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

XIAP upregulates expression of HIF target genes by targeting HIF1 alpha for Lys(63)-linked polyubiquitination

The cellular response to hypoxia is characterised by a switch in the transcriptional program, mediated predominantly by the hypoxia inducible factor family of transcription factors (HIF). Regulation of HIF1 is primarily controlled by post-translational modification of the HIF1α subunit, which can alter its stability and/or activity. This study identifies an unanticipated role for the X-linked inhibitor of apoptosis (XIAP) protein as a regulator of Lys63-linked polyubiquitination of HIF1α. Lys63-linked ubiquitination of HIF1α by XIAP is dependent on the activity of E2 ubiquitin conjugating enzyme Ubc13. We find that XIAP and Ubc13 dependent Lys63-linked polyubiquitination promotes HIF1α nuclear retention leading to an increase in the expression of HIF1 responsive genes. Inhibition of the Lys63-linked polyubiquitination pathway leads to reduced levels of nuclear HIF1α, promoter occupancy, HIF-dependent gene expression and cell viability. Our data reveals an additional and significant level of control of the HIF1 by XIAP, with important implications in understanding the role of HIF1 and XIAP in human disease

PBRM1 Cooperates with YTHDF2 to Control HIF-1α Protein Translation

PBRM1, a component of the chromatin remodeller SWI/SNF, is often deleted or mutated in human cancers, most prominently in renal cancers. Core components of the SWI/SNF complex have been shown to be important for the cellular response to hypoxia. Here we investigated how PBRM1 controls HIF-1alpha activity. We find that PBRM1 is required for HIF-1alpha transcriptional activity and protein levels. Mechanistically, PBRM1 is important for HIF-1alpha mRNA translation, as absence of PBRM1 results in reduced activly transalting HIF-1alpha mRNA. Interestingly, we find that PBRM1, but not BRG1, interacts with the m6A reader protein YTHDF2. HIF-1alpha mRNA is m6A modified, bound by PBRM1 and YTHDF2. PBRM1 is necessary for YTHDF2 binding to HIF-1alpha mRNA and reduction of YTHDF2 results in reduced HIF-1alpha protein expression in cells. Our results identify a SWI/SNF independent function for PBRM1, interacting with HIF-1alpha mRNA and the epitranscriptome machinery. Furthermore, our results suggests that the epitranscriptome associated proteins play a role in the control of hypoxia signalling pathway

PERK/eIF2 alpha signaling inhibits HIF-induced gene expression during the unfolded protein response via YB1-dependent regulation of HIF1 alpha translation

HIF1α (hypoxia inducible factor 1α) is the central regulator of the cellular response to low oxygen and its activity is deregulated in multiple human pathologies. Consequently, given the importance of HIF signaling in disease, there is considerable interest in developing strategies to modulate HIF1α activity and down-stream signaling events. In the present study we find that under hypoxic conditions, activation of the PERK branch of the unfolded protein response (UPR) can suppress the levels and activity of HIF1α by preventing efficient HIF1α translation. Activation of PERK inhibits de novo HIF1α protein synthesis by preventing the RNA-binding protein, YB-1, from interacting with the HIF1α mRNA 5′UTR. Our data indicate that activation of the UPR can sensitise tumor cells to hypoxic stress, indicating that chemical activation of the UPR could be a strategy to target hypoxic malignant cancer cells

Hypoxic stress suppresses RNA polymerase III recruitment and tRNA gene transcription in cardiomyocytes

RNA polymerase (pol) III transcription decreases when primary cultures of rat neonatal cardiomyocytes are exposed to low oxygen tension. Previous studies in fibroblasts have shown that the pol III-specific transcription factor IIIB (TFIIIB) is bound and regulated by the proto-oncogene product c-Myc, the mitogen-activated protein kinase ERK and the retinoblastoma tumour suppressor protein, RB. The principal function of TFIIIB is to recruit pol III to its cognate gene template, an activity that is known to be inhibited by RB and stimulated by ERK. We demonstrate by chromatin immunoprecipitation (ChIP) that c-Myc also stimulates pol III recruitment by TFIIIB. However, hypoxic conditions cause TFIIIB dissociation from c-Myc and ERK, at the same time as increasing its interaction with RB. Consistent with this, ChIP assays indicate that the occupancy of tRNA genes by pol III is significantly reduced, whereas promoter binding by TFIIIB is undiminished. The data suggest that hypoxia can inhibit pol III transcription by altering the interactions between TFIIIB and its regulators and thus compromising its ability to recruit the polymerase. These effects are independent of cell cycle changes

Increased migration and motility in XIAP-null cells mediated by the C-RAF protein kinase

Abstract The product encoded by the X-linked inhibitor of apoptosis (XIAP) gene is a multi-functional protein which not only controls caspase-dependent cell death, but also participates in inflammatory signalling, copper homeostasis, response to hypoxia and control of cell migration. Deregulation of XIAP, either by elevated expression or inherited genetic deletion, is associated with several human disease states. Reconciling XIAP-dependent signalling pathways with its role in disease progression is essential to understand how XIAP promotes the progression of human pathologies. In this study we have created a panel of genetically modified XIAP-null cell lines using TALENs and CRISPR/Cas9 to investigate the functional outcome of XIAP deletion. Surprisingly, in our genetically modified cells XIAP deletion had no effect on programmed cell death, but instead the primary phenotype we observed was a profound increase in cell migration rates. Furthermore, we found that XIAP-dependent suppression of cell migration was dependent on XIAP-dependent control of C-RAF levels, a protein kinase which controls cell signalling pathways that regulate the cytoskeleton. These results suggest that XIAP is not necessary for control of the apoptotic signalling cascade, however it does have a critical role in controlling cell migration and motility that cannot be compensated for in XIAP-knockout cells.</jats:p

Increased migration and motility in XIAP-null cells mediated by the C-RAF protein kinase.

The product encoded by the X-linked inhibitor of apoptosis (XIAP) gene is a multi-functional protein which not only controls caspase-dependent cell death, but also participates in inflammatory signalling, copper homeostasis, response to hypoxia and control of cell migration. Deregulation of XIAP, either by elevated expression or inherited genetic deletion, is associated with several human disease states. Reconciling XIAP-dependent signalling pathways with its role in disease progression is essential to understand how XIAP promotes the progression of human pathologies. In this study we have created a panel of genetically modified XIAP-null cell lines using TALENs and CRISPR/Cas9 to investigate the functional outcome of XIAP deletion. Surprisingly, in our genetically modified cells XIAP deletion had no effect on programmed cell death, but instead the primary phenotype we observed was a profound increase in cell migration rates. Furthermore, we found that XIAP-dependent suppression of cell migration was dependent on XIAPdependent control of C-RAF levels, a protein kinase which controls cell signalling pathways that regulate the cytoskeleton. These results suggest that XIAP is not necessary for control of the apoptotic signalling cascade, however it does have a critical role in controlling cell migration and motility that cannot be compensated for in XIAP-knockout cells

RAF inhibitors activate the integrated stress response by direct activation of GCN2

Paradoxical RAF activation by chemical RAF inhibitors (RAFi) is a well-understood on-target biological and clinical response. In this study, we show that a range of RAFi drive ERK1/2-independent activation of the Unfolded Protein Response (UPR), including expression of ATF4 and CHOP, that required the translation initiation factor eIF2-alpha. RAFi-induced ATF4 and CHOP expression was not reversed by inhibition of PERK, a known upstream activator of the eIF2-alpha-dependent Integrated Stress Response (ISR). Rather, we found that RAFi exposure activated GCN2, an alternate eIF2-alpha kinase, leading to eIF2-alpha-dependent (and ERK1/2-independent) ATF4 and CHOP expression. The GCN2 kinase inhibitor A-92, GCN2 RNAi, GCN2 knock-out or ISRIB (an eIF2-alpha antagonist) all reversed RAFi-induced expression of ATF4 and CHOP indicating that RAFi require GCN2 to activate the ISR. RAFi also activated full-length recombinant GCN2 in vitro and in cells, generating a characteristic bell-shaped concentration-response curve, reminiscent of RAFi-driven paradoxical activation of WT RAF dimers. Activation of the ISR by RAFi was abolished by GCN2 kinase dead mutations and M802A or M802G gatekeeper mutations, suggesting that RAFi bind directly to the GCN2 kinase domain; this was supported by mechanistic structural models of RAFi interaction with GCN2. Since the ISR is a critical pathway for determining cell survival or death, our observations may be relevant to the clinical use of RAFi, where paradoxical GCN2 activation may be a previously unappreciated off-target effect that may modulate tumour cell responses

An inactivating caspase 11 passenger mutation originating from the 129 murine strain in mice targeted for c-IAP1

A recent study revealed that ES (embryonic stem) cell lines derived from the 129 murine strain carry an inactivating mutation within the caspase 11 gene (Casp4) locus [Kayagaki, Warming, Lamkanfi, Vande Walle, Louie, Dong, Newton, Qu, Liu, Heldens, Zhang, Lee, Roose-Girma and Dixit (2011) Nature 479, 117–121]. Thus, if 129 ES cells are used to target genes closely linked to caspase 11, the resulting mice might also carry the caspase 11 deficiency as a passenger mutation. In the present study, we examined the genetic loci of mice targeted for the closely linked c-IAP (cellular inhibitor of apoptosis) genes, which were generated in 129 ES cells, and found that, despite extensive backcrossing into a C57BL/6 background, c-IAP1−/− animals are also deficient in caspase 11. Consequently, data obtained from these mice should be re-evaluated in this new context