced silencing complex (RISC) complex or specialized transcription machinery complex RNA induced transcriptional silencing (RITS) complex and target chromatin or complementary mRNAs for silencing. Thus as in other eukaryotes, fission yeast cells deploy RNAi mediated silencing machinery to regulate gene-expression and influence chromatin status. Several recent studies point to emerging new roles of RNAi and its association with other RNA processes (Woolcock et al., 2011; Bayane et al., 2008; Kallgren et al., 2014). Many recent reports suggest physical interactions of RISC or RITS and RNA dependent RNA polymerase complex (RDRC) with either some factors of the spliceosomal machinery, heterochromatin machinery (CLRC complex) and the exosome mediated RNA degradation machinery (Bayne et al., 2008 and Chinen et al., 2010 ; Hiriart et al., 2012; Buhler et al., 2008; Bayne et al., 2010 ). Thus we presume conditional alleles in spago1+ will facilitate future studies to probe the genetic network between these complexes as most analyses thus far rely on ago1∆ allele or have been based on proteomic pull down analyses of RISC or RITS complexes. In this study, we employed biophysical and modeling approaches described earlier to generate temperature sensitive mutants in spago1+ and spdcr1+. We tested several mutants for their ability to repress two reporter genes in a conditional manner. Our modeling studies on SpAgo1 PAZ domain indicated structural similarities with human Ago1 PAZ domain. We created site-directed missense mutants at predicted buried residues or in catalytic residues. We also analyzed the effects of random amino acid replacements in specific predicted buried or catalytic residues of SpAgoI. These ago1 mutants were screened as pools for their effects on silencing of GFP or of ura4+ reporter genes. These assays assessed post transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) activity of these mutants. We obtained three temperature sensitive SpAgo1 mutants V324G, V324S and L215V while the V324E replacement was a null allele. Based upon our modeling, a likely explanation for the phenotype of these mutants is structural distortion or mis-orientation of the functional groups caused due to these mutations, which affect activity in a temperature dependent manner. This distortion in the PAZ domain may affect binding of siRNA and thereby lead to heterochromatin formation defects that we observed. Our data on the SpAgo1 V324 mutant shows conditional centromeric heterochromatin formation confirmed by semi quantitative RT-PCR for dh transcripts levels that shows temperature dependent increase in these transcripts. We find reduced H3K9Me2 levels at dh locus by chromatin immunoprecipitation (ChIP) assay, linking the association of siRNAs for establishment of heterochromatin at this loci. The data on PTGS of GFP transcripts show SpAgo1 V324G mutation has decreased slicing activity as semi-quantitative RT-PCR for GFP transcripts show increased levels at non permissive temperature. These studies point out the importance of siRNA binding to the PAZ domain and its effect on slicing activity of SpAgo1. The mutations in Y292 showed residue loss of centromeric heterochromatin formation phenotype. Thus, we ascribe critical siRNA binding and 3’ end recognition functions to this residue of SpAgo1. These studies point out functional and structural conservation across hAgo1 and SpAgo1. Adopting the aforementioned biophysical mutational approach, we generated mutants in spdcr1+ and screened for those with conditional activity. Our modeling studies on SpDcr1 helicase domain shows it adopts the conserved helicase domain structure seen for other DEAD Box helicases. Our data on mutational analysis of a conserved buried residue I143 in the walker motif B created inactive protein. The data confirm critical functions for dicer in generation of siRNAs and also in recognition of dsRNA ends. Mutants in buried residues L1130 and I1228 of RNase IIIb domain were inactive and the proximity of these residues to the catalytic core suggest that the critical structural alignment of catalytic residues is indispensable for carrying out dsRNA cleavage to generate siRNAs. We also attribute critical catalytic functions to SpDcr1 D1185 residue for generation of siRNA and heterochromatin formation as measured by our transcriptional gene silencing assay. Our studies employing biophysical and computational approaches to design temperature-sensitive mutants have been successfully applied to an essential splicing factor SpPrp18, which was refractory for ts mutants by other methods. Using a missense mutant, we showed its intron-specific splicing function for subsets of transcripts and deduced that its ubiquitous splicing role is arguable. We have uncovered a link between the splicing substrates of SpPrp18 and direct evidence of splicing based cell cycle regulation, thus providing a mechanistic link to the cell cycle arrest seen in some splicing factor mutants. The same methodology was applied to another important biological pathway, the RNAi machinery, where central factors SpAgoI and SpDcrI were examined We report the first instance of conditional gene silencing tool by designing Ago1 ts mutants which will be useful for future studies of the global interaction network between RNAi and other RNA processing events.

Yes
The NAD(+)-dependent deacetylase SIRT1 is involved in diverse cellular processes, and has also been linked with multiple disease states. Among these, SIRT1 expression negatively correlates with cancer survival in both laboratory and clinical studies. Active regulator of SIRT1 (AROS) was the first reported post-transcriptional regulator of SIRT1 activity, enhancing SIRT1-mediated deacetylation and downregulation of the SIRT1 target p53. However, little is known regarding the role of AROS in regulation of SIRT1 during disease. Here, we report the cellular and molecular effects of RNAi-mediated AROS suppression, comparing this with the role of SIRT1 in a panel of human cell lines of both cancerous and non-cancerous origins. Unexpectedly, AROS is found to vary in its modulation of p53 acetylation according to cell context. AROS suppresses p53 acetylation only following the application of cell damaging stress, whereas SIRT1 suppresses p53 under all conditions analysed. This supplements the original characterization of AROS but indicates that SIRT1 activity can persist following suppression of AROS. We also demonstrate that knockdown of AROS induces apoptosis in three cancer cell lines, independent of p53 activation. Importantly, AROS is not required for the viability of three non-cancer cell lines indicating a putative role for AROS in specifically promoting cancer cell survival.