Exploring the Role of RNA Polymerase III Complex Assembly on Ribosomal DNA Silencing in Saccharomyces cerevisiae

The yeast rDNA region is host to a number of transcriptional regulatory elements, which work in conjunction to generate essential RNA subunits of ribosomes, as well as protecting the region from DNA damage. The role of RNA polymerase III complex binding at the 5S gene on rDNA silencing in the NTS2 region was investigated, both by use of a TY1:MET15 reporter insert and a MET15 gene integration at an endogenous SphI site. It was discovered that Pol III complexes do have an effect on reporter expression in the NTS2 region, though the specific effect was different based on the method of reporter integration. The ability of Reb1p and Pol III complex binding sites to block RNA polymerase II read through transcription was also explored. Reb1p was also found to be able to block Pol II read through transcription, while a 5S gene was only able to partially block Pol II read through transcription, and did so in an orientation dependent manner. Finally, a novel strategy for reducing leaky transcription from inducible promoters was designed, which may be of benefit to the greater research community. These results suggest an interesting possibility that DNA-bound Pol III complexes at 5S genes has an impact on rDNA silencing, and may have a greater impact in the regulation in the rDNA region than originally thought

Regulatory Studies of the Mammalian RNA Polymerase III Transcriptional Apparatus

Although many studies have shown that pol III transcription is strongly regulated in higher eukaryotes, it is poorly understood how this regulation is achieved. The basal pol III factors TFIIIB and TFIIIC have been implicated as common targets for regulation. I have developed reproducible purification protocols for yielding partially purified active human TFIIIB and human TFIIIC. The purity of hTFIIIB and hTFIIIC obtained are a significant improvement upon that of hTFIIIB and hTFIIIC typically used in our laboratory, allowing regulatory studies to be conducted with a much higher level of confidence than previously. One established repressor of pol III transcription is the tumour suppressor RB. Recently, the related proteins pi07 and pi30 have also been shown to inhibit pol III transcription. Here, I show that endogenous p107 and p130 cofractionate and coimmunoprecipitate with endogenous TFIIIB, suggesting that, like RB, p107 and p130 stably associate with TFIIIB under physiological conditions. I have also investigated why the binding of RB to TFIIIB inhibits pol III transcription. For several genes transcribed by pol II, RB represses transcription through the recruitment of the histone deacetylase HDAC1, which is thought to deacetylate histones at the promoter resulting in the formation of a more compact chromatin structure less accessible to transcription factors. However, the repression of pol III transcription in vitro by RB is unaffected by the presence of the histone deacetylase inhibitor trichostatin A. Using an immunoisolated pol III complex that contains pol III, TFIIIC and TFIIIB, I show that recombinant RB can specifically disrupt the interaction between TFIIIB and TFIIIC. The serine/threonine kinase CKII is identified as a novel activator of mammalian pol III transcription and is shown to stably interact with endogenous hTFIIIB. Significantly, CKII kinase activity appears to promote the binding of TFIIIB to TFIIIC. The receptor tyrosine kinase neu (erbB2) is also implicated in the regulation of pol III transcription. A rodent ovarian epithelial cell line transformed by an activated neu oncogene is found to display elevated pol III activity. TFIIIC2 B-block binding activity is specifically elevated. Using the purified TFIIIB and TFIIIC fractions, I show that TFIIIC is, limiting in the untransformed control cell line, indicating that upregulation of TFIIIC2 activity in response to neu transformation can at least partly account for the increase in pol III activity

Regulatory Impacts of Assembled Pol III Complexes on Neighboring Pol II Transcribed Genes in Saccharomyces cerevisiae

RNA polymerase III (Pol III) transcribes tRNAs as well as other small non-coding RNAs. tRNA genes contain internal promoter sequences (A- and B-boxes) which can be specifically recognized and bound by TFIIIC. Binding of TFIIIC facilitates TFIIIB recruitment, which in turn targets Pol III to be recruited and initiate transcription. In addition to typical tRNA genes, there are other chromosomal regions, referred as extra-TFIIIC (ETC) sites, that are only bound by TFIIIC. Apart from transcription of genes, both complete and partially assembled Pol III complexes perform extra-transcriptional functions such as influencing nearby Pol II transcription, displacement of nearby nucleosomes, as well as chromatin boundary activities. By analyzing transcriptome data from high throughput RNA sequencing, we observed numerous alterations in intergenic transcription in close proximity to tDNAs and other Pol III complex binding sites after TFIIIC binding was globally compromised. Reduction of TFIIIC binding activity was achieved by using a yeast strain containing a mutation in the Reb1p binding site within the TFC6 promoter, which drastically reduces the level of the TFIIIC component Tfc6p. Analysis of loci adjacent to Pol III complex binding sites reveal both 5’- and 3’-extended transcripts, readthrough transcripts, and increased intergenic cryptic transcription. Many of the effects of 5’-UTR extension and de-repression appear to be due to the release of bidirectional activity of neighboring promoters. Translation of affected mRNAs is greatly altered because of the usage of upstream transcriptional start site (TSSs) at both TFC6 and TRM12 loci. The results presented here add another type of boundary activity to the known list of extra-transcriptional functions – the blocking activity of transcription from bidirectional promoters. Also, such activities might explain a function of the conserved ETC sites in yeast Saccharomyces cerevisiae. Analysis of the TFC6 locus suggests regulatory effects of assembled Pol III complexes at ETC6 site. Also, Reb1p is confirmed to be the transcriptional factor that binds and activates the TFC6 promoter and it binds several base pairs upstream of the ETC6 site. Analysis of these two divergently transcribed genes (ESC2 and TFC6) reveals that both have 5’-UTR extensions after Reb1p is depleted or the Reb1 consensus binding site is mutated, which is consistent with results from our transcriptome data. Similarly, altered TSS results in a significant decrease of the translational level of both TFC6 and ESC2. By assessing nucleosome content within the intergenic region ESC2-TFC6, we find that nucleosome positioning is slightly altered, which could explain the observed 5’-extended transcripts of both genes. Taken together, both of these studies demonstrate significant effects of assembled Pol III complexes and Reb1p binding on the transcription of neighboring Pol II promoters and on the translation of their mRNA products

Regulation of RNA polymerase III transcription during differentiation

The differentiation of F9 embryonal carcinoma (EC) cells into parietal endoderm (PE)-like cells provides a tractable model for studying molecular events during early and inaccessible stages of murine development. It has been previously shown that PE formation is accompanied by a ~10-fold decrease in RNA polymerase (po1) III transcription. This down-regulation was attributed to a reduction in the activity of the basal po1 III factor TFIIIB; however, the mechanism involved was not established. The work presented here was aimed at elucidating the precise changes that lead to a decrease in po1 III transcriptional activity during F9 differentiation. The protein levels of all three components of the TFIIIB complex, Brf1, Bdp1 and TBP, were found to decrease. In the case of Brf1 and Bdp1, this decrease did not reflect a change in their stability, as proteasome inhibition did not have an effect on protein abundance. On the other hand, a specific down-regulation of the mRNAs encoding Brf1 and Bdp1 was observed. Temporal analysis of F9 differentiation, however, revealed that the decline in TFIIIB levels follows that in po1 III transcription in the same extracts. Furthermore, over-expression of Brf1 did not result in an increase in po1 III transcription in PE cells. These data point to TFIIIB playing a secondary role in the down-regulation of po1 III during differentiation. TFIIIB activity, and consequently po1 III transcription, can be regulated by a number of proteins. For example, c-Myc activates po1 III transcription, while the retinoblastoma protein (pRb) is a repressor. Time course analysis revealed that c- Myc down-regulation and pRb up-regulation closely follows the decline in po1 III transcription during differentiation. Furthermore, recombinant c-Myc efficiently restored transcription in PE extracts. These two proteins, therefore, provide likely candidates for the primary regulatory mechanism of po1 III transcription in response to differentiation, through their effect on TFIIIB

Regulation of RNA polymerase III transcription by the tumour suppressor p53

The tumour suppressor protein p53 is inactivated in a large proportion of human cancers. p53 controls growth and proliferation, through multiple mechanisms, including the ability to regulate transcription. p53 can function as a general repressor of RNA polymerase (pol) III transcription. Pol III is responsible for transcribing a variety of small stable RNAs including tRNA, 5S rRNA, and the adenoviral VA1 RNA. p53 targets TFIIIB, a TBP-containing factor that is essential for recruitment of pol III to its templates. This study investigates the TFIIIB-p53 interaction, and how it serves to regulate pol III-transcribed genes. p53 does not disrupt the interaction between the TFIIIB subunits, TBP and Brf1. It does, however, prevent association of TFIIIB with the pol III specific factor TFIIIC2, and pol III itself Immobilised template and chromatin immunoprecipitation analysis show that p53 prevents the recruitment of TFIIIB, but not TFIIIC2, to the tRNA promoter. Pol III repression cannot be attributed to one clearly defined region of p53. Sequence within both the N- and C-termini are essential, and the central core domain is also implicated in playing a role. Evidence is provided here that p53-mediated repression of tRNA genes occurs via a trichostatin A-sensitive histone deacetylase independent mechanism. p53 is deregulated or mutated in the vast majority of human cancers. Individuals who inherit mutant forms of p53 can suffer from Li-Fraumeni Syndrome (LFS), a familial cancer syndrome associated with a range of malignancies. Here is shown that pol III transcriptional activity is often highly elevated in primary fibroblasts from Li-Fraumeni patients, especially if the germline p53 mutation is followed by loss of the remaining allele. Deregulation of p53 function through the action of various oncoproteins can also contribute to carcinogenesis. E6 from human papilloma virus can bind to p53 and neutralise its function and E6 releases pol III from p53-mediated repression. Induction of the Mdm2 regulating protein p14ARF results in a p53-mediated repression of pol III activity. p53 does not interfere with normal cellular growth and development; it is, however, rapidly induced in response to cellular stress. Here it is shown that the DNA-damaging agent MMS provokes a p53 response that correlates with a dramatic pol III transcriptional repression. Collectively the data presented here support the idea that p53 can directly repress pol III transcription through interactions with the basal pol III machinery. p53 status can have a profound effect upon pol III activity; the precise circumstances under which such control becomes physiologically important however, remains to be determined

Mechanisms of RNA polymerase III transcriptional activation by c-Myc

The Myc family of proto-oncogenes encodes transcription factors that play a pivotal role in regulating cellular proliferation, cellular growth, differentiation and apoptosis. To regulate cellular growth, it can activate a number of RNA polymerase II- transcribed genes which encode ribosomal proteins, translation factors and other components of the biosynthetic apparatus. c-Myc can also directly activate transcription by RNA polymerases I and III, thereby stimulating the production of ribosomal (r)RNA and transfer (t)RNA. As such, c-Myc may possess the capacity to induce the expression of all the ribosomal components. The work in this project aimed to investigate the mechanisms behind the c-Myc-dependent activation of RNA polymerase III transcription. One mechanism by which activators of pol III transcription can stimulate the expression of class III genes is by promoting transcription complex formation. It had been previously demonstrated that c-Myc can interact with the pol Ill-specific transcription factor TFIIIB. Work in this thesis has further defined this interaction and demonstrated that activation of transcription by c-Myc can recruit this complex along with pol III to 5S rRNA and tRNA genes in vivo. Furthermore, the recruitment of TFIIIB and polymerase by c-Myc are distinct events, with a significant delay between TFIIIB and pol III binding, arguing against a pol III holoenzyme being recruited to the genes. Most recent work on the mechanisms of transcriptional activation by c-Myc has focussed on its ability to influence chromatin structure. Transcriptional activation of target genes by c-Myc may involve the remodelling of nucleosomes, since c-Myc has been shown to bind to the Snf5 subunit of the SWI/SNF complex, as well as the ATPase/helicases TIP48 and TIP49. In the present study, Snf5 and Brg1, both components of SWI/SNF, have been found at the promoters of pol Ill-transcribed genes. These may have a role in the regulation of pol III transcriptional activity. c-Myc can also recruit a variety of histone modifying enzymes to the promoters of its target genes. It can bind to the co-factor TRRAP, a 440 kDa protein that forms the scaffold of a variety of histone acetyltransferase complexes. It has been demonstrated that c-Myc can recruit these complexes to certain target genes, and the increase in histone acetylation correlates with gene expression. The TRRAP co-factor along with an associated HAT was found to be present in a c-Myc-sensitive manner on pol Ill- transcribed genes, and their presence correlated with histone acetylation and gene expression. In addition to these findings, depletion of endogenous TRRAP by RNAi in cultured cells resulted in a specific down-regulation of pol III transcription in vivo. In summary, this thesis has identified previously undescribed mechanisms by which c-Myc can activate transcription by pol III, and has identified novel co-activator proteins involved in the regulation of class III gene expression. This work has important implications in understanding the molecular basis of how activators can stimulate the expression of pol Ill-transcribed genes

Chromatin remodelers and their roles in chromatin organization

The DNA in the eukaryotic nucleus is organized into a complex DNA-protein structure called chromatin. The basic repeating unit of chromatin is the nucleosome, which consists of 147 bp of DNA wrapped around a histone protein octamer. The nucleosomes form a “beads on a string” structure, which can be folded into higherorder structures that allow an extensive degree of DNA compaction. This compaction is so effective that 2 meters of DNA can fit into the human cell nucleus with a diameter of only 10 m. Hence, nucleosomes condense and organize the genome, but at the same time they occlude many regulatory elements essential for transcription, replication, repair and recombination. To ensure dynamic access to packaged DNA, cells have evolved a set of proteins called chromatin remodeling complexes, which actively restructure chromatin. These enzymes use the energy from ATP hydrolysis to unwrap, slide, and eject nucleosomes. This thesis describes the roles of two families of ATP-dependent chromatin remodeling factors in chromatin regulation and organization in the model organism Schizosaccharomyces pombe (fission yeast). We show that the CHD remodeling factor, Hrp1, promotes incorporation of the H3 histone variant CENP-ACnp1 at centromeres and at a set of gene promoters. We suggest that Hrp1 participates in a remodeling process that evicts H3 from promoters, both in euchromatin and centromeric chromatin, which then facilitates CENP-A Cnp1 incorporation. Furthermore, we demonstrate that the Fun30 remodeling factor, Fft3, regulates the chromatin structure over insulator elements and tethers them to the inner nuclear membrane close to nuclear pores. This organizes the chromatin into different domains and ensures correct chromatin structure and gene expression at silent domains. Additionally, we have generated the first genome-wide map of nucleosome positions in S. pombe. This map revealed important differences from the related yeast Saccharomyces cerevisiae. The two yeasts showed differences in nucleosome spacing, the roles of DNA sequence features and in the regular nucleosome arrays. This argues against the existence of an evolutionarily conserved genomic code for nucleosome positioning. Instead, species-specific nucleosome positioning factors (e.g. chromatin remodeling complexes) appear to override the biophysical properties of the DNA sequence

The regulation of pol III transcription by mTOR

Regulation of protein synthesis is an important aspect of growth control. A major determinant of this process is the availability of tRNA and 5S rRNA, which are synthesised by RNA Polymerase (pol) III. Pol III transcription is tightly linked to growth conditions, decreasing when nutrients or serum factors are low and increasing upon mitogenic stimulation. Therefore, it seems inevitable that mechanisms have evolved to couple production of the biosynthetic machinery with the needs of the cell. The target of rapamycin (TOR) signalling pathway, in conjunction with signalling through the phosphoinositide 3-kinase (PI3K) pathway, is central to this process in a diverse number of organisms. Mammalian cell lines were investigated to assess if pol III transcription is under the control of the PI3K and mammalian TOR (mTOR) pathways. Levels of pol III transcripts were reduced in vivo and pol III transcription was reduced in vitro in response to inhibition of the pathways. Inhibition of the mTOR and PI3K pathways was not found to alter the abundance of the pol Ill-specific transcription factors TFIIIB and TFIIIC, or indeed of pol III itself. Moreover, the effects of inhibition of the pathways on pol III transcription were found to be independent of known regulators of pol III, such as c-Myc, Retinoblastoma protein (RB) or extracellular signal-regulated kinase (ERK) signalling, but were shown to be due, in part, to signalling through the translational effector kinase S6K1. When the mTOR pathway is blocked by rapamycin, the interactions between TFIIIB and TFIIIIC, and between TFIIIB and pol III are ablated, which correlates with the finding that mTOR activity is required for normal promoter occupancy at pol III promoters. These data may be explained by the finding that the mTOR pathway regulates phosphorylation of TFIIIB and TFIIIC subunits. Maf1, a known negative regulator of pol III transcription in yeast, was investigated, since it has been reported to be under the control of the TOR signalling pathway. Maf1 was found to repress transcription of all class III genes in mammalian cells, and this repression can be relieved by the addition of a purified fraction of TFIIIB. Direct interaction of Maf1 with pol III and with the Brf1 subunit of TFIIIB was demonstrated, and further investigation shows that Maf1, pol III and Brf1 follow the same pattern of promoter occupancy on tRNALeu" genes in response to stress. In vivo phospho-labelling and in vitro kinase assays demonstrated that Maf1 is a phosphoprotein, and the phosphorylation of Maf1 was found to be inhibited by both serum-starvation and rapamycin-treatment of cells. This suggests that Maf1 may receive signals from these signalling pathways to co-ordinate pol III activity, and hence the growth capacity of the cell, with nutrient availability

Deregulation of RNA polymerase III transcription in response to polyomavirus transformation

RNA polymerase (pol) III transcription is stimulated in response to a variety of factors. Numerous studies concerning the DNA tumour virus Simian Virus 40 (SV40) have served to identify mechanisms surrounding its ability to elevate pol III transcriptional activity. Polyomavirus, a close relative of SV40, has similarly been shown to induce abnormally elevated levels of pol III transcription; however, the mechanisms involved were not previously established. This study presents an analysis of the mechanisms employed by Polyomavirus, as well as providing further insight into those utilised by SV40. In untransformed fibroblasts, the basal pol III factor TFIIIB is repressed through association with the retinoblastoma protein RB; this restraint is overcome by the large T antigens of Polyomavirus and SV40. Furthermore, cells transformed by these papovaviruses overexpress the B" subunit of TFIIIB, at both the protein and mRNA levels. Despite the overexpression of B", the abundance of other TFIIIB components, TBP and BRF, is unperturbed following papovavirus transformation. In contrast, all five subunits of the basal factor TFIIIC2 are abnormally abundant in fibroblasts transformed by either Polyomavirus or SV40, as demonstrated by the elevated levels of their mRNAs. Thus, both papovaviruses stimulate pol III transcription by boosting production specifically of selected components of the basal machinery. However, Polyomavirus differs from SV40 in adopting an additional and apparently unique deregulatory mechanism. This study presents the first evidence of a direct increase in pol III itself following viral transformation, as pol III activity and an accompanying elevation in the abundance of pol III subunits are observed following transformation by Polyomavirus. Another important difference of Polyomavirus is its ability to encode a highly oncogenic middle T antigen that is localised outside the nucleus and activates several signal transduction pathways. Like the large T antigen, the middle T antigen can serve as a potent and specific activator of pol III in transfected cells. This may be mediated through the middle T-induced activation of the MAPK pathway, correlating with an increase in the expression of active ERK. Furthermore, an endogenous interaction between ERK and TFIIIB presents the possibility of a direct role for ERK in the stimulation of pol III transcription. Thus, a striking variety of distinct mechanisms contribute to the dramatically elevated levels of pol III transcription that accompany transformation by Polyomavirus and SV40

The regulation of RNA polymerase I and RNA polymerase III transcription by the pocket proteins

RB has been shown to repress pol III transcription by targeting the pol III-specific general factor TFIIIB. Work presented here demonstrates that RB binds to the BRF subunit of TFIIIB. The binding interaction between these two proteins has been shown to require the RB large pocket domain and the amino-terminal repeats of BRF. In addition, the mechanism whereby RB represses pol III transcription has been established; namely, RB does not compromise the interactions within TFIIIB, but instead disrupts the association between TFIIIB and TFIIIC and between TFIIIB and pol III. In human cancers, RB is generally inactivated by one of three means: hyperphosphorylation, mutation or viral oncoprotein sequestration. SV40-transformed cell lines displayed a diminished interaction between RB and BRF in comparison to the untransformed parental line. The association was also alleviated if RB was mutated or phosphorylated. In addition, mutant RB forms were unable to disrupt the interaction between TFIIIB and TFIIIC and between TFIIIB and pol III. RB has also been shown to repress pol I transcription. The current work demonstrated an interaction between RB and the pol I transcription factors UBF and SLl. The human and Xenopus UBF-binding domain in RB was identified as the large pocket, whilst xUBF was shown to bind RB through HMG boxes 1 and 2. The RB-related proteins p107 and p130 were investigated for their ability to undertake RB functions. Indeed, p107 and p130 were both able to interact with the pol I transcription factor UBF and the BRF subunit of the pol III transcription factor TFIIIB