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Hypoxia pathways are associated with the pathogenesis of both ischaemic and neoplastic diseases. In response to hypoxia the transcription factor hypoxia‐inducible factor (HIF) induces the expression of hundreds of genes with diverse functions. These enable cells to adapt to low oxygen availability. To date, pan-genomic analyses of these transcriptional responses have focussed on protein-coding genes and microRNAs. However, the role of other classes of non-coding RNAs, in particular lncRNAs, in the hypoxia response is largely uncharacterised. My thesis aimed at improving understanding of the transcriptional regulation of the non-coding transcriptome in hypoxia. I performed an integrated genomic analysis of both non-coding and coding transcripts by massively parallel sequencing. This was interfaced with pan-genomic analyses of DNAse hypersensitivity and HIF, H3k4me3 and RNApol2 binding in hypoxic cells. These analyses have revealed that hypoxia profoundly regulated all RNA classes. snRNAs and tRNAs are globally downregulated in hypoxia, whilst miRNAs, mRNAs and lncRNAs are both up- and downregulated with an overall trend towards slight upregulation. In addition, a significant number of previously non-annotated (and largely hypoxia upregulated) transcripts were identified, including novel intergenic transcripts and natural antisense transcripts. HIF bound close to genes for mRNAs, miRNAs and lncRNAs that were upregulated by hypoxia, but was excluded from binding at genes for RNA classes that showed global downregulation. This suggests that HIF acts as a transcriptional activator (but not repressor), of lncRNAs as well as mRNAs and miRNAs. Consistent with direct regulation by HIF, many of these hypoxia-inducible, HIF-binding lncRNAs were downregulated following HIF knockdown. Analysis of RNApol2 binding and DNAse HSS signals at HIF transcriptional target genes indicated that HIF-dependent transcriptional activation occurs through release of RNApol2 that is pre-bound to open promoters of lncRNAs as well as mRNAs. In these datasets, NEAT1 was the most hypoxia-upregulated, HIF-targeted lncRNA in MCF-7 cells and, despite binding of both HIF-1 and HIF-2 isoforms at its promoter, was selectively regulated by HIF-2 alone. Furthermore, NEAT1 was induced by hypoxia in a wide range of breast cancer cell lines and in hypoxic xenograft models. Functionally, NEAT1 is required for the assembly of nuclear paraspeckle structures. Increased nuclear paraspeckle formation was observed in hypoxia and was dependent on both NEAT1 and HIF-2. Knockdown of hypoxia-induced NEAT1 significantly reduced cell proliferation and survival and induced apoptosis. Finally, high expression of NEAT1 correlated with poor clinical outcome in a large cohort of breast cancer patients. These findings extend the role of the hypoxic transcriptional response in cancer into the spectrum of non-coding transcripts and provide new insights into molecular roles of hypoxia-regulated lncRNAs, which may provide the basis for novel therapeutic targets in the future.

Transcription lies at the centre of gene expression. In eukaryotes, transcription occurs not only at genes but also across the non-coding portion of the genome, an apparently pervasive process that gives rise to a wide array of different transcripts. In recent years, it has emerged that genes themselves are frequently subject to non-coding transcription of their antisense strand. This antisense transcription is evident in eukaryotes from yeast to mammals; however its general genome-wide role, if indeed it has one, remains elusive. Here, the nature of antisense transcription in the budding yeast Saccharomyces cerevisiae is explored on a genome-wide scale. Antisense transcription is ubiquitous and often abundant, and appears to be driven by a promoter architecture at the 3’ end of genes, one which shows evidence of regulation, and which mirrors that found at the 5’ end. Furthermore, antisense transcription shows evidence of changing gene behaviour. It is associated with a drastically altered chromatin environment at the 5’ promoter and across the gene body; however it is not associated with a change in the level of gene transcription itself. Rather, these chromatin changes appear to enforce a change in the mode of gene transcription, promoting rapid bursts of transcription re-initiation that result in noisier gene expression – a hitherto unknown role of antisense transcription. It is proposed that antisense transcription represents a fundamental layer of gene regulation, and that it should be considered a canonical feature of eukaryotic genes.

Genome-wide studies in Saccharomyces cerevisiae have revealed that the majority of the genome is transcribed on both strands, producing both coding and non-coding RNAs (ncRNAs). Initially, these ncRNAs were regarded as spurious transcripts but some have since been shown to have important roles as transcriptional regulators. Very little is understood about how ncRNAs are initiated, terminated and processed or how this influences their function. To address these questions, the expression, stability, and subcellular localization of the ncRNAs at the endogenous GAL locus was analysed. This revealed a complex interleaved transcript map, challenging the conventional view of a transcription unit (TU) flanked by 5’ sequences or promoters (P) that initiate transcription and 3’ regions, known as terminators (T), which control events such as transcript cleavage, polyadenylation, export and transcription termination. By creating conventional (PGAL-T) or unconventional (PGAL-P) hybrid TUs at the GAL locus, in which a promoter or terminator is positioned downstream of a galactose-inducible promoter, this work shows that both promoters and terminators are able to initiate antisense transcription to yield stable antisense transcripts. The data suggest that terminators contribute to efficient but variable expression from the promoter. An unconventional P-P TU, lacking a terminator, is transcribed on both strands but the sense transcript remains at low levels, through the repressive action of antisense transcription, and is retained in the nucleus. In contrast, the conventional P-T bi-directional TUs are plastic, with the Rrp6 component of the nuclear exosome and TATA-like sequences in the 3’ UTR determining whether the predominant transcript is antisense or sense. By relieving the repressive action of antisense transcription, this allows high levels of sense transcript to accumulate in the cytoplasm, contributing to gene expression, supporting a novel mode of gene regulation involving components of RNA quality control pathways acting through the 3’ region of genes.

Skeletal muscle hypertrophy is a complex process that involves a range of signaling pathways and transcriptional regulators. Many hormones and growth factors can activate key signaling pathways, such as the PI3K/AKT/mTOR, MAPK, and cAMP pathways, which play a crucial role in the regulation of muscle hypertrophy. In Chapter 1, we reviewed some of the hormones and growth factors known to be associated with skeletal muscle hypertrophy, as well as the function of these key signaling pathways, and revealed some unresolved issues. In Chapter 2, we explored the role of microRNA-1 (miR-1) in skeletal muscle hypertrophy and aimed to determine the underlying molecular mechanisms. We hypothesize that reducing miR-1 levels is necessary for skeletal muscle growth due to its interaction with essential pro-growth genes (e.g., IGF1). Using lentivirus to increase miR-1 levels, we observed a blunted hypertrophic response in mice undergoing a murine model of muscle hypertrophy. In addition, we identified a possible mechanism by which miR-1 regulates skeletal muscle hypertrophy. Our results clearly demonstrated the necessity of miR-1 regulation during muscle hypertrophy. Advisor: Ivan Vechetti.

Understanding molecular mechanisms which regulate bacterial virulence is essential for discovering new therapeutics. Alarmingly, some bacterial species are already unresponsive to antibiotic cocktails, and in the case of Shigella dysenteriae, will up-regulate virulence factors under the duress of antibiotic treatment. While molecular mechanisms involving the regulation of S. dysenteriae pathogenesis have been studied, many are still unknown, especially those of which are mediated by RNA. Ribo-regulators that have been characterized in Shigella, and published to date, have all been shown to impact virulence processes. Many other sRNAs have been discovered in Shigella and E. coli, yet, remain uncharacterized. One sRNA of interest, RyfA, was originally discovered in Escherichia and found to have nearly exact sequence identity to that of RyfA in Shigella flexneri. Interestingly, in S. dysenteriae, 95% identical twin copies of RyfA, designated RyfA1 and RyfA2 exist in tandem. Intriguingly, ryfA1-like copies of the gene are only found in pathogenic species of Escherichia and all species of Shigella. Non-pathogenic species of Escherichia contain a ryfA2-like copy. Upstream of both RyfA1 and RyfA2, additional sRNAs exist, termed RyfB1 and RyfB2. While RyfB1 and RyfB2 are only 60% identical to each other they each share large amounts of complementarity to RyfA1 and RyfA2 respectively, suggesting that these molecules may interact with each other and thus one sRNA may regulate the abundance and/or activity of the other. Indeed, overexpression of RyfB1 leads to reduced levels of RyfA1 in the bacterial cell but has no effect on the levels of RyfA2. Unexpectedly, it was it was found that RyfB2 alters the abundance of both RyfA1 and RyfA2 when produced in S. dysenteriae. Furthermore, overproduction of RyfA1 slightly inhibits RyfA2 levels, while RyfA2 overproduction strongly inhibits RyfA1 levels. In summary, sRNAs RyfA2, RyfB2, and RyfB1 all function to include tight control over RyfA1. The overproduction of RyfA1 results in a lack of cell to cell spread in S. dysenteriae, suggesting the number of sRNAs regulating RyfA1 is critical for optimal regulation. Lastly, overproduction of RyfB2 in S. dysenteriae results in increased plaque number. The complex interplay between these sRNAs is likely important for fine-tuned regulation of invasion and virulence in S. dysenteriae. Downstream targets of RyfA1 and RyfA2 may shed some light on genes/proteins impacted resulting in the above described phenotypes.

Meiosis is a specialized cell division that is required for sexual reproduction. Homologous chromosome pairing and meiotic recombination are two defining processes of prophase I of meiosis and are required for the generation of genetic variation. Core proteins involved in these processes are well studied, but it is not well understood how meiosis is regulated. In this dissertation, I shed light on how meiosis is regulated by long non-coding RNAs (lncRNAs) in Arabidopsis thaliana, as well as how meiotic recombination is regulated by chromatin in maize. Many long non-coding RNAs (lncRNAs) have been identified in various species but few have been functionally analyzed. Of the few lncRNAs that have been analyzed, they have been found to play roles in regulating gene expression and modifying chromatin. However, the functions of the vast majority of them are unknown and no lncRNAs have been identified in Arabidopsis meiosis. On the other hand, significantly more genes are expressed during meiosis than the number of genes actually required for meiosis completion, suggesting that there is a mechanism controlling gene expression post-transcriptionally, possibly involving lncRNAs. This situation prompted us to conduct a systematic analysis of lncRNAs in Arabidopsis meiosis. We generated a bioinformatics pipeline that utilizes RNA-seq data from Arabidopsis meiocytes and somatic tissues to identify novel lncRNAs that are specific to meiosis. We then established a multistep mutant screen to identify lncRNAs that are functional in meiosis and found that lncRNAs play a role in overall fertility in Arabidopsis thaliana. Chapter 3 of this dissertation focuses on understanding the role of chromatin in the regulation of meiotic recombination events that result in crossovers (COs) in maize. In this species, double strand breaks (DSBs) are evenly distributed along chromosomes, including pericentromeric and centromeric regions. However, CO distribution does not follow the DSB distribution. Instead, COs are enriched in distal chromosome regions and suppressed in pericentromeric and centromeric regions. We investigated the dynamics of recombination events in relationship to chromatin states in order to understand how this distribution is formed. We further studied the CO/non-crossover (NCO) decision through its relationship with chromatin. We found that DNA methylation is a major contributor to the decision of which DSBs become COs and this decision is progressively enforced when meiotic DSBs are repaired. Interestingly, this process proceeds differently in B73 and CML228, two maize inbreds with dramatically differing numbers of recombination events.

Cancer remains the second leading cause of death in the United States and the world, despite years of research and the development of different treatments. One reason for this is cancer cells are able to survive through adaptation to their environment and aberrantly activated growth signaling. As such, developing new therapies that overcome these hurdles are necessary to combat cancer. Previous work in our laboratory using RNA interference screening identified genes that regulate the survival of glioblastoma (GBM) or autophagy in chronic myelogenous leukemia (CML) cancer cells. One screen identified Phosphatidylinositol-4,5-bisophosphate 3-kinase catalytic subunit beta (PIK3CB) in the family of Phosphatidylinositol 3-kinases (PI3K) as a survival kinase gene in GBM. Work contained in this dissertation set out to study PIK3CB mediated GBM cell survival. We report that only PIK3CB, in its family of other PI3K genes, is a biomarker for GBM recurrence and is selectively important for GBM cell survival. Another screen identified the long non-coding RNA, Linc00467, as a gene that regulates autophagy in CML. Autophagy is a dynamic survival process used by all cells, benign and cancerous, where cellular components are broken down and re-assimilated to sustain survival. Work contained in this dissertation set out to characterize the role that Linc00467 serves in regulating autophagy in a myriad of cancers. Collectively our data have showed Linc00467 to actively repress levels of autophagy in cancer cells. Further, our data revealed an important role for Linc00467 in regulating the stability of the autophagy regulating protein serine-threonine kinase 11 (STK11). Because of the unique role that Linc00467 serves in regulating autophagy we renamed it as, autophagy regulating long intergenic noncoding RNA or ARLINC. Taken together the work in this dissertation unveils the inner-workings of two important cancer cell survival pathways and shows their potential for development into therapeutic targets to treat cancer.

Chlamydia trachomatis is an obligate intracellular bacterium that exhibits a unique biphasic developmental cycle that can be disrupted by growth in the presence of IFN-g and b-lactams, giving rise to an abnormal growth state termed persistence. Relatively little is known about the regulatory mechanisms that control temporal gene expression during the developmental cycle or the control of persistence and reactivation. Here we have examined the expression of a newly defined family of non-coding RNAs (ncRNAs) that are differentially expressed during the developmental cycle and the induction of persistence and reactivation (Using IFNγ and Carbenicillin). Non-coding RNAs were initially identified using an intergenic tiling microarray and were confirmed by Northern blotting. A group of 10 ncRNAs were mapped and characterized and compared to the previously described chlamydial ncRNAs (IhtA, pCHL antisense transcripts). The 5’ and 3’ ends of the ncRNAs were determined using an RNA circularization procedure. Promoter predictions indicated that all ncRNAs were expressed from s66 promoters and 9 ncRNAs contained non-templated 3’ poly-A or poly-AG additions. Expression of ncRNAs was studied by Northern blotting during i) the normal developmental cycle, ii) IFN-g-induced persistence, and iii) carbenicillin-induced persistence. Differential temporal expression during the developmental cycle was seen for all ncRNAs and distinct differences in expression were seen during IFN-g and carbenicillin-induced persistence and reactivation. Two of the studied ncRNAs were cis acting antisense molecules (CTIG270 and CTIG153). Expression of CTIG270 in a surrogate E. coli system along with its target gene ftsI proved that it was an antisense RNA, and it effectively controlled ftsI availability. A screening system was developed to determine the targets of potential trans-acting ncRNAs. The screen was composed of two compatible plasmids, a high copy effector plasmid expressing the ncRNA and a low/medium copy target plasmid expressing chlamydial library. Target plasmid contained translational fusion between chlamydial library and a positive/negative selection fusion system (ccdB/CAT fusion/TEV protease). The selection process to identify potential targets for ncRNAs was carried out over two stages. Stage one was to eliminate self ligated plasmids and non translational fusions, in this phase, only CAT+ ccdB resistant cells survived chloramphenicol selection i.e. cells representing true translational fusions survived antibiotic selection. Plasmids enriched for by stage one selection were subjected to stage two selection. In stage two ccdB sensitive cells were double transformed with both target plasmid and effector plasmid. Only in instances when ncRNA inhibits translation of ccdB would cells survive this selection. Stage two selection enriched for target(s) of ncRNA under test. We show in details the construction of this screening system and its functional aspect.