Characterisation of the epigenome of an in vitro model of chondrogenesis

PhD ThesisChondrogenesis, the differentiation of mesenchymal progenitor cells from the mesoderm germ layer during embryonic development, is partly regulated by epigenetic mechanisms such as histone modifications and DNA methylation. Histone proteins possess protruding N-terminal tails which may be post-translationally modified to alter the structure of chromatin resulting in a change in the accessibility of genes to the transcription machinery. In the genome, histone modifications mark cis-regulatory elements such as gene promoters and enhancers while DNA methylation occurs on cytosine residues at CpG sites and typically leading to transcriptional repression. The aim of this project was to characterise the epigenome during in vitro differentiation of human mesenchymal stem cells (hMSCs) into chondrocytes. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) was used to assess genome-wide a range of N-terminal post-transcriptional modifications (marks) to histone H3 lysines (H3K4me3, H3K4me1, H3K27ac, H3K27me3 and H3K36me3) in both hMSCs and differentiated chondrocytes. Chromatin states were characterised using the software ChromHMM and cis-regulatory elements were identified. Integration of DNA methylation data with chondrogenesis chromatin states revealed that enhancers marked by H3K4me1 and H3K27ac were hypomethylated during in vitro chondrogenesis. Similarity analysis between chondrogenesis chromatin states with epigenomes of cell types defined by the Roadmap Epigenomics project revealed that enhancers are more distinct between cell types compared to other chromatin states. SOX9 is regarded as the master transcription factor for chondrogenesis. An external mouse Sox9 ChIP-seq dataset was used to identify super enhancers in chondrocytes. Luciferase reporter assays showed that selected regions of super enhancers exhibit independent enhancer activity. In conclusion, we observed that CpG sites within enhancers are de-methylated during hMSC differentiation into chondrocytes and propose that gene transcription during chondrogenesis is regulated by epigenetic changes at enhancers. Epigenetic changes have been implicated in cartilage diseases and greater understanding of the chondrocyte epigenome may have potential therapeutic value.This PhD project was funded by the Centre for Integrated research into Musculoskeletal Ageing (CIMA)

Transcriptome analysis of ageing in uninjured human Achilles tendon

The risk of tendon injury and disease increases significantly with increasing age. The aim of the study was to characterise transcriptional changes in human Achilles tendon during the ageing process in order to identify molecular signatures that might contribute to age-related degeneration

Unraveling the functional role of the orphan solute carrier, SLC22A24 in the transport of steroid conjugates through metabolomic and genome-wide association studies.

Variation in steroid hormone levels has wide implications for health and disease. The genes encoding the proteins involved in steroid disposition represent key determinants of interindividual variation in steroid levels and ultimately, their effects. Beginning with metabolomic data from genome-wide association studies (GWAS), we observed that genetic variants in the orphan transporter, SLC22A24 were significantly associated with levels of androsterone glucuronide and etiocholanolone glucuronide (sentinel SNPs p-value <1x10-30). In cells over-expressing human or various mammalian orthologs of SLC22A24, we showed that steroid conjugates and bile acids were substrates of the transporter. Phylogenetic, genomic, and transcriptomic analyses suggested that SLC22A24 has a specialized role in the kidney and appears to function in the reabsorption of organic anions, and in particular, anionic steroids. Phenome-wide analysis showed that functional variants of SLC22A24 are associated with human disease such as cardiovascular diseases and acne, which have been linked to dysregulated steroid metabolism. Collectively, these functional genomic studies reveal a previously uncharacterized protein involved in steroid homeostasis, opening up new possibilities for SLC22A24 as a pharmacological target for regulating steroid levels

DNA hypomethylation during MSC chondrogenesis occurs predominantly at enhancer regions

Regulation of transcription occurs in a cell type specific manner orchestrated by epigenetic mechanisms including DNA methylation. Methylation changes may also play a key role in lineage specification during stem cell differentiation. To further our understanding of epigenetic regulation in chondrocytes we characterised the DNA methylation changes during chondrogenesis of mesenchymal stem cells (MSCs) by Infinium 450 K methylation array. Significant DNA hypomethylation was identified during chondrogenic differentiation including changes at many key cartilage gene loci. Integration with chondrogenesis gene expression data revealed an enrichment of significant CpGs in upregulated genes, while characterisation of significant CpG loci indicated their predominant localisation to enhancer regions. Comparison with methylation profiles of other tissues, including healthy and diseased adult cartilage, identified chondrocyte-specific regions of hypomethylation and the overlap with differentially methylated CpGs in osteoarthritis. Taken together we have associated DNA methylation levels with the chondrocyte phenotype. The consequences of which has potential to improve cartilage generation for tissue engineering purposes and also to provide context for observed methylation changes in cartilage diseases such as osteoarthritis

Pro-survival factor EDEM3 confers therapy resistance in prostate cancer

Prostate cancer is the most common cancer in men, and it is primarily driven by androgen steroid hormones. The glycosylation enzyme EDEM3 is controlled by androgen signalling and important for prostate cancer viability. EDEM3 is a mannosidase that trims mannose from mis-folded glycoproteins, tagging them for degradation through endoplasmic reticulum associated degradation. Here, we find that EDEM3 is upregulated in prostate cancer, and this is linked to poorer disease-free survival. Depletion of EDEM3 from prostate cancer cells induces an ER stress transcriptomic signature, and EDEM3 overexpression is cyto-protective against ER stressors. EDEM3 expression also positively correlates with genes involved in the unfolded protein response in prostate cancer patients and its expression can be induced through exposure to radiation. Importantly, the overexpression of EDEM3 promotes radio-resistance in prostate cancer cells and radio-resistance can be reduced through depletion of EDEM3. Our data thus implicate increased levels of EDEM3 with a role in prostate cancer pathology and reveal a new therapeutic opportunity to sensitise prostate tumours to radiotherapy

Pro-Survival Factor EDEM3 Confers Therapy Resistance in Prostate Cancer

Prostate cancer is the most common cancer in men, and it is primarily driven by androgen steroid hormones. The glycosylation enzyme EDEM3 is controlled by androgen signalling and is important for prostate cancer viability. EDEM3 is a mannosidase that trims mannose from mis-folded glycoproteins, tagging them for degradation through endoplasmic reticulum-associated degradation. Here, we find that EDEM3 is upregulated in prostate cancer, and this is linked to poorer disease-free survival. Depletion of EDEM3 from prostate cancer cells induces an ER stress transcriptomic signature, and EDEM3 overexpression is cyto-protective against ER stressors. EDEM3 expression also positively correlates with genes involved in the unfolded protein response in prostate cancer patients, and its expression can be induced through exposure to radiation. Importantly, the overexpression of EDEM3 promotes radio-resistance in prostate cancer cells and radio-resistance can be reduced through depletion of EDEM3. Our data thus implicate increased levels of EDEM3 with a role in prostate cancer pathology and reveal a new therapeutic opportunity to sensitise prostate tumours to radiotherapy

Developing physical activity interventions for adults with spinal cord injury. Part 2: Motivational counseling and peer-mediated interventions for people intending to be active

Objective: The majority of people with spinal cord injury (SCI) do not engage in sufficient leisure-time physical activity (LTPA) to attain fitness benefits; however, many have good intentions to be active. This paper describes two pilot interventions targeting people with SCI who are insufficiently active but intend to be active (i.e., intenders ). Method: Study 1 examined the effects of a single, telephone-based counseling session on self-regulatory efficacy, intentions, and action plans for LTPA among seven men and women with paraplegia or tetraplegia. Study 2 examined the effects of a home-based strengthtraining session, delivered by a peer and a fitness trainer, on strength-training task self-efficacy, intentions, action plans, and behavior. Participants were 11 men and women with paraplegia. Results: The counseling session (Study 1) yielded medium- to large-sized increases in participants\u27 confidence to set LTPA goals and intentions to be active. The home visit (Study 2) produced medium- to large-sized increases in task self-efficacy, barrier self-efficacy, intentions, action planning, and strength-training behavior from baseline to 4 weeks after the visit. Conclusions/Implications: Study 1 findings provide preliminary evidence that a single counseling session can impact key determinants of LTPA among intenders with SCI. Study 2 findings demonstrate the potential utility of a peer-mediated, home-based strength training session for positively influencing social cognitions and strength-training behavior. Together, these studies provide evidence and resources for intervention strategies to promote LTPA. among intenders with SCI, a population for whom LTPA interventions and resources are scarcely available. © 2013 American Psychological Association

Data-driven approach for highlighting priority areas for protection in marine areas beyond national jurisdiction

One of the aims of the United Nations (UN) negotiations on the conservation and sustainable use of marine biodiversity in areas beyond national jurisdiction (ABNJ) is to develop a legal process for the establishment of area-based management tools, including marine protected areas, in ABNJ. Here we use a conservation planning algorithm to integrate 55 global data layers on ABNJ species diversity, habitat heterogeneity, benthic features, productivity, and fishing as a means for highlighting priority regions in ABNJ to be considered for spatial protection. We also include information on forecasted species distributions under climate change. We found that parameterizing the planning algorithm to protect at least 30% of these key ABNJ conservation features, while avoiding areas of high fishing effort, yielded a solution that highlights 52,545,634 km2 (23.7%) of ABNJ as high priority regions for protection. Instructing the planning model to avoid ABNJ areas with high fishing effort resulted in relatively minor shifts in the planning solution, when compared to a separate model that did not consider fishing effort. Integrating information on climate change had a similarly minor influence on the planning solution, suggesting that climate-informed ABNJ protected areas may be able to protect biodiversity now and in the future. This globally standardized, data-driven process for identifying priority ABNJ regions for protection serves as a valuable complement to other expert-driven processes underway to highlight ecologically or biologically significant ABNJ regions. Both the outputs and methods exhibited in this analysis can additively inform UN decision-making concerning establishment of ABNJ protected areas