NON-CANONICAL TRANSLATION REGULATORY FUNCTION OF G9A IN CHRONIC INFLAMMATION ASSOCIATED DISEASES

We report a novel translation-regulatory function of G9a, a histone methyltransferase and well-understood transcriptional repressor, in promoting hyperinflammation and lymphopenia; two hallmarks of endotoxin tolerance (ET)-associated chronic inflammatory complications including sepsis, acute respiratory distress syndrome (ARDS) and COVID-19. Opening chapter provides an overview of sepsis and role of G9a in endotoxin tolerance. Then, in chapter 2, using multiple approaches, we demonstrate that G9a interacts with multiple translation regulators during ET, particularly the N6-methyladenosine (m6A) RNA methyltransferase METTL3, to co-upregulate expression of certain m6A-modified mRNAs that encode immune-checkpoint and anti-inflammatory response related proteins. Mechanistically, G9a promotes m6A methyltransferase activity of METTL3 at translational/post-translational level by regulating its expression, its methylation, and its cytosolic localization during ET. Additionally, from a broader view extended from the G9a-METTL3-m6A translation regulatory axis, our translatome proteomics approach identified numerous ‘G9a-translated’ proteins that unite the networks associated with inflammation dysregulation, T cell dysfunction, and systemic cytokine response. In sum, we identified a previously unrecognized function of G9a in protein-specific translation that can be leveraged to treat ET-related chronic inflammatory diseases.Interestingly, m6A-modification pathway is hijacked by several RNA viruses for their propagation and persistence, including SARS-CoV-2, making it an attractive host-directed therapeutic target for the development of broad-spectrum antivirals. As there are no clinically approved inhibitors for the m6A-modification pathway, we decided to target our newly identified G9a-mediated m6A RNA modification pathway to hinder SARS-CoV-2 replication. In Chapter 3, we combine results from ET macrophage and COVID-19 patient-derived PBMC cells to show that, indeed, G9a interacts with METTL3 and other translational regulators to promote expression and turnover of proteins involved in immune regulation, viral replication, and tissue damage. More importantly, drugs targeting G9a (UNC0642, YX59-126) and its associated protein EZH2 (UNC1999, tazemetostat) are potent inhibitors of SARS-CoV-2 replication with UNC0642/UNC1999 treatment reversing transcriptional, m6A-epitranscriptional, translational, and post-translational (phosphorylation or secretion) effects of coronavirus infection in human alveolar epithelial (A549-hACE2) and/or COVID-19 patient PBMC cells. Mechanistically, G9a facilitates SARS-CoV-2 mediated dysregulation of viral/host m6A epi-transcriptome to ultimately promote expression/translation of SARS-CoV-2 encoded transcripts and other viral lifecycle & host immune response related mRNAs in A549-hACE2 cells & COVID-19 patient PBMCs. Comparison of proteomic analyses of G9a/Ezh2 inhibitor-treated, SARS-CoV-2 infected cells or ex vivo culture of patient PBMCs with COVID-19 patient data revealed that G9a/Ezh2 inhibition reversed the patient proteomic landscapes that were highly correlated with COVID-19 pathology. This cell-to-patient conservation of G9a-translated, COVID-19 proteo-pathology makes G9a/Ezh2 inhibitors attractive candidates for drug repurposing. Concluding remarks are provided in the last chapter.Altogether, we extend G9a function(s) beyond transcription to translational regulation during sepsis/COVID-19 pathogenesis and show that targeting this master regulatory complex could complement existing immunomodulatory and antiviral therapies and fills a need for a new drug class that can be exploited to help combat drug resistance and infection.Doctor of Philosoph

m6A mRNA METHYLATION IN DEVELOPMENT AND DISEASE

Chemical modifications on mRNA have recently garnered attention as major regulators of cell behavior in embryonic development and many types of disease. In particular, N6-adenosine (m6A), is an abundant mRNA modification that mediates mRNA fate. Through distinct reader protein binding, m6A promotes various processing events such as mRNA degradation, alternative splicing, nuclear export, and translation initiation. While we have known of the existence of m6A for many years, the recent discovery of m6A demethylases has spurred interest in this dynamic modification as a regulatory system. In vitro work showed that m6A appears to be especially important in stem cell biology, where knockout of the m6A methyltransferase complex components causes major impairments in stem cell self-renewal and differentiation. In vivo work has been severely limited by the fact that full knockout of Mettl3 or Mettl14, which are central parts of the m6A methyltransferase complex, is embryonic lethal. We therefore used conditional knockout mice in which Mettl14 is knocked out in neural stem cells. My thesis has focused on the role of m6A in in vivo brain development, with studies on m6A in mammalian development and Fragile X Syndrome. We showed that m6A promotes mRNA degradation of transcripts that regulate the balance between stem cell self-renewal and neurogenesis. Loss of m6A slows the tempo of neurogenesis and also revealed that neural stem cells are normally pre-patterned with transcription of neural genes prior to differentiation. In parallel, I studied the role of m6A in hypoxic breast cancer cells because hypoxia induces the m6A demethylase, ALKBH5, to drive global changes in m6A methylation patterns. In this system, m6A promotes translation of modified transcripts to promote global translation, cell division, and oxidative metabolism. The study of m6A in multiple systems reveals the incredible cell-type specificity and dynamic nature of m6A

DEVELOPMENT OF COMPUTATIONAL TOOLS TO STUDY THE PATTERNING OF DNA AND RNA METHYLATION IN HEALTHY AND DISEASE STATES

Epigenetics can be defined as the set of sequence independent processes that produces heritable changes in cellular information. These chromatin-based events such as covalent modification of DNA and histone tails are laid down by the co-ordinated action of chromatin modifying enzymes, thus altering the organisation of chromatin and its accessibility to the transcriptional machinery. Our understanding of epigenetic intricacies has considerably increased over the last decade owing to rapid development of genomic and proteomic technologies. This has resulted in huge surge in the generation of epigenomics data. Integrative analysis of these epigenomics datasets provides holistic view on the interplay of various epigenetic components and possible aberration in patterns in specific biological or disease states. Although, there are numerous computational tools available catering individually to each epigenomic datatype, a comprehensive computational framework for integrated exploratory analysis of these datasets was missing. We developed a suite of R packages methylPipe and compEpiTools that can efficiently handle whole genome base-resolution DNA methylation datasets and effortlessly integrate them with other epigenomics data. We applied these methods to the study of epigenomics landscape in B-cell lymphoma identifying a putative set of tumor suppressor genes. Moreover, we also applied these methods to explore possible associations between m6A RNA methylation, epigenetic marks and regulatory proteins

Influence and regulation of PCBP2 and YTHDF2 RNA-binding proteins during self-renewal and differentiation of human induced pluripotent stem cells

2019 Fall.Includes bibliographical references.Embryonic stem cells (ESCs) are able to self-renew or differentiate into any cell type in the body, a property known as pluripotency that enables them to initiate early growth and development. However, the ethical implications of harvesting and manipulating ESCs hinders their use in basic research and the clinical applications. Thus, the discovery that somatic cells can be exogenously reprogrammed into induced pluripotent stem cells (iPSCs) offers new and exciting possibilities for gene therapy, personalized medicine and basic research. However, more research is needed into the mechanisms involved in regulating pluripotency in order for iPSCs to reach their full potential in the research lab and clinic. To maintain a state of self-renewal, yet also be able to rapidly differentiate in response to external signals, pluripotent stem cells need to exert tight control over gene expression through transcriptional and post-transcriptional mechanisms. There are several notable transcriptional networks that regulate pluripotency, but the post-transcriptional mechanisms remain poorly characterized. mRNA decay is one form of post-transcriptional regulation that can help to both maintain the steady-state of a transcriptome or facilitate its rapid remodeling. To this end, degradation rates are influenced by the elements contained in an mRNA and the RNA-binding proteins (RBPs) they associate with. Previous reports have indicated the RNA modification N6-methyladenosine (m6A) and C-rich sequence elements (CREs) can affect mRNA decay in pluripotent stem cells. Therefore, we sought to further understand the roles of m6A and CREs in mRNA decay in stem cells by characterizing the expression and mRNA targets of two RBPs that recognize these elements, YTHDF2 and PCBP2, respectively. In this thesis, I report YTHDF2 is differentially regulated in pluripotent and differentiated cells and that YTHDF2 contributes to pluripotency by targeting a group of mRNAs encoding factors important for neural development. The down-regulation of YTHDF2 during neural differentiation is consistent with increased expression of neural factors during this time. Moreover, YTHDF2 expression is regulated at the level of translation via elements located in the first 300 nucleotides of the 3' untranslated region of YTHDF2 mRNA. Based on these results, I propose that stem cells are primed for rapid differentiation by transcribing low levels of mRNAs encoding neural factors that are subsequently targeted for degradation, in part by YTHDF2, until differentiation is induced. On the other hand, PCBP2 is up-regulated upon differentiation of pluripotent stem cells and regulates several mRNAs associated with pluripotency and development, including LIN28B. Notably, expression of long non-coding RNAs (lncRNAs) that contain human endogenous retrovirus element H (HERV-H) is influenced by PCBP2. HERV-H lncRNAs are almost exclusively expressed in stem cells and play a role in maintaining a pluripotent state, although their functions are not fully understood. Intriguingly, some HERV-lncRNAs can also regulate PCBP2 expression, as altering the expression of LINC01356 or LINC00458 effects PCBP2 protein levels. Based on these results, I propose the reciprocal regulation of PCBP2 and HERV-H lncRNAs influences whether stem cells maintain a state of self-renewal or differentiate. Taken together, these findings demonstrate that YTHDF2 and PCBP2 post-transcriptionally regulate gene expression in stem cells and influence pluripotency

Metabolo-epigenetic interplay provides targeted nutritional interventions in chronic diseases and ageing

Epigenetic modifications are chemical modifications that affect gene expression without altering DNA sequences. In particular, epigenetic chemical modifications can occur on histone proteins -mainly acetylation, methylation-, and on DNA and RNA molecules -mainly methylation-. Additional mechanisms, such as RNA-mediated regulation of gene expression and determinants of the genomic architecture can also affect gene expression. Importantly, depending on the cellular context and environment, epigenetic processes can drive developmental programs as well as functional plasticity. However, misbalanced epigenetic regulation can result in disease, particularly in the context of metabolic diseases, cancer, and ageing. Non-communicable chronic diseases (NCCD) and ageing share common features including altered metabolism, systemic meta-inflammation, dysfunctional immune system responses, and oxidative stress, among others. In this scenario, unbalanced diets, such as high sugar and high saturated fatty acids consumption, together with sedentary habits, are risk factors implicated in the development of NCCD and premature ageing. The nutritional and metabolic status of individuals interact with epigenetics at different levels. Thus, it is crucial to understand how we can modulate epigenetic marks through both lifestyle habits and targeted clinical interventions -including fasting mimicking diets, nutraceuticals, and bioactive compounds- which will contribute to restore the metabolic homeostasis in NCCD. Here, we first describe key metabolites from cellular metabolic pathways used as substrates to “write” the epigenetic marks; and cofactors that modulate the activity of the epigenetic enzymes; then, we briefly show how metabolic and epigenetic imbalances may result in disease; and, finally, we show several examples of nutritional interventions - diet based interventions, bioactive compounds, and nutraceuticals- and exercise to counteract epigenetic alterations

Inside the stemness engine: mechanistic links between deregulated transcription factors and stemness in cancer

Cell identity is largely determined by its transcriptional profile. In tumour, deregulation of transcription factor expression and/or activity enables cancer cell to acquire a stem-like state characterised by capacity to self-renew, differentiate and form tumours in vivo. These stem-like cancer cells are highly metastatic and therapy resistant, thus warranting a more complete understanding of the molecular mechanisms downstream of the transcription factors that mediate the establishment of stemness state. Here, we review recent research findings that provide a mechanistic link between the commonly deregulated transcription factors and stemness in cancer. In particular, we describe the role of master transcription factors (SOX, OCT4, NANOG, KLF, BRACHYURY, SALL, HOX, FOX and RUNX), signalling-regulated transcription factors (SMAD, β-catenin, YAP, TAZ, AP-1, NOTCH, STAT, GLI, ETS and NF-κB) and unclassified transcription factors (c-MYC, HIF, EMT transcription factors and P53) across diverse tumour types, thereby yielding a comprehensive overview identifying shared downstream targets, highlighting unique mechanisms and discussing complexities

The regulation of PARP proteins by the m⁶A methyltransferase machinery

RNA methylation is an important regulator of RNA metabolism. The most common form of internal mRNA methylation is N6-methyladenosine (m⁶A), which is deposited by the m6A methyltransferase complex (MTC). This occurs co-transcriptionally, meaning the MTC must interact with components within the broader chromatin environment, in order to rapidly and selectively access nascent RNA. My thesis is a step towards a better understanding of those interactions. In the first part of my thesis, I examine the cellular response to UV-C irradiation, which has recently been demonstrated to induce dynamic m6A deposition. Not only do I find limited evidence to support this model, I also show this discrepancy partly arises from the cross-reactivity of m6A antibodies with poly (ADP-ribose) (PAR), which confounds imaging data. I then identify a previously uncharacterised regulatory relationship between the core MTC protein, METTL3, and the synthesis of PAR (PARylation). In the second part of the thesis, I utilise a range of experimental techniques in an attempt to describe how PARylation is affected by the loss of METTL3. These experiments give no single answer, but indicate several contexts in which PARylation and METTL3 may be linked. In the third section, I present a study of how PARP-1 and PARylation is regulated by METTL3 during the exit from pluripotency, and in the context of MEK/ERK signalling. At the heart of this section is a proteomic dataset that measures changes to the PARP-1 chromatin-associated interactome, in the presence and absence of METTL3. This identifies several interesting candidate proteins, on which further research can be based. In summary, I have identified, and begun the characterisation of, a regulatory relationship between two important processes: the m⁶A modification of RNA and PARylation. This may have important consequences for understanding several aspects of cell homeostasis and disease

Exploring links between 2-oxoglutarate-dependent oxygenases and Alzheimer's disease

Hypoxia, that is, an inadequate oxygen supply, is linked to neurodegeneration and patients with cardiovascular disease are prone to Alzheimer's disease (AD). 2-Oxoglutarate and ferrous iron-dependent oxygenases (2OGDD) play a key role in the regulation of oxygen homeostasis by acting as hypoxia sensors. 2OGDD also have roles in collagen biosynthesis, lipid metabolism, nucleic acid repair, and the regulation of transcription and translation. Many biological processes in which the >60 human 2OGDD are involved are altered in AD patient brains, raising the question as to whether 2OGDD are involved in the transition from normal aging to AD. Here we give an overview of human 2OGDD and critically discuss their potential roles in AD, highlighting possible relationships with synapse dysfunction/loss. 2OGDD may regulate neuronal/glial differentiation through enzyme activity-dependent mechanisms and modulation of their activity has potential to protect against synapse loss. Work linking 2OGDD and AD is at an early stage, especially from a therapeutic perspective; we suggest integrated pathology and in vitro discovery research to explore their roles in AD is merited. We hope to help enable long-term research on the roles of 2OGDD and, more generally, oxygen/hypoxia in AD. We also suggest shorter term empirically guided clinical studies concerning the exploration of 2OGDD/oxygen modulators to help maintain synaptic viability are of interest for AD treatment