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Thesis (Ph. D.)--Case Western Reserve University, 2008. / [School of Medicine] Department of Molecular Biology and Microbiology. Includes bibliographical references.

Flavopridol (FVP) is a well known pharmacological inhibitor of Cyclin Dependent Kinases (CDKs), with significant selectivity for Cyclin Dependent Kinase 9 (CDK9). Treatment of cells with FVP results in inhibition of transcription elongation. CDK9 is a serine/threonine kinase that associates with T-type cyclins. These complexes are designated transcription elongation factors (P-TEFb). P-TEFb controls transcription elongation by phosphorylating the carboxyl terminal domain (CTD) of RNA polymerase II (RNAPII) and negative elongation factors. Whether P-TEFb is required for the elongation of most genes transcribed by RNAPII or fraction of them is still debatable. The aim of my Thesis is to understand the early and late effects of FVP on primary response gene expression. Two different microarray analyses with RNA probes obtained from T98G and BJ-TERT cells were performed by Drs. Graña and Garriga to determine the effect of inhibiting CDK9 on global mRNA expression using a dominant negative mutant of CDK9 (dnCDK9) and FVP. These gene profiling experiments showed that FVP and dnCDK9 downregulate the expression of several genes. However, these studies also showed upregulation of a group of primary response genes (PRGs). The goal of this thesis was to bring some light into this unexpected phenomenon. I have found that several PRGs including FOS, JUNB, EGR1 and GADD45B, are rapidly and potently downregulated before they are upregulated upon FVP treatment in exponentially growing cells. In serum starved cells restimulated with serum, FVP also inhibits the expression of these genes, but subsequently, JUNB, GADD45B and EGR1 are upregulated in the presence of FVP. Chromatin Immunoprecipitation of RNAPII revealed that EGR1 and GADD45B are apparently transcribed at the FVP-treatment time points where their corresponding mRNAs accumulate. These results suggest a possible stress response triggered by CDK9 inhibition. I also show that serum starvation does not affect the localization of RNAPII immediately downstream of the promoter of a PRG where RNAPII remains paused in the absence of mitogenic stimulation, suggesting that initiation is not rate limiting for transcription of at least certain PRGs in the absence of mitogens and remains dependent on transcription elongation. In sum, I have shown that certain PRG/IRGs are transcribed in the presence of FVP and their transcription might be independent of CDK9 suggesting a possible alternative mechanism of their transcription. I also determined that transcription initiation is not affected by serum starvation, as paused RNAPII appears to remain bound downstream of a PRG promoter in quiescent cells independently of the length of mitogenic starvation. / Molecular Biology and Genetics

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Splicing mainly occurs co-transcriptionally, suggesting that transcription and premRNA splicing could be synchronized. The nature of this phenomenon suggests that transcription elongation rate may influence splicing outcomes and, indeed, there is evidence for effects on alternative splicing in mammals. To elucidate potential effects of transcription rate on splicing efficiency and fidelity, splicing of nascent transcripts was investigated in fast and slow elongating RNA polymerase II (RNAPII) mutants in Saccharomyces cerevisiae. High kinetic resolution 4-thio Uracil labelling of nascent RNA reveals that fast RNAPII accumulates unspliced pre-mRNA that represents reduced co-transcriptional splicing. Conversely, low levels of unspliced pre-mRNA were detected in the slow mutant due to increased co-transcriptional splicing. The highly stable association of nascent transcripts with elongating RNAPII permits co-transcriptional splicing to be measured by analysis of transcripts that co-purify with RNAPII. Measuring co-precipitation of the spliced mRNA and excised intron that are associated with RNAPII demonstrates that splicing is mostly co-transcriptional with the slow mutant, and the fast mutant reduces co-transcriptional splicing. How elongation rate affects splicing fidelity in budding yeast and whether faster and slower transcription have the opposite effect on splicing fidelity as might be predicted by the kinetic coupling model is an open question. Using deep RNA sequencing, splicing fidelity was determined in yeast transcription elongation mutants. Results show that both fast and slow transcription reduce splicing fidelity mainly in ribosomal protein coding transcripts. Analysis reveals that splicing fidelity depends largely on intron length, secondary structure and splice site score. These analyses also provide new insights regarding the effect of altering transcription rate on selection of transcription start sites. Together, these results indicate that optimal splicing efficiency and fidelity require finely-tuned transcription speed.

Relations among transcription, pre-mRNA processing and chromatin modifications are only partially understood. The human protein SNW1/SKIP belongs to factors which couple these processes. The protein plays role in pre-mRNA splicing and transcription on the level of both initiation and elongation. According to the hypothesis of K. Jones laboratory, it physically and functionally interacts with positive transcription elongation factor b during transcription elongation and influences methylation of histone H3 on lysine 4, a modification characteristic for active transcription (Bres et al., Genes Dev. 19:1211-26, 2005, Bres et al., Mol Cell. 36:75-87, 2009). The yeast ortholog of SNW1/SKIP, Prp45, was until now reported only in connection with splicing regulation. However, unpublished results from our Laboratory and others showed that it is employed in transcription elongation as well. The aim of the diploma project was to search for the relations between Prp45 and the factors regulating transcription. It was confirmed that the mutation prp45(1 169) results in the delay of PHO5 and PHO84 expression during transcriptional induction. Next, we discovered new genetic interactions between PRP45 and several genes encoding the effectors of chromatin modifications. How Prp45 influences the expression of PHO5 and PHO84...

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Abstract: Molecular and cellular insights into IKAP and Elongator functions As the first step in the complex process of gene expression, the transcription of genes from DNA to RNA by RNA polymerase II is subject to a multiplicity of controls and is thereby the endpoint of multiple cell regulatory pathways. We focused here on the molecular and cellular functions of IKAP and by extension of Elongator complex, initially found associated with the hyperphosphorylated RNA polymerase II during the elongation stage of transcription. IKAP is required for the assembly of Elongator subunits into a functional complex. Elongator has a histone acetyltransferase (HAT) activity associated with one of its subunits, named hELP3. In agreement with a potential role in transcript elongation, Elongator is associated with nascent RNA emanating from the elongating RNA polymerase II along the transcribed region of several yeast genes and chromatin immunoprecipitation experiments have also demonstrated an association of Elongator with genes in human cells. Different mutations in the human IKBKAP gene, encoding IKAP/hELP1, cause familial dysautonomia, a severe neurodevelopmental disease with complex clinical characteristics. Affected individuals are born with the disease and abnormally low numbers of neurons in peripheral nervous ganglions. To gain insight into the role played by IKAP and the Elongator complex in the transcription of genes and concomitantly learn about the molecular defects underlying the FD, an RNA interference approach was used to deplete the IKAP protein in human cells. In yeast, disruption of ELP1 (yeast homolog of human IKAP) is known to destabilize the ELP3 catalytic subunit, which leads to loss of Elongator integrity. Our experiments performed in human cells revealed that the levels of hELP3 protein is also affected by IKAP depletion after RNAi. We took advantage of this cellular loss-of-function model to identify genes whose transcription requires IKAP, by microarray experiments. Among the identified candidates, several were previously described to be involved in cell motility, or actin cytoskeleton remodelling. Because cell motility is of crucial importance for the developing nervous system, and therefore of obvious relevance to FD, the potential role of IKAP in cell motility was characterized at the cellular level. Several cell motility/migration assays demonstrated that the IKAP depletion has functional consequences so that IKAP-depleted cells showed defects in migration. Particularly, the reduced cell motility of neuronal-derived cell lines may be highly relevant to the neurodevelopmental disorder that affects FD patients. Whether or not the defects in cell migration resulted of impaired transcriptional elongation of the IKAP-dependent genes was investigated by chromatin immuno-precipitation technique. These experiments indicated that IKAP depletion leads to a decreased histone H3 acetylation in the transcribed region of its target genes in the context of Elongator complex. These acetylation defects are correlated with a decrease of the RNA polymerase II recruitment through the transcribed region of target genes, whereas the recruitment on the promoter is mostly unaffected. These results indicate that Elongator affects transcript elongation in vivo, but not the recruitment of the RNA polymerase II to the promoter. These very specific effects of IKAP/hELP1 depletion on histone acetylation and RNA polymerase II density across target genes are consistent with a direct effect of Elongator on transcriptional elongation in vivo and point to a function for Elongator in histone acetylation during transcript elongation. Résumé: Caractérisation des rôles biologiques de la protéine IKAP et du complexe Elongator La transcription des gènes de lADN en ARN est fondamentale pour lexpression des protéines et la capacité de nos cellules à sadapter à leur environnement. Ce processus finement régulé est catalysé par un enzyme, lARN polymérase II, vers lequel convergent une multitude de voies de signalisation. Dans le cadre de ce travail, nous nous sommes intéressés aux fonctions moléculaires et cellulaires de la protéine IKAP et du complexe Elongator. IKAP est la protéine qui assemble les sous unités dElongator en un complexe fonctionnel. Le complexe Elongator est associé à lARN polymérase II hyper-phosphorylée pendant létape délongation de la transcription et possède une activité histone acétyltransferase associée à une de ses sous unités, appelée ELP3. Chez la levure, Elongator est recruté an niveau des ARNs naissants, qui émanent directement de lARN polymérase II au niveau de la région transcrite des gènes étudiés. De plus, des expériences dimmunoprécipitation de la chromatine ont mis en évidence la présence du complexe Elongator au niveau de plusieurs gènes humains. Différentes mutations au niveau du gène IKBKAP, codant pour la protéine IKAP, sont responsables de la dysautonomie familiale, une maladie génétique qui affecte le développement du système nerveux périphérique. En effet, les individus affectés présentent une diminution de la densité de neurones au niveau des ganglions nerveux périphériques. Lobjectif de nos travaux est de comprendre davantage le rôle de la protéine IKAP et du complexe Elongator dans la transcription des gènes et ainsi, dinvestiguer les mécanismes moléculaires responsables dans la physiopathologie de la dysautonomie familiale. Un modèle de perte de fonction pour la protéine IKAP a dabord été généré par interférence dARN. Des travaux réalisés chez la levure indiquent que la protéine ELP1 (homologue de IKAP chez la levure) est essentielle pour la stabilité de la sous unité catalytique du complexe, la protéine ELP3. Les expériences réalisées sur notre modèle humain démontrent que le taux de la protéine ELP3 est également affecté par la déplétion dIKAP causée par linterférence dARN. Ce modèle de perte de fonction a été utilisé afin détablir la liste des gènes dont lexpression est contrôlée par la protéine IKAP, par des expériences de microarrays. Parmi les candidats identifiés, plusieurs ont été décrits comme impliqués dans la migration cellulaire et le remodelage du cytosquelette dactine. Le processus de migration des cellules est fondamental au cours du développement du système nerveux et par conséquent particulièrement relevant dans le contexte de la dysautonomie familiale. Limplication dIKAP dans la migration cellulaire a été investigué par différents tests de fonction qui montrent que la diminution dIKAP dans différentes lignées cellulaires entraîne une réduction significative de leur capacité migratoire. Ces résultats suggèrent que la diminution du nombre de neurones observée dans les ganglions périphériques des patients atteints de la dysautonomie familiale pourrait résulter dune altération de leur capacité à migrer au cours du développement. Enfin, des expériences dimmunoprécipitation de la chromatine ont été menées en utilisant notre modèle afin de déterminer dans quelle mesure le déficit de migration observé en labsence dIKAP serait la conséquence dun défaut de la fonction dElongator au niveau de lélongation de la transcription des gènes. Les résultats nous ont montré que la diminution dexpression dIKAP entraîne une réduction de lacétylation des histones H3 dans la région transcrite de ses gènes cibles. De plus, ce déficit dacétylation est directement corrélé avec un désengagement progressif de lARN polymérase II le long de la région transcrite de ces gènes. Par conséquent, ces résultats démontrent que le complexe Elongator affecte lélongation des transcrits in vivo, mais pas le recrutement de lARN polymérase II au niveau du promoteur. Ces effets très spécifiques de labsence dIKAP sur lacétylation des histones et lengagement de la polymérase II dans la transcription des gènes cibles montrent quElongator exerce un rôle direct au niveau de lélongation de la transcription de ces gènes. De plus, ces résultats suggèrent que la fonction dElongator serait dacétyler les histones au cours de lélongation transcriptionnelle in vivo.

RNA polymerase II (Pol II) is the enzyme responsible for the synthesis of all mRNAs in eukaryotic cells. As the central component of the eukaryotic transcription machinery, Pol II is the final target of regulatory pathways. While the role for different Pol II associated proteins, co-activators and general transcription factors (GTFs) in regulation of transcription in response to different stimuli is well studied, a similar role for some subunits of the core Pol II is only now being recognized. The studies reported in this thesis address the role of the fourth largest subunit of Pol II, Rpb4, in transcription and stress response using Saccharomyces cerevisiae as the model system. Rpb4 is closely associated with another smaller subunit, Rpb7 and forms a dissociable complex (Edwards et al., 1991). The rpb4 null mutant is viable but is unable to survive at extreme temperatures (>34ºC and <12ºC) (Woychik and Young, 1989). This mutant has also been shown to be defective in activated transcription and unable to respond properly in several stress conditions (Pillai et al., 2001; Sampath and Sadhale, 2005). In spite of wealth of available information, the exact role of Rpb4 remains poorly understood. In the present work, we have used genetic, molecular and biochemical approaches to understand the role of Rpb4 as described in four different parts below: i) Studies on Genetic and Functional Interactions of Rpb4 with SAGA/TFIID Complex to Confer Promoter- Specific Transcriptional Control To carry out transcription, Pol II has to depend on several general transcription factors, mediators, activators, and co-activators and chromatin remodeling complexes. In the present study, we tried to understand the genetic and functional relationship of Rpb4 with some of the components of transcription machinery, which will provide some insight into the role of Rpb4 during transcription. Our microarray analysis of rpb4∆ strain suggests that down regulated genes show significant overlap with genes regulated by the SAGA complex, a complex functionally related to TFIID and involved in regulation of the stress dependent genes. The analysis of combination of double deletion mutants of either the SAGA complex subunits or the TFIID complex with rpb4∆ showed that both these double mutants are extremely slow growing and show synthetic growth phenotype. Further studies, including microarray analysis of these double mutants and ChIP (chromatin immunoprecipitation) of Rpb4 and SAGA complex, suggested that Rpb4 functions together with SAGA complex to regulate the expression of stress dependent genes. ii) Study of Genome Wide Recruitment of Rpb4 and Evidence for its Role in Transcription Elongation Biochemical studies have shown that Rpb4 associates sub-stoichiometrically with the core RNA polymerase during log phase but whether recruitment of Rpb4 is promoter context dependent or occurs only at specific stage of transcription remains largely unknown. Having discovered that Rpb4 can recruit on both TFIID and SAGA dominated promoters, it was important to study the genome wide role of Rpb4. Using ChIP on chip experiments, we have carried out a systematic assessment of genome wide binding of Rpb4 as compared to the core Pol II subunit, Rpb3. Our analysis showed that Rpb4 is recruited on coding regions of most transcriptionally active genes similar to the core Pol II subunit Rpb3 albeit to a lesser extent. This extent of Rpb4 recruitment increased on the coding regions of long genes pointing towards a role of Rpb4 in transcription elongation of long genes. Further studies showing transcription defect of long and GC rich genes, 6-azauracil sensitivity and defective PUR5 gene expression in rpb4∆ mutant supported the in vivo evidence of the role of Rpb4 in transcription elongation. iii) Genome Wide Expression Profiling and RNA Polymerase II Recruitment in rpb4∆ Mutant in Non-Stress and Stress Conditions Structural studies have suggested a role of Rpb4/Rpb7 sub-complex in recruitment of different factors involved in transcription (Armache et al., 2003; Bushnell and Kornberg, 2003). Though only few studies have supported this aspect of Rpb4/Rpb7 sub-complex, more research needs to be directed to explore this role of Rpb4/Rpb7 sub-complex. To study if Rpb4 has any role in recruitment of Pol II under different growth conditions, we have studied genome wide recruitment of Pol II in the presence and absence of Rpb4 during growth in normal rich medium as well as under stress conditions like heat shock and stationary phase where Rpb4 is shown to be indispensable for survival. Our analysis showed that absence of Rpb4 results in overall reduced recruitment of Pol II in moderate condition but this reduction was more pronounced during heat shock condition. During stationary phase where overall recruitment of Pol II also goes down in wild type cells, absence of Rpb4 did not lead to further decrease in overall recruitment. Interestingly, increased expression levels of many genes in the absence of Rpb4 did not show concomitant increase in the recruitment of Pol II, suggesting that Rpb4 might regulate these genes at a post-transcriptional step. iv) Role of Rpb4 in Pseudohyphal Growth The budding yeast S. cerevisiae can initiate distinct developmental programs depending on the presence of various nutrients. In response to nitrogen starvation, diploid yeast undergoes a dimorphic transition to filamentous pseudohyphal growth, which is regulated through cAMP-PKA and MAP kinase pathways. Previous work from our group has shown that rpb4∆ strain shows predisposed pseudohyphal morphology (Pillai et al., 2003), but how Rpb4 regulates this differentiation program is yet to be established. In the present study, we found that disruption of Rpb4 leads to enhanced pseudohyphal growth, which is independent of nutritional status. We observed that the rpb4∆/ rpb4∆ cells exhibit pseudohyphae even in the absence of a functional MAP kinase and cAMP-PKA pathways. Genome wide expression profile showed that several downstream genes of RAM signaling pathway were down regulated in rpb4∆ cells. Our detailed genetic analysis further supported the hypothesis that down regulation of RAM pathway might be leading to the pseudohyphal morphogenesis in rpb4∆ cells.

The global re-emergence of TB and other mycobacterial infections have underscored the need for a thorough investigation of the biology of the causative agent, Mycobacterium tuberculosis, at the molecular level. The peculiar features of the bacterium such as slow growth rate, dormancy, unique cell wall composition and resistance towards phagocytosis by macrophages demands a detailed understanding of different essential molecular processes including transcription in this genus. Sequencing of several mycobacterial genomes provided an impetus for understanding the gene function and regulation of this formidable pathogen. Transcriptional regulation is one of the major mechanisms controlling gene expression. While a number of transcription units, promoters, sigma factors, and gene functions were identified and characterized, key features of transcription process are yet to be understood. The current study aims to understand some of the facets of transcription initiation and elongation in mycobacteria. The thesis is divided into five chapters. Chapter 1 introduces the bacterial transcription process. It starts with the description of the central molecule in transcription -the RNA polymerase (RNAP) and its catalytic mechanism. In the next section, each step of the transcription initiation, elongation and termination has been discussed. The mechanistic details as well as the different cellular factors involved in the regulation of the transcription have been discussed. The final part gives an overview of the transcription machinery of the mycobacteria, describing the promoter specificity and regulation of different sigma factors and other transcription factors known till date in mycobacteria. The scope and the objectives of the thesis are presented at the end of this chapter. In Chapter 2, a method of purification of RNAP from mycobacteria for optimized promoter -polymerase interactions is described. In vitro transcription analysis is important to understand the mechanism of transcription. Various assays for the analysis of initiation, elongation and termination form the basis for better understanding of the process. Purified RNAP with high specific activity is necessary to carry out a variety of these specific reactions. The RNAP purified from Mycobacterium smegmatis from exponential phase showed low σA-promoter specificity in promoter -polymerase interaction studies. This is due to the presence of a large number of sigma factors during exponential phase and under-representation of σA required for house - keeping transcription. In vivo reconstitution of RNAP holoenzyme with σA and its purification procedure which resulted in a holoenzyme with stoichiometric σA content is described in this chapter. The reconstituted holoenzyme showed enhanced promoter -specific binding and transcription activity compared to the enzyme isolated using standard procedure. Chapter 3 is aimed at the comparison of promoter - specific events during transcription initiation in mycobacteria. DNA -protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNAP binds to promoters in a sequence -specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. The first detailed kinetic analysis of promoter - RNAP interactions during transcription initiation in the σA-dependent promoters PrrnAPCL1, PrrnB and Pgyr of M. smegmatis are presented in this chapter. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to the distinct kinetic patterns, each one of the house -keeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation. Moving the focus of the thesis from transcription initiation to elongation, a transcript cleavage factor of M. tuberculosis has been characterized in Chapter 4. After initiation of transcription, a number of proteins participate during elongation and termination by modifying the properties of the RNAP. Gre proteins are one such class of transcription elongation factors which are conserved across bacteria. They regulate transcription by binding near the secondary channel of RNAP, projecting their N-terminal coiled-coil domain into the active center and stimulating hydrolysis of the newly synthesized RNA by RNAP in the backtracked elongation complexes. Rv1080c is a putative gre factor homolog (MtbGre) present in M. tuberculosis.The protein enhanced the efficiency of promoter clearance by lowering the abortive transcription and also rescued the arrested and paused elongation complexes efficiently in the GC rich mycobacterial template. The Gre factor of M. smegmatis encoded by the gene MSMEG_5263 also showed biochemical properties similar to the M. tuberculosis protein. Although the mycobacterial Gre is similar in domain organization and shared the key residues for catalysis and RNAP interaction with Escherichia coli Gre proteins, it could not complement the E. coli strain deficient in Gre factors. Moreover, MtbGre failed to rescue E. coli RNAP stalled elongation complexes, indicating the importance of specific protein - protein interactions for transcript cleavage. Decrease in the level of MtbGre also reduced the bacterial survival by several fold indicating its essential role in mycobacteria and suggesting that a single Gre copes up with the burden of transcription fidelity of the genome. Chapter 5 describes the studies carried out to identify Gre factor homologs in mycobacteria and deciphering their function during transcription. Gre factors are members of a growing family of proteins which regulate RNAP through secondary channel. Apart from the Gre factor, putative members of this class of proteins are identified in both M. smegmatis and M. tuberculosis.The closest homologue of the canonical Gre factor of M. tuberculosis in its genome is Rv3788. The protein has Gre factor like domain organization and possess the key acidic residues required for transcript cleavage activity and the putative hydrophobic RNAP interacting residues in the C-terminus similar to MtbGre. Despite having these common features, Rv3788 did not stimulate transcript cleavage. In contrast, it turns out to be a transcription inhibitor by preventing the binding of NTPs to the enzyme. The transcription inhibition is not promoter specific, and is mediated by its binding to RNAP through the secondary channel with its N-terminus coiled coil domain. Like M. tuberculosis, the fast growing non-pathogenic mycobacteria M. smegmatis also has an ORF (MSMEG_6292) which is homologous to its canonical Gre factor and it interacts with RNAP in a similar manner. However, this protein did not exert any transcript cleavage or inhibitory activities but could compete with the Gre factor for binding to RNAP. The Gre factor homologs in mycobacteria may be involved in regulation by inhibiting transcription or by blocking the RNAP secondary channel from other RNAP active site modulators.