Tasmanian Aborigines and DNA

The hyper-variable control region (HVI and HVII) of mitochondrial DNA (mtDNA) from Tasmanians with an unbroken, direct Tasmanian Aboriginal maternal lineage were DNA sequenced. The lineage dates back to the early 1800s, as evidenced by published family genealogies (Mollison 1978, Ryan 1981, Plomley 1966, 1971, 1987, 1990). Of the five distinct mtDNA haplotypes discovered, VI was found in living Tasmanian descendants traced back to a Port Phillip Aboriginal woman who lived with European sealers in Bass Strait. Haplotype T2 is two nucleotide transitions removed from 1/WD 2 of van Holst Pellekaan et al. 1998 and therefore related to this Australian mainland Aboriginal mtDNA haplotype. Vestiges of Tasmanian maternal mtDNA haplotypes from before white settlement, passing down through up to eight generations, still survive in Tasmania. The Tasmanian Aboriginal people are related and relatable to mainland Aboriginal people

Circular RNAs: New players in Ageing and Age-related Chronic Disease

Circular RNAs (circRNAs) are an emerging class of non-coding RNA that may regulate expression during normal and disease states. Although circRNAs accumulate in in vivo models of ageing, their role in this process and its physiological consequences remains largely unanswered. In the course of this thesis, I assessed dysregulation of circRNA expression in RNA samples from ageing human peripheral blood and examined associations of their expression with various ageing outcomes in human, mammalian longevity and senescence in human cell types of various lineages, and in blood and islet samples from patients with type 2 diabetes; an exemplar disease of ageing. Of the 15 circRNAs validated in this study, I identified 4 (circDEF6, circEP300, circFOXO3 and circFNDC3B) that were associated with ageing outcomes (parental longevity or hand grip strength) in the InCHIANTI population study of ageing. CircFOXO3 and circEP300 also demonstrated differential expression in one or more human senescent cell types. 4 ageing outcomes associated circRNAs appeared to be conserved in mouse of which circPlekhm1 nominally correlated with median strain lifespan. As type 2 diabetes is an exemplar chronic disease of ageing, I also aimed to examine the role of circRNA in this disorder. I first defined the circRNA repertoire in human pancreatic islets and assessed their differential expression in conjunction with type 2 diabetes status and genotype at T2D risk loci. Following this, I determined their responsiveness to diabetomimetic stimuli in the human EndoC-βH1 beta cell line, and the potential for use as biomarkers of T2D in human peripheral blood. 4 of the five most abundant circRNAs expressed in human pancreatic islets circCIRBP, circZKSCAN, circRPH3AL and circCAMSAP1, were associated with diabetes status in islets. CircCIRBP and circRPH3AL were also differentially expressed in β-cells in response to elevated fatty acid. Despite this, no associations with T2D diabetes risk loci was identified. Cumulatively, the data generated from my work suggest that circRNAs have potential as regulators of gene expression during ageing and age-related disease, raising the possibility that they may have future utility as biomarkers or therapeutic targets for the management of age-related chronic disease outcomes

Genetics and genomics of moso bamboo (Phyllostachys edulis) : Current status, future challenges, and biotechnological opportunities toward a sustainable bamboo industry

Sustainable goals for contemporary world seek viable solutions for interconnected challenges, particularly in the fields of food and energy security and climate change. We present bamboo, one of the versatile plant species on earth, as an ideal candidate for bioeconomy for meeting some of these challenges. With its potential realized, particularly in the industrial sector, countries such as China are going extensive with bamboo development and cultivation to support a myriad of industrial uses. These include timber, fiber, biofuel, paper, food, and medicinal industries. Bamboo is an ecologically viable choice, having better adaptation to wider environments than do other grasses, and can help to restore degraded lands and mitigate climate change. Bamboo, as a crop species, has not become amenable to genetic improvement, due to its long breeding cycle, perennial nature, and monocarpic behavior. One of the commonly used species, moso bamboo (Phyllostachys edulis) is a potential candidate that qualifies as industrial bamboo. With its whole-genome information released, genetic manipulations of moso bamboo offer tremendous potential to meet the industrial expectations either in quality or in quantity. Further, bamboo cultivation can expect several natural hindrances through biotic and abiotic stresses, which needs viable solutions such as genetic resistance. Taking a pragmatic view of these future requirements, we have compiled the present status of bamboo physiology, genetics, genomics, and biotechnology, particularly of moso bamboo, to drive various implications in meeting industrial and cultivation requirements. We also discuss challenges underway, caveats, and contextual opportunities concerning sustainable development.Peer reviewe

Beyond transcription : a post-transcriptional role of 3D chromatin crosstalk in oncogene regulation

This thesis explores how stochastic chromatin fibre interactions, chromatin organization in the 3D nuclear architecture, and environmental signals collaborate to regulate MYC oncogene expression in human colon cancer cells. In Paper I, we employ the ultra-sensitive Nodewalk technique to uncover the dynamic and stochastic nature of chromatin networks impinging on MYC. The analyses revealed that the MYC interactome mainly consists of stochastic pairwise interactions between MYC and its flanking enhancers in two neighbouring topologically associated domains (TADs), which are insulated self-interacting genomic domains. The limits of Nodewalk were also pushed to enable the detection of interactions in very small cell populations, corresponding to the genomic content of ~7 cells. Comparing the frequency of interactions detected in such small input samples with ensemble interactomes of large cell populations uncovered that the enhancer hubs of the ensemble interactomes that appear to simultaneously interact with MYC likely represent virtual events, which are not present in reality at the single cell level. These data support a model where MYC interacts with its enhancers in a mutually exclusive way, with MYC screening for enhancer contacts, rather than the other way around. Paper II provides a detailed understanding of a novel post-transcriptional mechanism of enhancer action on MYC expression. We have thus uncovered that the cancer-specific recruitment of the MYC gene to nuclear pores and ensuing rapid nuclear export of MYC transcripts - a process that increases MYC expression by enabling the escape of MYC mRNAs from rapid decay in the nucleus - require a CTCF binding site positioned within the colorectal oncogenic super-enhancer. Genetic editing by CRISPR-Cas9 was thus commissioned to establish two clones of human colon cancer cells with a mutated sequence in the OSE-specific CTCFBS. Comparing the mutant cells to the parental cell line, we uncovered that the WNT-dependent increase in the nuclear export rate of MYC transcripts was abrogated in the CTCFBS mutant clones, providing the first genetic evidence of super- enhancer-mediated gene gating in human cells. In line with this finding, the OSE-specific CTCFBS thus conferred a significant growth advantage to the parental colon cancer cells, compared to the mutant clones. Moreover, we found that WNT-dependent CCAT1 eRNA transcription is mediated by the OSE-specific CTCFBS that is required for recruitment of AHCTF1 to the OSE to mediate the positioning of the OSE to the nuclear periphery, enabling the subsequent facilitation of MYC mRNA export. A multistep molecular process including WNT signalling and the OSE-specific CTCFBS thus underlies the gene gating of MYC in human colon cancer cells, and could potentially be targeted for diagnostic or therapeutic uses. In summary, this thesis explores the dynamics of the stochastic interactomes impinging on the MYC oncogene, and provides new insights on the role of 3D chromatin orchestration in the transcriptional regulation of MYC. Our analyses uncovered the molecular factors involved in the gene gating of MYC, and thus increase our understanding of tumour development. These findings could potentially be beneficial for future diagnostic approaches, or for targeted therapeutic strategies in the treatment of cancer

Human genetics and clinical aspects of neurodevelopmental disorders

This chapter traverses contemporary understandings of the genetic architecture of human disease, and explores the clinical implications of the current state of knowledge. Many different classes of genetic mutations have been implicated as being involved in predisposition to certain diseases, and researchers are continually uncovering other means by which genetics plays an important role in human disease, such as with somatic genetic mosaicism. Putative “de novo” mutations can represent cases of parental mosaicism (including in the germline), which could be revealed by careful genotyping of parental tissues other than peripheral blood lymphocytes. There is an increasingly rich literature regarding rare mutations with seemingly large phenotypic effects. Privacy concerns have added to the difficulties of implementing genomics-guided medicine. With the advent of exome and whole genome sequencing (WGS), one needs to focus again on families over several generations, so as to attempt to minimize genetic differences, locus heterogeneity and environmental influences

The role of small RNAs in susceptibility and tolerance to cassava mosaic disease

A dissertation presented by Sarah Jane Rogans to The Faculty of Science, University of the Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Molecular and Cell Biology. 2016Cassava (Manihot esculenta, Crantz) is considered to be an important food security crop consumed by over a billion peoples globally, many who subsist on it. Cassava mosaic disease (CMD) is one of the main biotic and economically important constraints to cassava cultivation in sub-Saharan Africa. Geminiviruses are the casual agents of CMD and cause disease to many staple food and cash crops of great economic importance worldwide. There are currently 11 species of Begomoviruses that belong to the Geminiviridae family. South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite (DNA A and DNA B components) begomovirus belonging to the family Geminiviridae, and is one of the causal agents of cassava mosaic disease (CMD) endemic to southern Africa. Various strategies to control CMD are currently being investigated, one of which is cis-genics, which involves manipulation of endogenous host genes to combat viral pathogens. In order to achieve this, it is imperative to elucidate molecular mechanisms involved in host-virus interactions. Endogenous small RNAs (sRNAs), including microRNAs (miRNAs), have been found associated with gene regulatory mechanisms in response to virus infection. Amongst the non-coding host sRNAs targeting viruses are small interfering RNAs (siRNAs) associated with posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS), which are involved in the host RNA silencing pathway. The RNA silencing pathway is a highly conserved basal immunity pathway involved in host defence against plant viruses. The aim of this study was to identify siRNAs and miRNAs associated with gene regulatory mechanism in response to SACMV infection and to determine if they a play a role in the susceptible or recovery phenotype observed in SACMV tolerant cassava landrace TME3 or T200, respectively. Furthermore, virus-derived siRNA (vsRNA) populations targeting the DNA A and B components of SACMV were also investigated. MicroRNAs (miRNAs) are an important class of endogenous non-coding single-stranded small RNAs (21-24 nt in length), which serve as post-transcriptional negative regulators of gene expression in plants. Despite the economic importance of Manihot esculenta Crantz (cassava) only 153 putative cassava miRNAs (from multiple germplasm) are available to date in miRBase (V.21). Therefore, both conserved and novel miRNAs needed to be identified in cassava before we could determine what association they had with SACMV infection. In this part of the study, mature sequences of all known plant miRNAs were used as a query for homologous searches against cassava EST and GSS databases, and additional identification of novel and conserved miRNAs were gleaned from next generation sequencing (NGS) of two cassava landraces (T200 from southern Africa and TME3 from West Africa) at three different growth stages post explant transplantation and acclimatization. EST and GSS derived data revealed 259 and 32 conserved miRNAs in cassava, and one of the miRNA families (miR2118) from previous studies has not been reported in cassava. NGS data collectively displayed expression of 289 conserved miRNAs in leaf tissue, of which 230 had not been reported previously. Of the 289 conserved miRNAs identified in T200 and TME3, 208 were isomiRs. Thirty-nine novel cassava-specific miRNAs of low abundance, belonging to 29 families, were identified. Thirty-eight (98.6%) of the putative new miRNAs identified by NGS have not been previously reported in cassava. Several miRNA targets were identified in T200 and TME3, highlighting differential temporal miRNA expression between the two cassava landraces. This study contributes to the expanding knowledge base of the micronome of this important crop. MicroRNAs play a crucial role in stress response in plants, including biotic stress caused by viral infection. Viruses however can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of miRNAs. This study aimed to understand the regulation of miRNAs in tolerant (TME3) and susceptible (T200) cassava landraces infected with SACMV. Next-generation sequencing was used for analysing small RNA libraries from infected and mock-inoculated cassava leaf tissue collected at 12, 32 and 67 dpi (days post-inoculation). The total number of differentially expressed miRNAs (normalized against mock-inoculated samples) across all three time points was 204 and 209 miRNAs, in TME3 and T200 infected plants, respectively, but the patterns of log2fold changes in miRNA families over the course of infection differed between the two landraces. A high number were significantly altered at 32 dpi when T200 and TME3 plants showed severe symptoms. Notably, in T200 69% and 28 (100%) of miRNA families were upregulated at 12 and 32 dpi, respectively. In contrast, TME3 showed an early pre-symptomatic response at 12 dpi where a high number (87%) of miRNAs showed a significant log2fold downregulation. Endogenous targets were predicted in the cassava genome for many of the identified miRNA families including transcription factors, disease resistance (R)-genes and transposable elements. Interestingly, some of the miRNA families (miR162, miR168 and miR403) that were significantly affected in both T200 and TME3 upon SACMV infection were shown to target proteins (DCL1, AGO1 and AGO2) that play important roles in the RNA silencing pathway. From results, we suggest that the early (12 dpi) miRNA response to SACMV in TME3 appears to involve PTGS-associated AGO1, DCL2 and a cohort of R genes belonging to the miR395 family which may prime the plant for tolerance and recovery downstream, while in T200, SACMV suppresses AGO1, AGO2 (at 32 and 67 dpi), and DCL2 (32 dpi) mediated RNA silencing, leading to severe persistent disease symptoms. This study provides insights into miRNA-mediated SACMV cassava interactions and may provide novel targets for control strategies aimed at developing CMD-resistance cassava varieties Endogenous small RNAs (sRNAs) associated with gene regulatory mechanisms respond to virus infection, and virus-derived small interfering RNAs (vsRNAs) have been implicated in recovery or symptom remission in some geminivirus-host interactions. Transcriptional gene silencing (TGS) (24 nt vsRNAs) and post transcriptional gene silencing (PTGS) (21-23 nt vsRNAs) have been associated with geminivirus intergenic (IR) and coding regions, respectively. In this Illumina deep sequencing study, we compared for the first time, the small RNA response to South African cassava mosaic virus (SACMV) of cassava landrace TME3 which shows a recovery and tolerant phenotype, and T200, a highly susceptible landrace. Interestingly, different patterns in the percentage of SACMV-induced normalized total endogenous sRNA reads were observed between T200 and TME3. Notably, in T200 there was a significant increase in 21 nt sRNAs during the early pre-symptomatic response (12 dpi) to SACMV compared to mock, while in TME3, the 22 nt size class increased significantly at 32 dpi. While vsRNAs of 21 to 24 nt size classes covered the entire SACMV DNA- A and DNA-B genome components in T200 and TME3, vsRNA population counts were significantly lower at 32 (symptomatic stage) and 67 dpi in tolerant TME3 compared with T200 (non-recovery). It is suggested that the high accumulation of primary vsRNAs, which correlated with high virus titres and severe symptoms in susceptible T200, may be due to failure to target SACMV-derived mRNA. In contrast, in TME3 low vsRNA counts may represent efficient PTGS of viral mRNA, leading to a depletion/sequestration of vsRNA populations, supporting a role for PTGS in tolerance/recovery in TME3. Notably, in TME3 at recovery (67 dpi) the percentage (expressed as a percentage of total vsRNA counts) of redundant and non-redundant (unique) 24 nt vsRNAs increased significantly. Since methylation of the SACMV genome was not detected by bisulfite sequencing, and vsRNA counts targeting the IR (where the promoters reside) were very low in both the tolerant or susceptible landraces, we conclude that 24 nt vsRNA-mediated RNA directed genome methylation does not play a central role in disease phenotype in these landraces, notwithstanding recognition for a possible role in histone modification in TME3. This work represents an important step toward understanding variable roles of sRNAs in different cassava genotype-geminivirus interactions. Also, by comparing the differences between a tolerant and susceptible host the aim is to achieve better understanding of the effect of pathogens on host sRNAome, an area that is deserving of me attention in plant systems. The expectation is that these findings presented in the PhD will contribute to the long-term goals of devising new methods of disease control against SACMV and understanding the complex interconnected mechanisms involved in virus-host interactome.LG201

Functional analysis of long non-coding RNAs in cancer

For years the functional role of noncoding RNAs was greatly underestimated. Whole genome RNA sequencing projects that unraveled pervasive transcription emitting from large parts of the human genome changed those perspectives and prompted scientists to further look into the involvement of noncoding RNAs (ncRNAs) in development and disease. To date, we have significantly advanced our understanding of ncRNAs, however only a fraction of them has been functionally characterized. The subject of this thesis is to investigate the functional role of long noncoding RNAs (lncRNAs) and their involvement in cancer, particularly in cutaneous malignant melanoma. Previously, the tumor suppressor gene PTEN has been reported to be post-transcriptionally regulated by its pseudogene. In paper I, we investigated an antisense RNA (PTENpg1 asRNA) that is transcribed from the PTEN pseudogene (PTENpg1). We uncovered various PTENpg1 asRNA isoforms and designated two of them as α and β. The role of the PTENpg1 asRNA β isoform is to form an RNA:RNA duplex with the PTENpg1 transcript. This interaction stabilizes and assists the PTENpg1 transcript out to the cytoplasm. On the other hand, the PTENpg1 asRNA α has a very different function, namely mediating epigenetic changes by recruiting EZH2 and DNMT3a to the PTEN promoter. In paper II, we further sought out to understand the recruitment of PTENpg1 asRNA α to the PTEN promoter. We observed promoter-associated/5´UTR transcript emitting from PTEN, which binds and facilitates the recruitment of PTENpg1 asRNA α to the PTEN promoter. In return, PTENpg1 asRNA recruits DNMT3a to the promoter, which leads to epigenetic silencing of PTEN. In paper III we investigated the role of PTENpg1 asRNA in vemurafenib resistance of melanoma. We observed increased PTENpg1 asRNA expression and consequently low PTEN levels caused by enrichment of EZH2 and H3K27me3 at the PTEN promoter. Further, we found that C/EBPβ transcriptionally induced PTENpg1 asRNA in vemurafenib-resistant melanoma cell lines. In addition, manipulation of key components of the PTENpg1 asRNA network caused re-sensitization of the resistant melanoma cells to vemurafenib, and high PTENpg1 asRNA expression was found to correlate with shorter survival in melanoma patients. In paper IV, we investigated the effect of the C/EBPβ antisense (C/EBPβ-AS) transcript on transcriptional regulation of C/EBPβ. We found that C/EBPβ auto-regulates its own and also regulates C/EBPβ-AS expression. In return, C/EBPβ-AS inhibits C/EBPβ positive feedback loop by modulating epigenetic changes at the C/EBPβ promoter in melanoma cell lines. Interestingly, knockdown of C/EBPβ-AS caused re-sensitization to vemurafenib. This thesis highlights the dynamics of lncRNAs in epigenetic silencing and their involvement in cancer and therapy resistance

The U1A/U2B /SNF Family of RNA Binding Proteins: Evolution of RNA Binding Specificity and Contributions of Heterotropic Linkage to snRNP Protein Partitioning

The U1A/U2B /SNF is a family of RNA binding proteins that is a highly conserved throughout eukaryotes. These proteins are found in the U1 and/or U2 splicing snRNPs (small nuclear ribonucleoprotein particles). In humans, U1A and U2B specifically bind to the U1 and U2 snRNAs, respectively. The Drosophila genome codes for SNF, an essential protein that localizes to both the U1 and U2 snRNP. While a specific splicing functions for these proteins have not been determined, their conserved snRNP localization suggests an important splicing-related function. The difference in protein number and partitioning between Drosophila and humans suggested that these proteins may use different RNA binding mechanisms to function in their cellular contexts. This work begins by exploring some of the differences amongst human U1A, U2B , and Drosophila SNF. The thermodynamics of the RNA-protein interactions also reveal substantial differences in the RNA binding mechanisms of these proteins. Further studies investigate the evolution of this protein family in metazoans. Reconstructing the protein phylogeny permitted resurrection of ancestral proteins. This led to the discovery that the last common ancestor of humans and Drosophila had a single U1A/U2B /SNF family homolog. This protein had RNA binding properties that most closely resemble those of Drosophila SNF. Evolution of protein motions and RNA binding specificity toward the defining characteristics of modern vertebrate proteins is also examined. Finally, linkage effects between protein-protein and protein-RNA interactions are analyzed. U2A\u27; is a U2 snRNP-specific protein that binds to U2B ; in humans and SNF in Drosophila. In Drosophila, large, positive linkage was only seen between U2A\u27-SNF and SNF-U2 snRNA binding. The RNA dependence of enhancement for SNF binding to U2A\u27 can explain the observed protein partitioning of U2A\u27 in vivo. For the more complicated human system, which contains two SNF homologs, substantial contributions to protein partitioning come from differences in both intrinsic RNA-protein binding affinities and differences in protein-U2A\u27 binding affinities. RNA dependence of the linkage parameter also contributes to protein partitioning. The binding parameters can explain U2A\u27 protein partitioning, and the presence of U2A\u27 reinforces U1A and U2B partitioning to their respective snRNAs. These linkage studies have important implications for the assembly of RiboNucleoprotein Particles, macromolecular complexes that are fundamental to many cellular activities

The association of circular RNAs with hypertension

This thesis contains six chapters in total with chapter 1 being a literature overview and chapter 2 being a summary of material and methods. The three results chapters contain all my own work unless stated otherwise and lastly chapter 6 is discussion and conclusions linking together the work undertaken in this thesis.Doctor of Philosoph