Mechanisms of DNA demethylation

While every cell in an organism is genetically identical, there are marked phenotypic differences between tissues and organs that are controlled by epigenetic modifications. These epigenetic modifications provide an important role in controlling the machinery of gene expression. DNA methylation is the most stable epigenetic modification and is necessary for cellular activities. Many cancers show dysregulation of DNA methylation, with significant global loss of methylation. While the phenomenon of DNA methylation is well described in mammals, the mechanisms of reversal of methylation are incompletely understood. In previous studies, our laboratory observed rapid DNA demethylation in Jurkat cells (T cell leukaemia) after exposure to oxidative stress. This rapid effect indicated active demethylation and suggested an activation of TET (Ten Eleven Translocation) proteins may be occurring. My research investigates the mechanism of changes in DNA methylation using a barcoded hairpin-bisulfite sequencing technique. This high throughput hairpin-bisulfite assay provides a direct assessment of the methylation status of the DNA strands by linking the complementary strands together with a barcoded DNA hairpin linker. We established and developed a bioinformatic workflow to analyse hairpin-bisulfite sequencing data, initially using the Galaxy online platform and later using UNIX command line tools. We demonstrated hemi-methylation of densely methylated promoter regions, as early as 2 h after DNA replication in cells treated with decitabine, and this hemi-methylation increased with decitabine treatment. With the power of the hairpin-bisulfite sequencing assay, we were able to describe the kinetics and pattern of DNMT1 inhibitor treatment. Even though these results were predicted by previous studies, the kinetics of decitabine-induced hemi-methylation has not been demonstrated before in such detail and clarity. We observed an increase (40%) in unmethylated hairpin reads of the PCDHGA12 promoter after treatment with ascorbate/decitabine compared to untreated controls. This observed rapid demethylation implicates a novel mechanism of active demethylation that has not yet been recognised by researchers in the field. Importantly, we demonstrated that decitabine is capable of erasing the epigenetic memory of somatic cells by full removal of methyl groups from both complementary DNA strands. This study is the first to report active demethylation in somatic cells treated with decitabine. Furthermore, in both single-gene and global investigations, we found loss of DNA methylation in different leukaemia cell lines (Molt4, Nalm6 and HL60) following ascorbate and DNMT inhibitor treatment, as well as an increase in 5-hydroxymethyl cytosine in Jurkat cells treated with decitabine and combined ascorbate/decitabine. We performed a low-coverage methylation sequencing assay (PBAT) to detect changes in global DNA methylation. We observed a gradual loss in global DNA methylation in synchronised Jurkat cells treated with decitabine and combined ascorbate/decitabine, confirming the results from locus-specific demethylation. The clinical relevance of our study is that it supports the premise that ascorbate is necessary to enhance the efficacy of decitabine by promoting the function of TET. It is likely that the demethylation pathways that we are studying operate during the onset of cancer and the existence of molecules that alter TET activity may have implications for modification of this process. Cancer patients are often markedly ascorbate (vitamin C) deficient, so the addition of ascorbate to treatment protocols may increase the clinical efficacy (or toxicity) of such drugs in patients with leukaemia

Transient Relaxation of DNA Methylation at the Onset of Meiosis

Meiotic prophase I (MPI) is a unique phase of the cell cycle, specific to germ cells and defining of sexual reproduction. MPI is a period of extensive and specialized homologous chromosome interactions and genetic exchange. Proper progression of MPI requires elaborate epigenetic control, deficiencies in which often lead to infertility. Changes in DNA methylation during MPI can endanger genome integrity by activating transposable elements (TEs) that when mobilized induce DNA breaks and mutations. Therefore, MPI was thought to be under strict surveillance by DNA methylation, whose levels were assumed to be high and stable throughout MPI. Interestingly, expression of LINE retrotransposons, specifically LINE-1 (L1)-encoded protein ORF1p has been observed in MPI germ cells of wild-type male mice. Since tight epigenetic regulation is associated with transposon silencing, we hypothesized that L1 expression in MPI may indicate relaxation of epigenetic silencing in meiotic germ cells. Thus, we investigated the dynamics of CpG DNA methylation during MPI. We enriched and isolated individual MPI stages by Fluorescence Activated Cell Sorting (FACS) and profiled individual MPI germ cells using whole-genome bisulfite sequencing, and RNA-sequencing. Using this approach we uncovered transient and stage-specific changes in DNA methylation dynamics. In contrast to the prevailing view, we show that male germ cells undergo genome-wide transient relaxation of DNA methylation (TRDM) during early MPI. Specifically, we find that a transition from pre-meiotic spermatogonia to meiotic onset in preleptotene spermatocytes is accompanied by genome-wide hypomethylation. Gradual, but uneven remethylation of the genome creates hypomethylated domains throughout meiotic prophase, with pre-meiotic levels of DNA methylation achieved only by late MPI. Our data are most consistent with a DNA replication- coupled mechanism of DNA demethylation in pre-meiotic S-phase. Intriguingly, a TRDM- independent set of hypomethylated domains emerges in mid to late MPI and is enriched in transcriptionally upregulated spermatogenic genes. Using Mael -/- mice defective in piRNA pathway, we show that early MPI offers an opportunity for TE expression and reactivation. We demonstrate that if germ cells enter MPI with insufficient levels of DNA methylation at L1 elements, then during TRDM, meiotic onset can be hijacked to reactivate potentially active L1s. Cumulatively, we demonstrate that early MPI is epigenetically relaxed, exhibits dynamic DNA methylation pattern in MPI and that transient genome-wide DNA hypomethylation at meiotic onset might have implications in gamete quality control

High-throughput Human Cell Reprogramming through Substrate and Microfluidics Integration

Human cells and tissues are key systems to study human biology and physiology, and to develop new strategies and targeting drugs for human diseases. Since the study and testing on human beings may not be acceptable due to exposure to risks and practical and ethical concerns, in vitro strategies are of paramount importance to rely on human organism and avoid non-fully predictive animal models. The demand of research in clinical and industrial fields for effective, representative and affordable strategies is undoubtedly increasing. Conventional cell culture systems and drug discovery are normally performed in vessels with a characteristic dimension in the order of centimeters. Nutrients are delivered to cells through liquid media containing balanced saline buffers and oligo-elements. A reasonable amount of medium is necessary to homogeneously cover a cell layer and must exchanged with fresh media to maintain a proper amount of available nutrients and remove released waste products. Many studies and applications require expensive reagents and are subjected to limited data throughput. The discovery of reprogramming process by 2012 Nobel Prize Yamanaka opened breakthrough new perspective on research and clinical applications. Basically, from a patient’s skin biopsy it is now possible to derive induced pluripotent stem cells (iPSC) and to obtain new tissues for an ad hoc self-repair. So far, human iPSC (hiPSC) have not been applied to clinics due to some unexplored aspects on their derivation, non clinical-grade methods and the significative cost of hiPSC derivation per patient. The down-scale of reprogramming process could provide an unique opportunity to derive cost-effective hiPSC and obtain valuable human in vitro tissues. The aim of this thesis is the development of a comprehensive platform for the reprogramming of human cells at the microscale. To this end, we focused on the development of cell microenvironment which is composed by both soluble and solid components. During this thesis, synthetic and biodegradable hydrogels were developed. The large-scale production of mechanically-tunable poly-acrylamide-based substrates were fundamental to reveal the interaction occurring between substrate stiffness and cell behavior and fate. Engineering of biodegradable hydrogels has revealed the potential to develop in vitro functional tissues and to integrate them at a later stage in patients. Chemical modifications were transferred to topological substrate control and in turn in microfluidic platforms. Microfluidic chip environment and management was designed in order to allow long-term adhesion, culture and biologically relevant cell behaviors. Adhesion proteins fundamental for cell attachment and growth were modified and integrated with the micronized substrates. Since medium for microfluidic cell culture relies on perfusion, continuous or periodic flow could be applied. Thus, we studied the management of media delivery in order to determine the best strategy for long-term cell cultures. The achievements obtained with both substrate and microfluidic cell culture development was applied to the generation of a new platform for hiPSC derivation, differentiation and testing at the microscale. For the first time, it is possible to obtain human iPSC clones in microfluidics with a remarked reduction of minimum requirements (materials, reagents, overall expenses). The production of cost effective hiPSC can lead to a mass production of characterized and functional tissues that can be either integrated in 3D developed constructs and serve as valuable tissue source derivation for drug development. Our platform opens new perspectives in studying and treating both abundant and rare diseases involving both scientists and entrepreneur

Metabolic regulation during early embryo development

The preimplantation embryo must satisfy dynamic changes in energy demand during development to the blastocyst stage. Energy is provided through regulated metabolic pathways including glycolysis, β-oxidation and oxidative phosphorylation. Oxygen consumption rate (OCR), representing overall oxidative metabolism, has been reported in several species but few studies have examined the bioenergetics of embryo development.Several methods were optimised to measure components of OCR by individual embryos. On average, 66% of blastocyst OCR was coupled to ATP synthesis, the majority being complex I-dependent. A further 13% was of non-mitochondrial origin, while maximal OCR was 189% of basal, providing a spare respiratory capacity of +89%. This profile allows re-interpretation of existing data to estimate ATP production by the bovine embryo.The endogenous triglyceride store of the oocyte is increasingly considered a vital energy source in preimplantation development. In the present study, β-oxidation was manipulated during embryo culture. Inhibition of β-oxidation led to i) increased OCR ii) increased lipid content, iii) increased pyruvate uptake and iv) decreased lactate release at the blastocyst stage. Enhancing β-oxidation caused i) OCR at blastocyst stage to fall, ii) decreased lipid content during early cleavage, iii) decreased pyruvate consumption and iv) increased lactate release. Neither treatment affected blastocyst development rate or differential cell count, while both led to mitochondrial depolarisation.These metabolic observations were hypothesised to have legacy effects on gene expression. Groups of 10 blastocysts with similar metabolic profiles were analysed using transcription and DNA methylation microarray platforms. Following manipulation of β-oxidation, gene transcripts involved in mitochondrial function, metabolism, key signalling cascades, recognition of pregnancy, stress response, protein modification and transcription were differentially expressed. Genes involved in transcription, protein modification, key signalling cascades and disease were differentially methylated, potentially linking dysregulated β-oxidation to deleterious conditions in later development.These data highlight the plasticity of metabolic regulation in the embryo, allowing successful preimplantation development despite an apparently deleterious phenotype, yet indicate that metabolic activity has subtle effects on development

Optically Induced Nanostructures

Nanostructuring of materials is a task at the heart of many modern disciplines in mechanical engineering, as well as optics, electronics, and the life sciences. This book includes an introduction to the relevant nonlinear optical processes associated with very short laser pulses for the generation of structures far below the classical optical diffraction limit of about 200 nanometers as well as coverage of state-of-the-art technical and biomedical applications. These applications include silicon and glass wafer processing, production of nanowires, laser transfection and cell reprogramming, optical cleaning, surface treatments of implants, nanowires, 3D nanoprinting, STED lithography, friction modification, and integrated optics. The book highlights also the use of modern femtosecond laser microscopes and nanoscopes as novel nanoprocessing tools

Metabolic regulation during early embryo development

The preimplantation embryo must satisfy dynamic changes in energy demand during development to the blastocyst stage. Energy is provided through regulated metabolic pathways including glycolysis, β-oxidation and oxidative phosphorylation. Oxygen consumption rate (OCR), representing overall oxidative metabolism, has been reported in several species but few studies have examined the bioenergetics of embryo development. Several methods were optimised to measure components of OCR by individual embryos. On average, 66% of blastocyst OCR was coupled to ATP synthesis, the majority being complex I-dependent. A further 13% was of non-mitochondrial origin, while maximal OCR was 189% of basal, providing a spare respiratory capacity of +89%. This profile allows re-interpretation of existing data to estimate ATP production by the bovine embryo. The endogenous triglyceride store of the oocyte is increasingly considered a vital energy source in preimplantation development. In the present study, β-oxidation was manipulated during embryo culture. Inhibition of β-oxidation led to i) increased OCR ii) increased lipid content, iii) increased pyruvate uptake and iv) decreased lactate release at the blastocyst stage. Enhancing β-oxidation caused i) OCR at blastocyst stage to fall, ii) decreased lipid content during early cleavage, iii) decreased pyruvate consumption and iv) increased lactate release. Neither treatment affected blastocyst development rate or differential cell count, while both led to mitochondrial depolarisation. These metabolic observations were hypothesised to have legacy effects on gene expression. Groups of 10 blastocysts with similar metabolic profiles were analysed using transcription and DNA methylation microarray platforms. Following manipulation of β-oxidation, gene transcripts involved in mitochondrial function, metabolism, key signalling cascades, recognition of pregnancy, stress response, protein modification and transcription were differentially expressed. Genes involved in transcription, protein modification, key signalling cascades and disease were differentially methylated, potentially linking dysregulated β-oxidation to deleterious conditions in later development. These data highlight the plasticity of metabolic regulation in the embryo, allowing successful preimplantation development despite an apparently deleterious phenotype, yet indicate that metabolic activity has subtle effects on development

Flow sorting in the study of teratocarcinoma cell differentiation

Flow cytometry is a technique by which particles (cells, subcellular fragments, bacteria) in aqueous suspension are passed one by one through a sensing region where optical (or electrical) signals are generated. These signals for each individual cell are collected and processed, and may be stored to yield the distr1bution of the property measured for the population of cells analyzed. Most often cells are fluorescently labelled (with fluorescent dyes, particles or antibodies) and fluorescence signals are measured. Instruments called flow sorters in addition have a sorting capability which allows physical separation of a desired subpopulation for further analysis. This thesis is concerned with the cell biological application of such an instrument with particular emphasis on the use of the sorting capabilities. The process of cell specialization which occurs when a multicellular organism develops from one cell into the mature organism is called cell differentiation. Mouse teratocarcinoma cells share many properties with early embryonic cells and can be used as an tn vitro cell model system to study the early events of cell differentiation. Some aspects of teratocarcinoma cell differentiation may especially profitable be studied by making use of the sorting capabilities of the cell sorter and this is what the main part of this thesis is about. Teratocarcinoma cells can be induced to differentiate by treatment with chemical inducers. Analysis of properties changing upon differentiation can yield information about their role in the differentiation process. One of these properties is the "fluidity" of the plasma membrane. A part of this thesis describes the flow cytophotometric measurement of this change after induction of differentiation