Dissertations / Theses on the topic 'Epigenetics'

Les dérivés de la cytosine sont d’importantes modifications épigénétiques dont le rôle dans l’évolution de la leucémie lymphoïde chronique (LLC) n’est pas totalement exploré. Dans ce contexte, notre première étude vise à examiner le niveau global de la 5-methylcytosine (5-mCyt), 5-hydroxymethylcytosine (5-hmCyt), 5-carboxylcytosine (5-CaCyt) et 5-hydroxymethyluridine (5-hmU) dans des lymphocytes B purifiés de patients LLC (n=56) et d’individus sains (n=17). Les principaux acteurs de la régulation épigénétique (DNMT1/3A/3B, MBD2/4, TET1/2/3, SAT1) ont été évalués par PCR quantitative en temps réel. L’analyse a permis de mettre en exergue trois groupes de patients. En premier lieu, un groupe de patients stables (délai médian de progression [PFS] et délai au premier traitement [TFT] >120 mois), avec un profil épigénétique similaire au groupe contrôle. Deuxièmement, un groupe intermédiaire (PFS=84; TFT=120 mois) qui présente une augmentation de la déméthylation de l’ADN expliquée par l'induction SAT1 / TET2 pendant la progression de la maladie. Troisièmement, un groupe de patients avec une forme active de la maladie (PFS=52; TFT=112 mois) qui présentent une hyperlymphocytose, une réduction du temps de doublement des lymphocytes et des modifications épigénétiques majeures. Au sein de ce groupe, une réduction est observée pour la 5-mCyt, 5-hmCyt, 5-CaCyt et serait associée à une diminution des DNMTs, TETs et MBDs au cours de la progression de la maladie. Les profils épigénétiques mis en évidence sont indépendants du statut mutationnel IGHV mais sont associés avec les anomalies cytogénétiques. Nous nous sommes également intéressés à cette association et nous avons montré dans la deuxième étude que les modifications des dérivées de la cytosine peuvent affiner le pouvoir pronostic des anomalies cytogénétiques. En conclusion, nos résultats suggèrent que les variations de la méthylation ainsi que des intermédiaires de la déméthylation de l’ADN sont impliqués dans la progression de la LLC
Cytosine derivatives are important epigenetic modifications whose role in the pathogenesis and evolution of chronic lymphocytic leukemia (CLL) is not fully explored. In this context, our first study aims to examine the global DNA level of 5-methylcytosine (5-mCyt), 5-hydroxymethylcytosine (5-hmCyt), 5-carboxylcytosine (5-CaCyt) and 5-hydroxymethyluridine (5-hmU) in purified B lymphocytes of CLL patients (n = 56) and healthy individuals (n = 17). The main actors in epigenetic regulation (DNMT1 / 3A / 3B, MBD2 / 4, TET1 / 2/3, SAT1) were evaluated by quantitative real time PCR. The analysis highlighted three groups of patients. First, a group of patients with stable disease (median time to progression [PFS] and time to first treatment [TFT]> 120 months), with an epigenetic profile similar to the control group. Secondly, an intermediate group (PFS = 84, TFT = 120 months) which shows an increase in DNA demethylation explained by SAT1 / TET2 induction during disease progression. Third, a group of patients with an active form of the disease (PFS = 52, TFT = 112 months) who have hyperlymphocytosis, a short lymphocyte doubling time, and major epigenetic changes. Within this group, a reduction is observed for 5-mCyt, 5-hmCyt, 5-CaCyt which is associated with a decrease in DNMTs, TETs and MBDs during disease progression. The identified epigenetic profiles are independent of IGHV mutational status but are associated with cytogenetic abnormalities. We were also interested in this association and we showed in the second study that modifications of cytosine derivatives levels can refine the prognostic power of cytogenetic abnormalities.In conclusion, our results suggest that methylation variations as well as DNA demethylation intermediates are involved in the progression of CLL

Virtually all multicellular organisms are capable of developing differently in response to environmental variation. At the molecular level, such developmental plasticity requires interpretation and perpetuation of environmental signals without changing the underlying genotype. Such non-genetic, heritable information is known as epigenetic information. This dissertation examines epigenetic information among social insects, and how differences in such information relate to phenotypic caste differences. The studies included herein primarily focus on one form of epigenetic information: DNA methylation. In particular, these studies explore DNA methylation as it relates to and impacts (i) alternative phenotype and particular gene expression differences in two social insect species, (ii) histone modifications, another important form of epigenetic information, in insect genomes, and (iii) molecular evolutionary rate of underlying actively transcribed gene sequences. We find that DNA methylation exhibits marked epigenetic and evolutionary associations, and is associated with alternative phenotype in multiple insect species. Thus, DNA methylation is emerging as one important epigenetic mediator of phenotypic plasticity in social insects.

A typical gene expression data set consists of measurements of a large number of gene expressions, on a relatively small number of subjects, classified according to two or more outcomes, for example cancer or non-cancer. The identification of associations between gene expressions and outcome is a huge multiple testing problem. Early approaches to this problem involved the application of thousands of univariate tests with corrections for multiplicity. Over the past decade, numerous studies have demonstrated that analyzing gene expression data structured into predefined gene sets can produce benefits in terms of statistical power and robustness when compared to alternative approaches. This thesis presents the results of research on gene set analysis. In particular, it examines the properties of some existing methods for the analysis of gene sets. It introduces novel Bayesian methods for gene set analysis. A distinguishing feature of these methods is that the model is specified conditionally on the expression data, whereas other methods of gene set analysis and IGA generally make inferences conditionally on the outcome. Computer simulation is used to compare three common established methods for gene set analysis. In this simulation study a new procedure for the simulation of gene expression data is introduced. The simulation studies are used to identify situations in which the established methods perform poorly. The Bayesian approaches developed in this thesis apply reversible jump Markov chain Monte Carlo (RJMCMC) techniques to model gene expression effects on phenotype. The reversible jump step in the modelling procedure allows for posterior probabilities for activeness of gene set to be produced. These mixture models reverse the generally accepted conditionality and model outcome given gene expression, which is a more intuitive assumption when modelling the pathway to phenotype. It is demonstrated that the two models proposed may be superior to the established methods studied. There is considerable scope for further development of this line of research, which is appealing in terms of the use of mixture model priors that reflect the belief that a relatively small number of genes, restricted to a small number of gene sets, are associated with the outcome.

There is a need to identify factors that are able to influence health in old age and to develop interventions that could slow down the process of aging and its associated pathologies. Lifestyle modifications, and especially nutrition, appear to be promising strategies to promote healthy aging. Their impact on aging biomarkers has been poorly investigated. In the first part of this work, we evaluated the impact of a one-year Mediterranean-like diet, delivered within the framework of the NU-AGE project in 120 elderly subjects, on epigenetic age acceleration measures assessed with Horvath’s clock. We observed a rejuvenation of participants after nutritional intervention. The effect was more marked in the group of Polish females and in subjects who were epigenetically older at baseline. In the second part of this work, we developed a new model of epigenetic biomarker, based on a gene-targeted approach with the EpiTYPER® system. We selected six regions of interest (associated with ELOVL2, NHLRC1, SIRT7/MAFG, AIM2, EDARADD and TFAP2E genes) and constructed our model through a ridge regression analysis. In controls, estimation of chronological age was accurate, with a correlation coefficient between predicted and chronological age of 0.92 and a mean absolute deviation of 4.70 years. Our model was able to capture phenomena of accelerated or decelerated aging, in Down syndrome subjects and centenarians and offspring respectively. Applying our model to samples of the NU-AGE project, we observed similar results to the ones obtained with the canonical epigenetic clock, with a rejuvenation of the individuals after one-year of nutritional intervention. Together, our findings indicate that nutrition can promote epigenetic rejuvenation and that epigenetic age acceleration measures could be suitable biomarkers to evaluate their impact. We demonstrated that the effect of the dietary intervention is country-, sex- and individual-specific, thus suggesting the need for a personalized approach to nutritional interventions.

Epigenetic processes are known to play an important role in the regulation of embry-onic development and gene expression. Here we utilise next-generation sequencing and bioinformatics methodologies to investigate the role of epigenetics in two different sys-tems, heart and brain. In heart, the endocardium is a distinct understudied epithelial population of cells that is involved in directing morphogenesis of the myocardium, valve leaflets and trabeculae. We generate whole-genome bisulphite sequencing data for the endocardium and endothelium and compare these data to transcriptomic profiles of these cells. We identify a plethora of differentially expressed genes and differentially methylated genomic regions. Through motif analysis we identify the ETS family of transcriptional activators as likely to play a role in the development of the endocardium. In brain, we investigate the role of the CTCF and cohesin DNA binding factors in imprinted gene expression by performing high depth allele-specific ChIP-seq for these two factors. We develop a novel bioinformatics approach for performing allele-specific mapping of next-generation sequencing reads and we compare our results with existing data for mouse liver and embryonic stem cells. We note that embryonic stem cells have fewer unique CTCF binding sites consistent with their undifferentiated profile. We examine CTCF and cohesin binding in the vicinity of imprinted loci and note that CTCF and/or cohesin bind to a subset of imprinted regions, suggesting a heterogeneous mechanism for imprinting. Collectively, our studies examine the role of epigenetics and their interplay with tran-scription in two distinct systems and identify a variable role for these processes in gene expression and development.

Thousands of genetic and epigenetic variants have been identified for many common diseases including cancer through genome-wide association studies (GWAS) and epigenome-wide association studies (EWAS). To advance the complex interpretation of both GWAS and EWAS results, I developed new software tools (FORGE2 and eFORGE) for the analysis and interpretation of GWAS and EWAS data, respectively. Both tools determine the cell type-specific regulatory component of a set of target regions (either GWAS-identified genetic variants or EWAS-identified differentially methylated positions). This is achieved by detecting enrichment of overlap with histone mark peaks or DNase I hypersensitive sites across hundreds of tissues, primary cell types, and cell lines from the ENCODE, Roadmap Epigenomics, and BLUEPRINT projects. Application of both tools to publicly available datasets identified novel disease-relevant cell types for many common diseases, a stem cell-like signature in cancer EWAS, and also demonstrated the ability to detect cell-composition effects for EWAS performed on heterogeneous tissues. To complement these bioinformatics efforts and validate selected variants predicted by FORGE2, eFORGE and additional analyses, I performed conformation capture using 4C-seq to fine-map the 3D context of the genomic regions involved, uncovering novel interactions for autoimmunity-associated variants and IKZF3.

The dominantly held view in evolutionary theory focuses on gradual or punctuated change, primarily via natural selection, as the mechanism by which novel traits arise and evolution occurs. Noticeably absent from this portrayal of evolution is mention of the conservation of general characteristics, such as homologous morphological features or conserved nucleotide sequences, commonly observed across even distantly related groups at both the molecular and organismal levels. This raises at least the following questions: a) How does the evolution of conserved traits fit into an evolutionary theory that emphasizes change? b) What components of an evolving system provide the capacity for adaptation in spite of this apparent conservation of general traits? And c) How do these components affect the evolution of lineages? Here I suggest that heritable traits such as DNA methylation and histone modifications provide one place to look when addressing these questions. Current quantitative and population genetic models reflect the dominant view of evolution described above, and act as the foundation for both formal and informal descriptions and predictions of evolutionary change. Using results from recent work in molecular epigenetics, I consider the evolutionary implications for these traits, and show how current models of evolution fail to accurately capture this influence. In doing so, I also address some of the philosophical implications for how we conceptualize evolution, and what potential changes might be necessary for a more complete theory.
Master of Arts

Breast cancer it is the most common type of cancer and leading cause of cancer mortality among women worldwide. Women who inherit mutations in the breast cancer 1 susceptibility gene (BRCA1) are five times more likely to develop breast cancer than women who do not. However, only ~5-10% of breast cancer cases are due to germline mutations in tumor suppressor genes. There are currently no targeted therapies available triple negative breast cancers (TNBC), which often lack BRCA1 expression. BRCA1 is epigenetically silenced by the activated aryl-hydrocarbon receptor (AhR), suggesting that dietary antagonists of the AhR may inhibit BRCA1 silencing. Genistein is an isoflavone abundant in soy foods and its high consumption levels is thought to underlie the lower prevalence of breast cancer in Asian countries compared to Western countries. The hypothesis of this work is that genistein antagonizes AhR-dependent epigenetic silencing of BRCA1. To test this hypothesis we first determined the capacity of genistein to prevent AhR-dependent silencing of BRCA1 in estrogen receptor-alpha (ERα) expressing cells, with wild-type BRCA1 and inducible AhR (MCF-7). We also determined the effectiveness of genistein in reversing silencing of BRCA1 in ERα-negative cells with hypermethylated BRCA1 and constitutively active AhR (UACC-3199). The effect of genistein on BRCA1 promoter methylation and markers of cell proliferation was also determined in both cell lines.

In light of human-mediated environmental change, a fundamental goal for biologists is to determine which phenotypic characteristics enable some individuals, populations or species to be more adept at coping with such change, while rendering others more vulnerable. Studying ongoing range expansions provide a unique opportunity to address this question by allowing documentation of how novel environments shape phenotypic variation on ecological timescales. At range-edges, individuals are exposed to strong selective pressures and population genetic challenges (e.g. bottlenecks and/or founder effects), which make genetic adaptation difficult. Nevertheless, certain species, such as the house sparrow (Passer domesticus), seem to thrive in their introduced ranges, despite genetic challenges, resulting in a genetic paradox. Increasing evidence suggests that rapid phenotypic differentiation at range-edges may be facilitated by phenotypic plasticity among individuals. Further, a role for epigenetic mechanisms as molecular drivers of such plasticity—particularly in genetically depauperate populations—has recently garnered empirical support across a broad range of taxa. For my dissertation, I investigated the role of epigenetic mechanisms (i.e. DNA methylation) as a potential mediator of range expansion success in vertebrates. Specifically, I proposed that success or failure at range-edges may be underlain by variation in the capacity for epigenetically-mediated plasticity (i.e. epigenetic potential) and used extant literature on an inherently plastic and highly integrated physiological system (i.e. the HPA-axis) to support this hypothesis (Chapter I). I then tested these ideas empirically by examining the relative contribution of genetic and epigenetic variation to immunological variation in Kenyan house sparrows (Chapter II) and explored whether mediators of neural plasticity (i.e. BDNF) and epigenetic potential (i.e. DNA methyltransferases; DNMTs) varied among populations of Senegalese house sparrows, including the potential for covariation among BDNF, DNMTs and corticosterone (CORT) within individuals (Chapter III). Flexibility in the regulation of glucocorticoids (GCs) via the HPA-axis is crucial for survival at range-edges because (i) GCs act as integrators capable of coordinating diverse physiological and/or behavioral responses and (ii) the HPA-axis contains multiple regulatory checkpoints which may help to buffer organisms from maladaptive responses (via redundancy) while simultaneously allowing for the fine-tuning of phenotypic responses to future stressors contingent on current and past experiences. GC regulatory flexibility can be influenced by (and in some cases have an effect on) variation in the capacity for epigenetic mechanisms to regulate environmentally-induced phenotypic changes (i.e. epigenetic potential). DNMTs are capacitators of epigenetic change, thus provide one such example of how variation in epigenetic potential could arise via genetic (e.g. variation in coding regions of DNMT genes) and/or environmental (e.g. developmental programming of DNMT expression) factors. For my first chapter, I conducted a literature review to explore where within the HPA-axis epigenetic potential was most likely to occur and to demonstrate how such variation could promote/constrain range expansion success via its impact on GC regulatory flexibility. Results from the literature search revealed that within the HPA-axis, evidence for epigenetic regulation was highest for receptors, suggesting that variation in epigenetic potential of these targets may be most impactful for variation in GC regulatory flexibility. Using a physiological regulatory network (PRN) framework, I showed how variation in epigenetic potential can modify plasticity of PRN states by altering the regulatory relationships (e.g. connectivity) between HPA elements (e.g. GCs as central hubs) and other physiological/behavioral traits (e.g. subnetworks). As such, I portrayed how genetic forms of epigenetic potential can dictate the upper/lower limits of an individual’s homeostatic range, while environmental forms can act to further titrate GC regulatory flexibility through plasticity of PRN states or stabilization of PRN states. The concept of epigenetic potential in the HPA-axis demonstrates how plasticity at the molecular level can influence plasticity at the whole-organism level, which is likely to be important when coping with novel challenges at range-edges. Among the strongest of selective pressures faced by range-edge populations is exposure to parasites, particularly those with which individuals have little to no evolutionary history. Previous work from our lab on house sparrows in Kenya—site of an ongoing range expansion—revealed that range-edge birds had higher expression of Toll-like receptor 4 (TLR4—a microbial surveillance gene) than birds from the range-core. Moreover, extensive inter-individual variation in genome-wide DNA methylation was found among Kenyan house sparrows, including an inverse relationship between epigenetic diversity and genetic diversity across populations. For my second chapter, I investigated whether these two observations were related, asking whether and how DNA methylation and/or genetic variation within the putative promoter of the TLR4 gene contributed to variation in TLR4 expression. I found that DNA methylation status at CpG1, which varied from only ~73-100%, was a strong predictor of TLR4 expression within individuals. Interestingly, other studies have shown that similar magnitudes of variation in DNA methylation of TLR4 can result in differences in the susceptibility/resistance to bacterial pathogens, thus, it’s plausible that the variation we observed could have functional implications for host defense. I also discovered four genetically linked polymorphisms within the TLR4 promoter that grouped into two general genotypes. We revealed a trend that suggests that genotype differences may influence TLR4 expression, confirmation of which may be possible with increased representation from individuals with the rare genotype. Given that DNA methylation did not vary systematically among populations and evidence for extensive genetic admixture at the Kenyan range-edge, it seems likely that individual-level factors (e.g. genotype, early-life experience, infection history, etc.) may be more predictive of variation in DNA methylation of TLR4 than population-level processes. Coping with novel challenges often requires coordinated adjustments to environmentally-sensitive (i.e. plastic) traits. Findings from my first dissertation chapter, as well as previous research from the Martin lab, revealed that CORT regulation, exploratory behavior and epigenetic mechanisms likely contribute to range expansion success in house sparrows. Within the hippocampus, mediators of neural plasticity such as brain-derived neurotrophic factor (BDNF), play a unique role in the bidirectional regulation of CORT and exploratory behavior, with important implications for hippocampal-dependent learning and memory. Moreover, evidence suggests that the regulatory capacity of CORT and BDNF to influence learning and memory relies heavily on the catalytic capacity of epigenetic modification enzymes—including DNA methyltransferases (DNMTs). For my third chapter, I explored whether previous CORT/behavioral/epigenetic patterns contributed to population-level differences in hippocampal BDNF expression and/or hippocampal expression of DNMTs (mediators of epigenetic potential), including potential covariation among CORT, BDNF and DNMTs within individuals. I collected house sparrows from three populations in Senegal—site of an ongoing range expansion—and measured stressor-induced CORT, hippocampal BDNF, DNMT1 and DNMT3a expression. Given the potential importance of neural plasticity and epigenetic potential for coping with novel challenges, I hypothesized that BDNF and DNMT expression would be highest at the range-edge, while positive covariation would occur between CORT, BDNF and/or DNMT expression within individuals. I found that intermediate levels of CORT resulted in the highest BDNF expression within individuals, suggesting that interactions between CORT and BDNF are likely important for balancing homeostatic and progressive (e.g. cognitive) changes within the hippocampus in response to environmental challenges. I also found that CORT positively covaried with DNMT1 expression in one, but not both, range-edge populations, while the reverse was true at the range-core. These findings suggest that in newly established population, CORT may promote epigenetic potential, allowing for rapid and fine-tuned organism-wide responses to novel stressors, while at the range-core, where stressors are presumably less novel, CORT may inhibit epigenetic potential as a means of diverting resources away from cognitive processes and towards maintaining homeostasis. Altogether, my dissertation has demonstrated how inherently plastic sub-organismal level traits (i.e. molecular, physiological, and neurological) may interact and contribute to range expansion success in an introduced bird. Specifically, my research has not only shown that epigenetic variation can influence an ecologically-relevant trait, but also that variation in the regulatory potential of epigenetic mechanisms can be mediated by intrinsic and extrinsic factors. These studies have expanded our understanding about how epigenetic mechanisms act as regulatory mediators of plasticity at the molecular level and can influence (and be influenced by) variation at multiple phenotypic levels, with implications for whole-organism performance in natural populations. I hope that my work contributes to the field of ecological epigenetics by providing the framework for epigenetic potential as an additional tool for assessing how epigenetic processes contribute to phenotypic outcomes in the face of rapid environmental change.

Epigenetic variability is a new mechanism for the study of human microevolution, because it creates both phenotypic diversity within an individual and within population. This mechanism constitutes an important reservoir for adaptation in response to new stimuli and recent studies have demonstrated that selective pressures shape not only the genetic code but also DNA methylation profiles. The aim of this thesis is the study of the role of DNA methylation changes in human adaptive processes, considering the Italian peninsula and macro-geographical areas. A whole-genome analysis of DNA methylation profile across the Italian penisula identified some genes whose methylation levels differ between individuals of different Italian districts (South, Centre and North of Italy). These genes are involved in nitrogen compound metabolism and genes involved in pathogens response. Considering individuals with different macro-geographical origins (individuals of Asians, European and African ancestry) more significant DMRs (differentially methylated regions) were identified and are located in genes involved in glucoronidation, in immune response as well as in cell comunication processes. A "profile" of each ancestry (African, Asian and European) was described. Moreover a deepen analysis of three candidate genes (KRTCAP3, MAD1L and BRSK2) in a cohort of individuals of different countries (Morocco, Nigeria, China and Philippines) living in Bologna, was performed in order to explore genetic and epigenetic diversity. Moreover this thesis have paved the way for the application of DNA methylation for the study of hystorical remains and in particular for the age-estimation of individuals starting from biological samples (such as teeth or blood). Noteworthy, a mathematical model that considered methylation values of DNA extracted from cementum and pulp of living individuals can estimate chronological age with high accuracy (median absolute difference between age estimated from DNA methylation and chronological age was 1.2 years).

Epigenetic variability is a new mechanism for the study of human microevolution, because it creates both phenotypic diversity within an individual and within population. This mechanism constitutes an important reservoir for adaptation in response to new stimuli and recent studies have demonstrated that selective pressures shape not only the genetic code but also DNA methylation profiles. The aim of this thesis is the study of the role of DNA methylation changes in human adaptive processes, considering the Italian peninsula and macro-geographical areas. A whole-genome analysis of DNA methylation profile across the Italian penisula identified some genes whose methylation levels differ between individuals of different Italian districts (South, Centre and North of Italy). These genes are involved in nitrogen compound metabolism and genes involved in pathogens response. Considering individuals with different macro-geographical origins (individuals of Asians, European and African ancestry) more significant DMRs (differentially methylated regions) were identified and are located in genes involved in glucoronidation, in immune response as well as in cell comunication processes. A "profile" of each ancestry (African, Asian and European) was described. Moreover a deepen analysis of three candidate genes (KRTCAP3, MAD1L and BRSK2) in a cohort of individuals of different countries (Morocco, Nigeria, China and Philippines) living in Bologna, was performed in order to explore genetic and epigenetic diversity. Moreover this thesis have paved the way for the application of DNA methylation for the study of hystorical remains and in particular for the age-estimation of individuals starting from biological samples (such as teeth or blood). Noteworthy, a mathematical model that considered methylation values of DNA extracted from cementum and pulp of living individuals can estimate chronological age with high accuracy (median absolute difference between age estimated from DNA methylation and chronological age was 1.2 years).

Alterazione dell’espressione genica sono alla base di numerose patologie, incluse quelle del sistema cardiovascolare. Meccanismi simili a quelli che regolano l'espressione genica nello sviluppo del cuore sono alla base dei meccanismi che si verificano nella ipertrofia e scompenso cardiaco. Pertanto, studiare i meccanismi regolatori che governano lo sviluppo e l'adattamento del cuore a stimoli fisiopatologici é di particolare importanza. L'epigenoma -la somma di modificazioni chimiche su DNA e proteine istoniche - sta emergendo come un meccanismo di regolazione chiave durante lo sviluppo del cuore e nelle malattie cardiache. Lo scopo della tesi è stato di chiarire il ruolo delle modificazioni epigenetiche, con particolare attenzione alla metilazione del DNA, nella regolazione del programma trascrizionale cardiaco. Pertanto usando un approccio genome-wide abbiamo studiato il ruolo della metilazione e idrossimetilazione del DNA nel cuore. Abbiamo trovato che l’ ipertrofia cardiaca é associata con alterazioni del profilo genomico di metilazione del DNA, e,in particolare, con un incremento di metilazione del DNA sui promotori di geni coinvolti nel rimodellamento metabolico del cuore. Screening dell’espressione di geni coinvolti nella metilazione del DNA ha rivelato che il gene Uhrf1 - che codifica per un cofattore epigenetico assente in tessuti sani e in cellule post-mitotiche - è fortemente riespresso in cardiomiociti ipertrofici. Silenziamento in vivo di Uhrf1 con vettori adeno-associati (AAV9) ha portato a una ridotta la disfunzione cardiaca e mitocondriale in topi sottoposti a sovraccarico pressorio. Infine abbiamo valutato il profilo di idrossimetilazione del DNA di cardiomiociti embrionali, neonatali, adulti e ipertrofici. Questo studio ha definito l’importanza della modulazione dinamica del DNA idrossimetilato nel regolare il programma trascrizionale dei cardiomiociti sia nello sviluppo che nell’ipertrofia.

Mechanical ventilation, a lifesaving intervention for patients with acute respiratory distress syndrome (ARDS), also unfortunately contributes to excessive mechanical stress and impaired lung physiological and structural integrity. We have elsewhere established the pivotal role of increased nicotinamide phosphoribosyltransferase (NAMPT) transcription and secretion as well as its direct binding to the toll-like receptor 4 (TLR4) in the progression of this devastating syndrome; however, regulation of this critical gene in ventilator-induced lung injury (VILI) is not well characterized. On the basis of an emerging role for epigenetics in enrichment of VILI and CpG sites within the NAMPT promoter and 5'UTR, we hypothesized that NAMPT expression and downstream transcriptional events are influenced by epigenetic mechanisms. Concomitantly, excessive mechanical stress of human pulmonary artery endothelial cells or lipopolysaccharide (LPS) treatment led to both reduced DNA methylation levels in the NAMPT promoter and increased gene transcription. Histone deacetylase inhibition by trichostatin A or Sirt-1-silencing RNA attenuates LPS-induced NAMPT expression. Furthermore, recombinant NAMPT administration induced TLR4-dependent global H3K9 hypoacetylation. These studies suggest a complex epigenetic regulatory network of NAMPT in VILI and ARDS and open novel strategies for combating VILI and ARDS.

Dysregulation of placental and fetal epigenetics can affect gene expression patterns, including the parent-of-origin dependent expression in imprinted genes. While defects of imprinted genes have been implicated in some adverse pregnancy outcomes, little is currently known about the role of epigenetics in regulating normal or pathological human pregnancy and development. The objective of this thesis is to provide fundamental DNA methylation profiles of human fetal and placental development so as to offer insights into the etiology of human disease and adverse pregnancy outcomes. Taking advantage of the unbalanced parental genomic constitutions in triploidies, 45 novel imprinted genes were identified by comparing the genome-wide DNA methylation profiles between 10 diandries and 10 digynies. A comparison of DNA methylation profiles between placentas of different gestations and other somatic tissues showed tissue-specific and gestational age-specific DNA methylation changes in many imprinted genes. To gain insight into the genomic pattern of tissue-specific methylation, DNA methylation profile was evaluated in 5 somatic tissues (brain, kidney, lung, muscle and skin) from eight normal second-trimester fetuses. Tissue-specific differentially methylated regions (tDMRs) were identified in 195 loci, suggesting that tissue-specific methylation is established early in the second trimester. Importantly, only 17% of the identified fetal tDMRs were found to maintain this same tissue-specific methylation in adult tissues, implicating an extensive epigenetic reprogramming between fetus and adult. Besides intra-individual differences, there is also substantial DNA methylation variation between individuals. While many sites show a continuous pattern of DNA methylation variation between different placentas, WNT2, TUSC3 and EPHB4 were identified to have epipolymorphisms at their promoter region. The methylation status at the TUSC3 promoter showed an association with preeclampsia, suggesting a role of DNA methylation change in adverse pregnancy outcomes. A further investigation of DNA methylation profiles in 26 placentas from preeclampsia, IUGR and control subjects showed 34 loci were hypomethylated in the early-onset preeclamptic placentas, with TIMP3 having a potential of being a biomarker for the disorder. These results provided comprehensive DNA methylation profiles for both normal and abnormal fetal and placental tissues, which contribute to the biological and clinical aspects of the pathogenesis of fetal and placental disorders.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.
Cataloged from PDF version of thesis. Vita.
Includes bibliographical references.
The importance and significance of a model organism in biological research cannot be overstated. The mouse in particular has been very useful in understanding questions in many areas of research such as developmental biology, cancer biology, neuroscience and genetics. However, even though the methods to make transgenic mice and gene knockin and knockouts have been successful, they are very inefficient, labor intensive and costly. Therefore, in this thesis we developed a novel methodology to rapidly and efficiently modify the mouse genome. Using CRISPR/Cas9, a novel genome-engineering technology developed from bacteria, we were able to genetically modify mouse embryonic stem cells and make mice that carried genetic modification by zygotic injections. Using CRISPR/Cas9 we were able to make mice in as little as three weeks that contained multiple gene knockouts, single nucleotide modifications, GFP and mCherry reporter alleles, epitope-tagged alleles, and conditional alleles. Another interesting area of research in mouse genetics is epigenetic regulation, specifically how DNA methylation regulates development, gene expression, and cell state. Multiple studies have shown that this epigenetic modification plays an important regulatory role in these processes; however, the technology that has existed so far to investigate DNA methylation has only been able to look at snapshots of methylation patterns in fixed cell populations. In this thesis we have developed a novel technology named Reporter of Genomic Methylation (RGM), which allows for the investigation of methylation dynamics at single cell-resolution in vivo. The RGM technology was developed using a minimal synthetic secondary DMR promoter that drives the expression of a florescent protein. Using CRISPR/Cas9 the RGM reporter can be integrated into any genomic locus where it can report on the methylation state of its surroundings. We further show that the RGM reporter activity reflects the methylation state of non-coding regulatory elements such as promoters and enhancers. Furthermore, we show that the RGM technology allows for the dynamics of methylation and demethylation to be observed at these non-coding loci as cells transition between a pluripotent and differentiated state.
by Chikdu Shakti Shivalila.
Ph. D.

Ischaemic stroke rates are expected to rise significantly in the next decades due to an aging population. This increases the demand for new stroke biomarkers for early detection of patients at risk and new targets for treatment. It has been hypothesized that epigenetics may be important in the aetiology of stroke. The study consisted of three types of investigation: analysis of candidate gene polymorphism, candidate gene methylation analysis and epigenome-wide methylation analysis (EWAS) of pooled stroke and control samples. The stroke types studied were large vessel disease (LVD), small vessel disease (SVD) and cardioembolic stroke (CE). DNA from peripheral blood samples was used for EWAS and methylation analysis. Significant increases in rare allele frequency were observed in the EHMT2 and DNMT3B genes for all stroke cases; MBD2, DNMT3B and DNMT3L polymorphisms were associated with LVD. IL10, SOD3, LINE1 and PITX2 were significantly hypomethylated in LVD. IL10 and ALOX15 were hypomethylated in CE compared to controls. Methylation levels of following genes were associated with age (LINE1, IL10, MTHFR, TNFα, and PITX2), gender (SOD3 and LINE1), total cholesterol level (SOD3) and systolic blood pressure (IL10). HDAC9 genetic polymorphism was associated with the MTHFR methylation level. A distinctive methylation pattern for each stroke subtype was found by EWAS. The CE pool was hypomethylated at genome, chromosome and gene level, while LVD and SVD pools had regions with higher and lower methylation levels compared to the controls. GNAS was identified as new candidate gene by EWAS. The results suggested that genetic polymorphism and DNA methylation levels of candidate genes were associated with ischaemic stroke. Stroke subtypes had distinct methylation profiles suggesting differences in underlying aetiology. Variations in methylation levels detected in this study could lead to identification of specific biomarkers. Replication on a large number of subjects is required before final conclusions can be drawn.

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease associated with a wide range of clinical features involving different organs and the prognosis is also highly variable. It is a complex genetic trait in which more than 80 susceptibility loci show robust genetic association with disease risk. Firstly, I preformed eQTL mapping, incorporating co-localization analysis of GWAS in the functional immune cells, in order to identify likely causal genes. The results indicate that eQTLs present in a diverse set of immune cells, encompassing both the innate and adaptive immune responses for more than half of the loci. I then integrated genetics, epigenetics and gene expression to delineate the regulatory map of SLE risk loci across human cells and tissues. Using GARFIELD and GoShifter, I demonstrated that SLE genetic associations displayed a marked enrichment pattern for areas of open chromatin in blood, including B cells, T cells, monocytes, and NK cells. Moreover, I found that a large proportion (66%) of the SLE eQTLs showed to overlap with areas of open chromatin, denoting extensive local coordination of genetic influences on gene expression and epigenetics. SLE patients with renal involvement have more severe clinical outcomes and an increased mortality risk. By calculating the genetic risk score (GRS) using a list of SNPs that are reported to be significantly associated with SLE, I found a significant correlation between GRS and patients with renal involvement – the higher the GRS, the higher probability of getting renal disease. The GRS also correlated inversely with age of SLE onset. As part of the functional study of post-GWAS, I investigated the protein expression of selected SLE-susceptibility gene products, namely Ikaros family members and OX40L, in a range of immune cells, using multi-parameter flow cytometry. The results reveal some cellular specificity in gene expression in disease. In particular, IKZF3 expression on activated regulatory T cell subsets was decreased, while OX40L expression on B cell subsets was increased in SLE. In summary, I combined eQTLs and epigenomes to identify the functional tissues and causal genes. In addition, I measured the expression of OX40L and Ikraos family to help understand the cell effects at protein level. Finally, I found that the genetic risk factors that influence the severity of SLE are through a quantitative way but not qualitative way, suggesting that the GRS approach may become a useful factor in predicting outcome in this clinically heterogeneous disease.

Myelodysplastic syndromes (MDS) are a class of myeloid malignancy characterized by peripheral blood cytopenias and impaired hematopoietic differentiation. Our understanding of the molecular basis of MDS has improved enormously in recent years due to clinical research efforts to characterize the spectrum of acquired mutations found in patients. This work has revealed that mutations in TET2 are common lesions in MDS and other myeloid malignancies. TET2 function has only recently been elucidated: TET proteins convert 5’-methylcytosine (mC) first to 5’-hydroxymethylcytosine (hmC), apparently the first step in an active DNA demethylation program that leads to the replacement of 5-mC with unmodified cytosine. My thesis work focuses on a characterization of TET2 loss on DNA methylation, and on how TET2 mutations impact patient response to treatment with hypomethylating agents. We examined DNA methylation in a matched set of TET2-WT and -mutant MDS samples, and found that loss of TET2 results in global hypermethylation. This global increase is due to gains in intragenic methylation, specifically localized to intron-exon boundaries. We then used clonal TF1 cell lines with CRISPR/Cas9-engineered TET2 mutations to examine global DNA hydroxymethylation. Loss of TET2 results in a global loss of 5-hmC. By aligning our methylation data with hydroxymethylation data from TET2-WT cells, we were able to identify direct TET2 targets. Because changes in mC/hmC with loss of TET2 appeared to localize to intron-exon boundaries, we investigated the effect of aberrant methylation on mRNA splicing in our TF1 cell system. TET2 loss resulted in an overall increase in exon skipping, consistent with published data on the effect of methylation on splicing, and hypermethylated regions were enriched for alternate splicing events. These findings suggest that the alterations in hematopoietic differentiation seen in TET2-mutant models are due to shifts in the expression of different mRNA isoforms rather than wholesale changes in gene expression. Our data show that loss of TET2 function results in region-specific gains in DNA methylation, and that these alterations affect mRNA splicing by promoting exon skipping. Finally, we have found that presence of TET2 mutations are positively associated with response to HMA therapy.
Medical Sciences

Body fluids such as blood are amongst the most important biological evidence recovered from crime scenes. Identification of the donor can be achieved through STR profiling; however, extracting additional information regarding the tissue type or the donor’s physical appearance such as age could prove very useful in police investigations. Firstly, the performance of existing tissue-specific mRNA-based systems was assessed via collaborative exercises. All proposed methods have shown to be highly sensitive; however, issues regarding markers’ specificity, especially for the vaginal detection, were observed. Analysing complex casework samples revealed the need for interpretation guidelines and the use of a scoring system when implementing mRNA profiling in casework. It was understood that developing DNA-based testing would overcome the limitations of existing methods so the main aim of this study was to evaluate the applicability of DNA methylation profiling in forensics. Using three approaches various tissue-specific differentially methylated CpG sites in 18 different loci were evaluated by analysing various forensically relevant body fluids and tissues. As a result, a set of suitable blood- and semen-specific markers were validated using aged and mock casework samples; however, the identification of other tissues like saliva, vaginal fluid and menstrual blood seemed to be challenging. Regarding age prediction, a set of age-associated CpG sites were selected from genome-wide DNA methylation studies and the correlation of their blood methylation levels with age was assessed on two sequencing platforms. Using a subset of 16 CpG sites and taking advantage of artificial neural networks’ capabilities, age could be accurately predicted in 1,156 blood samples (mean error of 4.1 years). The applicability of the proposed prediction model was also tested by means of next generation sequencing. Although further research is required prior to implementing these results in casework, it can be concluded that epigenetics could shed light on the proposed forensic applications.

Histone acetylation is an epigenetic post-translational modification recognised by the bromodomain, a protein module that forms part of multi-component complexes affecting transcription. This interaction plays fundamental cellular roles, and shows association with particular diseases including inflammation and cancer. The biological roles of bromodomains and the progress of ligands developed so far has been summarised in introductory Chapter 1. Work within the group has led to the development of a nanomolar ligand for BRD4, a BET bromodomain implicated in cancer and numerous diseases. Evaluation in an NCI-60 cancer cell screen indicated antiproliferative activity in a variety of cancer types. However, metabolic predictions indicate that this compound is unoptimised for use in vivo. Chapter 2 describes synthesis of a collection of analogues to improve the physical and pharmacokinetic properties of this series of compounds. This work identified compounds with equivalent affinity but greater predicted metabolic stability, as well as more potent derivatives. This research will direct the design of potent and metabolically stable derivatives that can be used in animal models. Chapter 3 describes work carried out towards the development of small molecules to target bromodomains for which there are no known ligands, using the FALZ bromodomain as an initial target. A fragment-based approach has identified a number of compounds that bind to different non-BET bromodomains. These fragments will be a useful starting point for the development of more potent and selective non-BET bromodomain ligands. As well as acetylated lysines, a number of other acylation post-translational modifications occur on lysine residues. Chapter 4 describes work carried out to investigate the interaction of other acylated lysine residues with bromodomains. This work highlighted that other acylated lysines can interact with bromodomains, and selectivity for particular bromodomains can also be achieved. These modified lysines could be incorporated into cognate peptides to improve in vitro peptide displacement assays, aiding the development of small molecular bromodomain probes.

Isogenic cells exhibit a large degree of cell-to-cell heterogeneity in proliferation, with a subpopulation of cells growing at a substantially lower rate. We conducted a high throughput microscopy screen to determine the proliferation distributions for a collection of wild isolates and 1592 single gene deletions in S. cerevisiae. We found that mitochondrial impairment is a primary cause of slow growth within an isogenic population and that high mitochondrial membrane potential is predictive of reduced growth and respiratory deficiency. We showed that respiratory deficiency can be recovered and that temporary respiratory deficiency is a common trait present in many genetic backgrounds. We developed a mathematical model that predicts the dynamics of the respiratory capacity of single cells within a population as a function of mtDNA state. Finally, we showed that growth in the antifungal agent fluconazole enriches for temporarily respiratory deficient cells, suggesting that this phenotype may be a form of bet-hedging in which a slow growing drug-resistant respiratory deficient cell with low mtDNA content can produce progeny with fast growth and fully functioning mitochondria.
Les cèl·lules isogèniques mostren un gran grau d' heterogenietat cel·lular en la seva taxa de proliferació, amb una subpoblació de cèl·lules creixent de manera substancialment lenta. Hem realitzat un assaig de microsopia d’alt rendiment (high-throughput) i hem determinat les distribucions de proliferació d'un conjunt de més de 1590 delecions de gens individuals i de soques wild type de Saccharomyces cerevisiae. Hem trobat que la disfunció mitocondrial és una causa primària del creixement lent dins d'una població isogènica i hem observat que un potencial alt de la membrana mitocondrial ens permet predir la reducció del creixement i la deficiència respiratòria. Hem mostrat que aquesta reducció es pot revertir en determinades circumstàncies i que la deficiència respiratòria temporal és un tret comú en moltes soques. També hem relacionat la dinàmica de la capacitat respiratòria d'una cèl·lula amb l’estat del seu ADN mitocondrial. Finalment, hem mostrat que el creixement en presència d'agents antifúngics augmenta el nombre de cèl·lules amb deficiència temporal respiratòria, suggerint que aquest fenotip pot ser una forma de protecció (subpoblació resistent als fàrmacs amb ADN mitocondrial intacte).

Resistance to antimicrobials is a well-known phenomenon leading to difficulties in the treatment of infectious diseases. The genetic determinants of such resistance are in general well understood: plasmids, transposons, insertion sequences and integrons are the most frequently related genetic elements. The word epigenetics refers to changes in the phenotype or in the gene expression caused by mechanisms other than underlying DNA sequence. In some cases these changes can remain for generations. Serratia marcescens is an enterobacterium characterized by its natural (intrinsic) resistance to most antibiotics. It is also a relevant opportunistic pathogen which has been involved in several pathologies such as urinary tract infections, prostheses infections, cellulitis, bacteremia and others. P. aeruginosa is a Gram-negative bacterium considered one of the major nosocomial pathogens worldwide. It causes several infections such as wound and burn infections as well as respiratory tract infections mostly affecting cystic fibrosis patients. An increasing prevalence of infections caused by multidrug-resistant (MDR) isolates has been reported in many countries and is actually a cause of concern. Both, P. aeruginosa and S. marcescens are relevant nosocomial pathogens. Some of the classic antimicrobials used to treat these pathogens are out-of-date and several of the new drugs available have already become targets for bacterial mechanism of resistance. Environmental conditions exert high pressure not only in the selection of genes encoding resistance to antibiotics or integron fixation in bacterial genomes or plasmids and other mobile elements transmission, but also in the expression of these potentialities that leads to resistance. Thus the role of epigenetics remains to be investigated. In addition it is well known that bacteria causing infections are naturally forming part of biofilms instead the planktonic way of life normally assumed to be in laboratory conditions. The aim of this thesis was the study of unconventional mechanisms of antimicrobial resistance contributing to MDR phenotypes in both S.marcescens and P. aeruginosa. Also the exploration of changes in antimicrobial susceptibilities of Serratia marcescens in the last 50 years by comparing isolates collected between 1945 and 1950, and current isolates. ¬The main conclusions obtained from this study are: 1. The resistome of Serratia marcescens did not change significantly during the antibiotic era. 2. Antibiotic withdrawing tends to restore original susceptible phenotypes, irrespective to the molecular mechanism involved in resistance. 3. None of Serratia strains studied presented integrons, any extended spectrum ß-lactamases. 4. Phenotypically determination of susceptibilities of old strains inactive during the last 60 years have confirmed results obtained by metagenomics i.e. the genes of resistance already existed before antibiotics discovery and use. 5. Multiresistant Pseudomonas aeruginosa harbored class 1 integrons containing a cassette encoding aminoglycoside adenylyltransferase (aadB). 6. Multiresistant Pseudomonas aeruginosa overexpressed MexAB-OprM and MexXY efflux machinery. 7. CCCP use should be avoided in experiments performed with P. aeruginosa and probably in other aerobic bacteria. 8. Meropenem induces the formation of aberrant long rods which can survive, accumulate less antibiotic than normal bacteria, and can revert to normal forma when antibiotic pressure disappears. 9. Colistin, the last therapeutic option to fight against Pseudomonas infections in cystic fibrosis patients, is normally active although cases of resistance have arisen recently. 10. Resistance to colistin seems to be mediated by lipopolysaccharide singular properties. 11. Colistin induces injuries in lipid bilayers, which can be studied by means of planar black lipid bilayer techniques. Preliminary results showed the ability of colistin to induce transient channels in the bilayers, with some dependence to voltage. 12. Recovery of susceptibility to imipenem is slower than acquision of resistance, since the selective advantage conferred by imipenem resistance in the presence of the antimicrobial is strong whereas OprD expression is likely evolutionarily advantageous only under certain and unknown environmental conditions.
Es van explorar els mecanismes no convencionals de resistència als agents antimicrobians que contribueixen a l’aparició de fenotips multiresistents en els bacteris Gram-negatius Serratia marcescens i Pseudomonas aeruginosa. Es van examinar també els canvis en la susceptibilitat als agents antimicrobians en S. marcescens durant els darrers 50 anys, comparant soques aïllades entre els anys 1945-1950 y soques actuals. ¬Les principals conclusions obtingudes d’aquest estudi són les següents: 1. El resistoma de Serratia marcescens no ha canviat significativament des de l’era pre-antibiòtica fins l’actualitat. 2. La retirada dels antibiòtics tendeix a recuperar els fenotips de susceptibilitat originals, independentment del mecanisme molecular implicat en la resistència. 3. Cap de las soques de Serratia estudiades va presentar integrons ni tampoc ß-lactamases d’espectre estès. 4. La determinació fenotípica de les susceptibilitats de las soques “antigues” de Serratia inactives durant 60 anys ha confirmat els resultats obtinguts mitjançant metagenòmica, és a dir, els gens de resistència als antibiòtics ja existien amb anterioritat al descobriment i ús dels antibiòtics. 5. En les soques clíniques de P. aeruginosa multiresistents es va detectar un integró de classe 1 que contenia el cassette gènic aadB, que codifica l’enzim aminoglicòsid 2’-O- adeniltransferasa, que confereix resistència a gentamicina, tobramicina i kanamicina. 6. Les soques multiresistents de P. aeruginosa van sobreexpressar els sistemes de reflux MexAB-OprM i MexXY-OprM. 7. L’ús de l’inhibidor de bombes de reflux CCCP s’hauria d’evitar als experiments realitzats amb P. aeruginosa i probablement amb altres bacteris de metabolisme aeròbic. 8. El meropenem indueix la formació de llargs bacils aberrants capaços de sobreviure en presència de l’antibiòtic. Aquests bacils acumulen menys antibiòtic que els bacteris normals, i poden revertir a la forma normal quan s’elimina la pressió selectiva. 9. La colistina, la última alternativa terapèutica per lluitar contra P. aeruginosa en pacients amb fibrosis quística, és normalment efectiva, encara que recentment han sorgit casos de resistència a aquest agent antimicrobià. 10. La resistència a colistina sembla estar causada per propietats singulars del lipopolisacàrid. 11. La colistina produeix danys en les membranes lipídiques que poden ser estudiats mitjançant tècniques de Black lipid bilayer. Estudis preliminars van mostrar la capacitat de la colistina para induir canals transitoris en les bicapes lipídiques, amb certa dependència de voltatge. 12. La recuperació de la susceptibilitat a l’imipenem en P. aeruginosa és més lenta que l’adquisició de resistència, donat que l’avantatge selectiva conferida per la resistència a l’imipenem en presència de l’agent antimicrobià és forta, mentre que l’expressió d’ OprD és probablement avantatjosa solament sota certes i desconegudes condicions ambientals.
Se exploraron los mecanismos no convencionales de resistencia a agentes antimicrobianos que contribuyen a la aparición de fenotipos multiresistentes en las bacterias Gram-negativas Serratia marcescens y Pseudomonas aeruginosa. Se examinaron también los cambios en la susceptibilidad a los agentes antimicrobianos en S. marcescens en los últimos 50 años, comparando cepas aisladas entre los años 1945-1950 y cepas actuales. ¬Las principales conclusiones obtenidas de este estudio son las siguientes: 1. El resistoma de Serratia marcescens no ha cambiado significativamente desde la era pre-antibiótica hasta la actualidad. 2. La retirada de los antibióticos tiende a recuperar los fenotipos de susceptibilidad originales, independientemente del mecanismo molecular implicado en la resistencia. 3. Ninguna de las cepas de Serratia estudiadas presentó integrones ni tampoco ß-lactamasas de espectro extendido. 4. La determinación fenotípica de las susceptibilidades de las cepas “antiguas” de Serratia inactivas durante 60 años ha confirmado los resultados obtenidos mediante metagenómica, es decir, los genes de resistencia a los antibióticos ya existían con anterioridad al descubrimiento y uso de los antibióticos. 5. En las cepas clínicas de P. aeruginosa multiresistentes se detectó un integrón de clase 1 que contenía el cassette génico aadB, que codifica la enzima aminoglicósido 2’-O- adeniltransferasa, que confiere resistencia a gentamicina, tobramicina y kanamicina. 6. Las cepas multiresistentes de P. aeruginosa sobreexpresaron los sistemas de reflujo MexAB-OprM y MexXY-OprM. 7. El uso del inhibidor de bombas de reflujo CCCP se debería evitar en los experimentos realizados con P. aeruginosa y probablemente con otras bacterias de metabolismo aeróbico. 8. El meropenem induce la formación de largos bacilos aberrantes capaces de sobrevivir en presencia del antibiótico. Estos bacilos acumulan menos antibiótico que las bacterias normales, y pueden revertir a la forma normal cuando se elimina la presión selectiva. 9. La colistina, la última alternativa terapéutica para luchar contra P. aeruginosa en pacientes con fibrosis quística, es normalmente efectiva, aunque recientemente han surgido casos de resistencia a este agente antimicrobiano. 10. La resistencia a colistina parece estar mediada por propiedades singulares del lipopolisacárido. 11. La colistina produce daños en las membranas lipídicas que pueden ser estudiados mediante técnicas de Black lipid bilayer. Estudios preliminares mostraron la capacidad de la colistina para inducir canales transitorios en las bicapas lipídicas, con cierta dependencia de voltaje. 12. La recuperación de la susceptibilidad al imipenem en P. aeruginosa es más lenta que la adquisición de resistencia, dado que la ventaja selectiva conferida por la resistencia al imipenem en presencia del agente antimicrobiano es fuerte, mientras que la expresión de OprD es probablemente ventajosa sólo bajo ciertas y desconocidas condiciones ambientales.

Anal squamous cell carcinoma (SCC) which is strongly associated with human papilloma virus (HPV) infection is a rare cancer but its incidence is increasing throughout the world. Even though it represents just 0.4% of all new cancer cases in the US, the mortality rate is estimated at 14%, which is comparable to both breast and prostate cancer mortality rates. To decrease the high rate of mortality and morbidity of anal cancer there is an enormous need for early detection and prevention strategies. Besides understanding the role of HPV infection, we also need to comprehend the basics of genetics and epigenetics involved in anal cancer progression. With both the highest incidence rate and a lower survival rate among African-American men, we are interested in understanding the relationship of HPV, miRNAs and somatic mutations associated with the African-American population in anal cancers. This was accomplished by (1) identifying and determining HPV genotypes associated with anal condylomas, pre-malignant/dysplastic lesions and malignant anal SCC through type specific genotyping, (2) profiling miRNAs in anal SCC based on gender and type of HPV infection to identify novel biomarkers using Nanostring technology, and (3) by identifying oncogenic mutations associated with anal lesions, transformation and progression using novel next generation sequencing methods. Common HPV genotypes associated with our samples included HPV-11, 16, 6, 32, 35, 51, 58, 59, and 68, of which HPV-32, 51, 59 and 68 are not protected by the current FDA approved nonavalent vaccine. Furthermore, 10 of 800 known human miRNAs were significantly dysregulated in SCC samples; these miRNAs (miR-451a, miR-1185-13p, miR-637, miR-4525a-5p, miR-1275, miR-1303, miR-600, miR-892b, miR-297 and miR-944) target tumor suppressor and oncogenes and potentially play an oncomir role in cancer progression. TP53, PIK3CA, PDGFRA, HRAS, and RET were some of the most frequently found somatic mutations in the sample set and it was observed that the accumulation of mutations begin at the condyloma stage. In conclusion, it was determined that three key factors determine the possible progression of anal cancer and can therefore aid in future development of novel targeted therapy approaches: type of HPV infection, epigenetic factors involving miRNAs, and genetic factors such as ‘driver’ somatic mutations that an individual accumulates over their lifetime.

This long-standing nature versus nurture debate is cited in behavioral and physical expressions of disease dysfunctions, resiliencies, and recovery. Their purposes are noted both in scientific pursuits as well as literature. This discourse has been particularly intense in the fields of psychology, psychiatry, and biology where there is a long history of scientists’ attempts to disprove or discredit others’ intellectual and professional measures. Interestingly, recent advances in the neurosciences and genetic technologies have brought these fields closer together with a new focus – the interactional relationship between nature and nurture – epigenetics.

Cancer is the 2nd most common cause of death in our society and is associated with high morbidity and costs. The word ‘cancer’ amalgamates the complex interplay between cells which have acquired genetic alterations leading to uncontrolled proliferation, i.e. the malignant cells, and genetically ‘normal’ host cells, i.e. stromal cells, vascular cells and inflammatory cells which all acquire modified biological phenotypes in the presence of malignant cells. This community of cells and their secreted proteins defines the tumour microenvironment. Stromal cells in the tumour microenvironment display characteristic biological changes which promote cancer growth. Little is known on the underlying regulatory mechanisms defining this phenotype. Epigenetics describes inheritable changes not encoded by the nucleic acid sequence. Epigenetic regulation has been described to occur in stromal cells in the tumour microenvironment, but little is known about its role on myofibroblasts. In this work I describe how oesophageal cancer derived stromal cells, i.e. cancer associated myofibroblasts (CAMs) accelerate tumour growth in vivo. I observed that CAMs not only affect the local tumour microenvironment but might also accelerate tumour growth at a distant site. I also show how myofibroblasts play an important role in early tumour niche formation in xenograft models and describe their disappearance and replacement by murine stromal cells during tumour progression. Oesophageal CAMs were shown to be epigenetically distinct from matched adjacent tissue myofibroblasts (ATMs). They exhibited a global DNA hypo- methylation compared to ATMs. We identified distinct DNA methylation signatures between oesophageal cancer CAMs and ATMs with the use of the Illumina 450k bead chip methylation array. The methylation array data showed altered methylation signatures of genes implicated Wnt/β-catenin signalling pathway. The transcription factor paired like homeodomain (PITX) 2 and the regulatory protein secreted frizzled like protein (SFRP) 2 both showed altered methylation signatures and expression patterns between oesophageal cancer CAMs and ATMs. I found that upregulation of SFRP2 in myofibroblasts induces angiogenesis and I hypothesise that epigenetic modification regulates myofibroblasts-derived SFRP2 expression which may play an important role in tumour neovascularisation. Based on these findings I conclude that ATMs and CAMs are epigenetically distinct and altered protein expression is at least partially regulated by altered DNA methylation. This work also presents a model for epigenetic modification of tumour stroma cells: exposure of myofibroblasts to the DNA methyl transferase inhibitor 5’Aza-3’deoxycytosine (DAC) lead to a mild decrease of global DNA methylation and induced persistent biological changes in myofibroblasts. These epigenetically modified myofibroblasts induced an accelerated xenograft growth when injected together with oesophageal cancer cells. Based on these experiments I conclude that DAC epigenetically modifies myofibroblasts which induces an activation of normally silenced genes leading to a biologically more active cell.

Genetic analyses of increasing power are now regularly incorporated into wildlife management assessments of threatened and endangered species. Genetic data provide valuable information regarding taxonomy, kinship, and population size and structure. Recently transformed by the advent of powerful technologies that expand our view from single genes to the entire genome, the field of conservation may be on the verge of another revolution with the emergence of epigenetics as a promising means of surveying environmental response in natural populations. In this dissertation, I present my doctoral research upon population genetics and epigenetics of pronghorn (Antilocapra americana). Considerable effort has been undertaken to conserve pronghorn, particularly in the periphery of its range in the southwestern United States and northwestern Mexico. Translocation is regularly used to supplement and re-establish populations of the wide-ranging A. a. americana subspecies while captive breeding has been established for two endangered pronghorn subspecies, A. a. sonoriensis found in Arizona and Sonora, Mexico and A. a. peninsularis of the Baja Peninsula. The primary goal of my doctoral work was to provide pronghorn managers with current estimates of genetic diversity, relatedness, and structure within and between pronghorn subspecies in the desert southwest. My work shows that conservation measures for A. a. sonoriensis have successfully maintained genetic diversity within this endangered subspecies. My estimates of population structure within A. a. americana in northern Arizona reveal the influence of translocation and habitat fragmentation and demonstrate the successful reestablishment of gene flow following the removal of highway fences. With the purpose of guiding future release of captive pronghorn, I explored the subspecies status of pronghorn extirpated from a portion of their range in southern California and northern Baja California. My analyses of museum specimens indicate that the historical range of A. a. peninsularis may have extended as far north as the international border while specimens collected just north of the border share more genetic identity with A. a. sonoriensis. To follow my interests in epigenetics, I also conducted the first ever conservation epigenetics study with Arizona pronghorn. I found that pronghorn are more epigenetically than genetically diverse and this is an indicator that further epigenetic study will reveal the signature of response to environmental factors, as it has with other species demonstrating this pattern.

La plasticité cérébrale existe à l’âge adulte mais est particulièrement forte au cours de périodes critiques de développement pendant lesquelles l’anatomie et la physiologie du système nerveux sont extrêmement sensibles à l’expérience sensorielle. Les mécanismes contrôlant le début et la durée de ces périodes critiques sont multiples. Parmi eux, la régulation épigénétique des gènes de transcription a récemment été mis en évidence comme étant un nouveau mécanisme fondamental impliqué dans la plasticité cérébrale. Ainsi, dans le but d’identifier de nouveaux acteurs sensibles à ces périodes de forte plasticité, nous avons émis l’hypothèse que l’analyse des profils de méthylation d’ADN pourrait être une méthode intéressante. L’étude 1 a révélé que le profil de méthylation de CYP2E1 différait chez le nouveau-né en fonction de la consommation d’antidépresseurs de sa mère pendant la grossesse. L’étude 2 a recherché si des marqueurs de méthylation d’ADN étaient associés à la capacité de discrimination de consonnes chez le foetus. L’étude 3, pour sa part, a suggéré que la régulation du méthylome était dynamique et pouvait être différente d’un mois à un autre. L’étude 4 a identifié certains candidats qui pourraient participer à avoir l’oreille absolue. L’étude 5 a identifié F2RL2, un recepteur de thrombine, comme nouveau candidat impliqué dans la période critique du système visuel. Ensemble, ces résultats ont montré l’importance du rôle de la méthylation d’ADN dans diverses périodes critiques de plasticité
Inadequate sensory input and/or drug exposure during specific developmental stage, called critical periods of plasticity, can affect their timing and can lead to long-term developmental outcomes. The aim of this thesis was to understand the role played by epigenetic mechanisms in this form of plasticity in using a DNA methylation microarray approach. Study 1 showed the impact of selective reuptake inhibitors prenatal exposure in the methylome of neonates. Study 2 investigated whether DNA methylation patterns could be involved in the speech perception critical period. Study 3 demonstrated that methylation patterns for some CpGs could be different within a month suggesting the existence of a dynamic regulation mechanism for them. Study 4 identified CpGs that could be involved in absolute pitch and CpGs that might be associated with valproate exposure in young healthy adults. Study 5 identified a thrombin receptor F2RL2 as a new candidate gene involved in visual cortical plasticity. Together the results of these studies have highlighted the importance of DNA methylation in several critical periods of plasticity

The utilisation of pluripotent cells in regenerative medicine requires the cells to be faithfully maintained in a pluripotent state. One epigenetic feature associated with pluripotency in the embryo is the relative global hypomethylation of cytosine (at CpG dinucleotides, 5meC) that occurs within the nucleus of the inner cell mass and epiblast. It is surprising therefore that the global levels of 5meC in embryonic stem cells (ESCs) propagated under conventional methods are as high as many differentiated somatic cells. Using the newly developed antigen retrieval method that incorporates both acid and trypsin based epitope retrieval processes this project assessed the effects of culture conditions on global levels and localization of 5meC in ESCs. The study shows that the loss of global DNA methylation accompanied increased pluripotency achieved by ESC in 2i media or in suspension culture of EBs. It shows for the first time that this was accompanied by the recruitment of 5meC to heterochromatin. It also showed that global hypermethylation is an early response to conditions that favour the loss of pluripotency, and occurs before evidence of the loss pluripotency or morphological differentiation. This finding provides a new framework for further investigation of the epigenetic requirements of the pluripotent state.

The human GI tract has evolved to simultaneously absorb nutrients and be the frontline in host defence. These seemingly mutually exclusive goals are achieved by a single cell thick epithelial barrier, and a complex resident immune system which lives in symbiosis with the intestinal microflora and is also able to rapidly respond to invading pathogens. An immunological balance is therefore required to permit tolerance to the normal intestinal microflora, but also prevent the dissemination of pathogenic micro-organisms to the rest of the host. Inappropriate immune responses in genetically susceptible individuals are the hallmark of human inflammatory bowel disease (IBD) and are thus targeting effector immune cells and their cytokines remains the mainstay of treatment. However despite vigorous efforts to delineate the genetic contribution to IBD disease susceptibility using large multinational cohorts, the majority of disease heritability remains unknown. Epigenetics describes heritable changes in chromatin that are not conferred by DNA sequence. These incorporate changes to histones, chromatin structure and DNA methylation, which confer changes to gene transcription and thus gene expression and cellular function. Methylbinding proteins (MBD) have the ability to bind to methylated DNA and recruit large chromatin remodeling complexes that underpin a variety of epigenetic modifications. Methyl- CpG-binding domain protein 2 (MBD2) is one such MBD that is required for appropriate innate (dendritic cell) and adaptive (T cell) immune function, though its role has not been investigated in the GI tract. We hypothesized that alterations in chromatin are central to the reprogramming of normal gene expression that occurs in disease states. By defining the phenotype of immune cells in the absence of MBDs we hope to understand the mechanisms of chromatin-dysregulation that lead to immune-mediated diseases such as IBD. We therefore aimed to assess the role of MBD2 in colon immune cells in the steady state and in murine models of GI tract inflammation, thereafter identifying the culprit cell types and genes responsible for any observed changes. We envisaged that investigating heritable, epigenetic changes in gene expression that are inherently more amenable to environmental manipulation than our DNA code, may provide novel insight to a poorly understood mechanism of disease predisposition. In addition identifying the cellular and gene targets of Mbd2 mediated changes to immune homeostasis that may provide exciting and novel approaches to therapeutic modulation of pathological inflammatory responses. In chapter 3 we assessed the expression of Mbd2/MBD2 in the murine/human GI tract. Consistent with existing mouse data, levels of Mbd2 mRNA increased between anatomical divisions of small (duodenum, ileum, terminal ileum) and large intestine (caecum, colon, rectum). In addition MBD2 mRNA was greater in the rectum versus ileum, with active IBD associated with lower rectal MBD2 mRNA compared to quiescent IBD controls. Thus we sought to understand the role of Mbd2 in the colon, where mRNA levels were the highest in the GI tract and where appropriate immune function is central to prevent damaging inflammation. To address these aims required the development of existing methods of cell surface marker expression analysis using flow cytometry techniques to simultaneously identify multiple innate and adaptive immune populations. Using naïve Mbd2 deficient mice (Mbd2-/-) we observed CD11b+ CD103+ DCs were significantly reduced in number in Mbd2 deficiency. To understand the role of Mbd2 in colonic inflammation we employed a mouse model of chemical (DSS) and infectious (T. gondii) colitis comparing Mbd2-/- and littermate controls (WT). Mbd2-/- were extremely sensitive to DSS and T. gondii mediated colonic inflammation, characterized by increased symptom score, weight loss and histological score of tissue inflammation (DSS) and increased antibody specific cytokine responses (T. gondii) in Mbd2 deficient animals. Flow cytometry analysis of colon LP cells in both infectious and chemical colitis revealed significant accumulation of monocytes and neutrophils in Mbd2-/-. Indeed monocytes and neutrophils were the principal myeloid sources of IL-1b and TNF in DSS colitis and the number of IL-1b/TNF+ monocytes/neutrophils was significantly greater in Mbd2-/-. Lastly we employed our colon LP isolation techniques to analyse immune populations in active and quiescent IBD and healthy controls, using endoscopically acquired biopsy samples. Analysis revealed that as in murine colitis, active human IBD is characterized by the accumulation of CD14High monocyte-like cells, with an associated increased ratio of macrophage:monocyte-like cells. In Chapter 4 we sought to understand the cellular sources of Mbd2 that may explain the predisposition of Mbd2-/- to colitis. Firstly we restricted Mbd2 deficiency to haematopoietic cells using grafting Mbd2-/- bone marrow (BM) into lethally irradiated WT mice. These animals treated with DSS displayed increased weight loss, symptom score, neutrophil accumulation and histopathology score compared to mice irradiated and grafted with WT BM. Given the accumulation of monocytes in Mbd2-/- DSS treated mice, and existing literature supporting a pathogenic role in this model, we then investigated the role of Mbd2 in monocyte function. Colon monocytes sorted from Mbd2-/- and WT DSS treated mice displayed similar expression for many pro-inflammatory genes (Il6, Il1a, Il1b, Tnf), but demonstrated significantly dysregulated expression for some others (Regb, Lyz1, Ido1, C4a). To investigate this in a more refined model, we lethally irradiated WT mice and repopulated them with a WT:Mbd2-/- BM mix. This enabled the analysis of WT and Mbd2-/- haematopoietic cells in the same animal. Colon WT and Mbd2-/- monocyte recruitment and cytokine production in DSS treated mixed BM chimeras was equivalent between genotypes suggesting that Mbd2 deficiency in monocytes alone did not explain the increased susceptibility of Mbd2-/- to DSS colitis. We then restricted Mbd2 deficiency to CD11c expressing cells, given the known role for Mbd2 in their function, and for CD11c+ cells in DSS, using a CD11cCreMbd2Fl/Fl system. DSS treated mice with Mbd2 deficient CD11c+ cells demonstrated increased weight loss, symptoms score, histolopathology score, monocyte and neutrophil colon accumulation compared to controls. To further explore the role of Mbd2 in colon CD11c+ cells, macrophage and DCs from DSS treated WT and Mbd2-/- mice were purified and their gene expression analysed. Mbd2-/- versus WT macrophages demonstrated significantly altered expression of both pro- (Il1a, C6, Ido1, Trem2) and antiinflammatory (Tgfbi, Retnla) pathways that we hypothesized was a method for attempted host control of excessive colon damage in Mbd2-/- mice. DC gene expression analysis was hampered by small sample size, but demonstrated a large number of small expression changes, including IL-12/IL-23 (Jak2) and autophagy (Lrrk2) pathways. Lastly levels of costimualtory molecules (CD40/CD80) were increased in Mbd2-/- but not CD11cΔMbd2 colon LP DCs/macrophages suggesting that non-CD11c+ cellular sources of Mbd2 were required to produce increased activation phenotype in these cells. Finally in Chapter 5 we explored the role for Mbd2 in non-haematopoietic cells, namely the colonic epithelium. Here we first developed a novel method for identifying and purifying these cells using flow cytometry. Mbd2 deficient colonic epithelium demonstrated increased expression of activation markers MHC II and LY6A/E in the steady state and in DSS / T. muris mediated colonic inflammation. Indeed FACS purified colon epithelial cells from naive and DSS treated, Mbd2-/- and WT mice revealed conserved dysregulated gene expression independent of inflammation: Both naïve and inflamed Mbd2 deficient epithelium displayed significantly increased expression of genes responsible for antigen processing/presentation (MHC I, MHC II, immunoproteasome) and decreased expression of genes involved in cell-cell adhesion (Cldn1, Cldn4). Lastly we investigated whether the observed differences in Mbd2-/- cell types conferred alterations in the makeup of the intestinal microflora. Interestingly independent of co-housing of Mbd2-/- and WT animals, Mbd2 deficiency consistently predicted the microbial composition, with increased levels of Clostridales and decreased levels of Parabacteroides bacteria. Collectively we have identified CD11c+ cells, monocytes and colon epithelial cells as key cell types for Mbd2 mediated changes in gene expression that affect mucosal immune responses. These data thus identify Mbd2 gene targets within these cell types as exciting new areas for investigation and therapeutic modulation to limit damaging GI tract inflammation.

Epigenetics plays an important role in carcinogenesis with heritable changes in DNA methylation and histone modifications intricately linked to the initiation, promotion, and progression of cancer. Evidence shows that a number of chemical and physical agents can induce epigenetic changes during carcinogenesis. Two such agents, estrogen and ionizing radiation, are generally recognized as being carcinogenic. Yet the epigenetic repercussions of these carcinogens remain relatively unknown. More importantly, the combined effect of these carcinogens has never been addressed in vivo from an epigenetic standpoint. Therefore, we focused on the effect of estrogen and ionizing radiation applied separately or in conjunction. We have found that the exposure to estrogen, either alone or in combination with radiation, induced pronounced morphological alterations, which was paralleled by modifications to the epigenomic landscape in the mammary gland. The results obtained from these rodent models can potentially be extrapolated to humans.
xiv, 190 leaves : ill. (chiefly col.) ; 29 cm

Dosage compensation serves to equalise the levels of X-linked gene products between males and females. In mammals this occurs through the transcriptional silencing of the majority of the genes on one of the two female X chromosomes. The inactive X chromosome (Xi) differs from its active homologue in a number of ways, including the hypoacetylation of core histones, a common property of genetically inactive chromatin. This study has used Xi to explore the functional significance of hypoacetylation and patterns of histone methylation in silent chromatin. Xi was shown to be depleted for di- and tri-methylated lysine 4 of H3, but retained di-methylated lysine 9 of H3. I have examined the temporal order of these modifications as they become established using an in vitro model system for X inactivation; differentiating female embryonic stem cells. The results showed that the loss of tri-methylated lysine 4 of H3 preceded the loss of its di-methylated equivalent, which occurs during a time period of concurrent core histone deacetylation supporting a functional role to the level of lysine methylation. I have used cases of X;autosome translocation to examine how these modifications relate to late replication and transcriptional silencing. Results show that whilst the spread of X inactivation can occur in the absence of both of these properties, histone modifications are a more reliable indicator of the extent of spread of X inactivation than late replication. To explore mechanisms that drive changes in histone modification I have analysed the distribution of histone deacetylases across a region of defined histone deacetylation. The results showed a ubiquitous distribution that did not correlate with acetylated H3 or H4 suggesting that the global association of the Hdacs might serve to provide a rapid return the basal level of histone acetylation following specific targeting events.

The risk of breast cancer transiently increases immediately following pregnancy. Hispanic women have one of the highest rates of postpartum breast cancers of all racial/ethnic minority groups in the US. The biology that underlies this risk window and the effect on the natural history of the disease is unknown. MicroRNAs (miRNAs) are small non-coding RNAs that have been shown to be dysregulated in breast cancer. In this study, we measured the miRNA expression of 56 tumors from a case series of multiparous Hispanic women and assessed the pattern of expression by time since last full-term pregnancy. A data-driven splitting analysis on the pattern of 355 miRNAs separated the case series into two groups: a) an early group representing women diagnosed with breast cancer ≤ 5.2 years postpartum (n=12), and b) a late group representing women diagnosed with breast cancer ≥ 5.3 years postpartum (n=44). We identified 15 miRNAs that are differentially expressed between the early and late postpartum groups; 60% of these miRNAs are encoded on the X chromosome. Ten miRNAs had a two-fold or higher difference in expression; miR-138, miR-660, miR-31, miR-135b, miR-17, miR-454, and miR-934 were overexpressed in the early versus the late group; while miR-892a, miR-199a-5p, and miR-542-5p were under expressed in the early versus the late postpartum group. The DNA methylation of three out of five tested miRNAs (miR-31, miR-135b, and miR-138) was lower in the early versus late postpartum group, and negatively correlated with miRNA expression. Taken together, the results of this study show that miRNAs are differentially expressed and differentially methylated between tumors of the early versus late postpartum, suggesting that potential differences in epigenetic dysfunction may be operative in postpartum breast cancers.

This review will attempt to bring together several current fields of research on the topic of the olfactory system. The neurobiology of the system is fairly well understood in many different species, from insects to humans, which advanced significantly with the characterization of olfactory G-protein coupled receptors. These receptors bind odorant molecules and the sensory neurons carry the signal of that odor to the brain for further identification. Each olfactory sensory neuron only expresses a single type of receptor. The mechanisms for how this is done are not well understood. Epigenetics have been identified as a possible mechanism of inheritance of neurobiological and behavioral changes in response to odor fear-conditioning. Many different scientific disciplines will need to combine knowledge and further studies in order to discover how olfaction has evolved in varying ways across species having many different lifestyles.

Nature has evolved an additional level of genetic regulation by-passing direct changes in genetic code through the means of posttranslational modifications (PTMs) of nucleobases and histone proteins. Acetylation, methylation, phosphorylation, O-GlcNAcylation, ubiquitination, sumoylation, and ADP ribosylation are few common examples of various histone modifications. Identification of these modifications and subsequent access to homogeneously modified histone proteins are key for understanding the functional consequence of these PTMs. In this doctoral thesis, the role of PTMs of histone proteins in epigenetics was investigated with emphasis on understanding the role of O-GlcNAcylation in particular. In the second chapter, the functional consequence of O-GlcNAcylation at histone protein, H2B-Ser112 was explored. Homogeneously GlcNAcylated histones and nucleosomes were synthesized using protein chemical reactions. Mass Spectrometry (MS) based quantitative interaction proteomics revealed a direct interaction between GlcNAcylated nucleosomes and the Facilitates Chromatin Transcription (FACT) complex. Preferential binding of FACT to GlcNAcylated nucleosomes provides a molecular mechanism for FACT-driven transcriptional control. In the third chapter, the physical effect of O-GlcNAcylation on the nucleosome structure is described. Homogeneously GlcNAcylated histone protein, H2A-Thr101 was synthesized. The modified protein was used to reconstitute histone sub-complexes and nucleosomes. Various biophysical studies involving circular dichroism and native mass spectrometry revealed that H2A-T101 GlcNAcylation regulates the stability of the nucleosome structure, suggesting a role in transcriptional activation. In the fourth chapter, we discuss an interesting scenario where two PTMs - O-GlcNAcylation and phosphorylation - can compete for the same modification site of histone protein, H2B-Ser36. The resulting outcome is possibly a competitive antagonism or cross-talk, which can modulate the overall control of chromatin regulation. Using a "Tag-and-modify" approach, modified histone proteins bearing both modifications was synthesized, and was later used for nucleosome reconstitution. Quantitative interaction proteomics experiments with the modified nucleosome revealed key interacting protein partners for both the modifications.

Epigenetic regulation is a mechanism by which heritable changes in gene expression are controlled by chromatin status rather than primary DNA sequence. Changes in chromatin structure affect accessibility of DNA elements to the transcriptional machinery and thus affect transcription activity of the gene. A key event in this process is reversible modification of core histones, which is catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDs, HDAs, or HDACs). In general, histone deacetylation is related to transcriptional gene silencing, whereas acetylation is associated with gene activation.To study the role of histone deacetylase in plant gene regulation and development, we generated constitutive antisense histone deacetylase 1 (CASH) transgenic plants. AtHD1 is a homolog of RPD3 protein, a global transcriptional regulator in yeast. Expression of the antisense AtHD1 caused dramatic reduction in endogenous AtHD1 transcription, resulting in accumulation of acetylated histones. Down-regulation of histone deacetylation caused a variety of growth and developmental abnormalities and ectopic expression of tissue-specific genes. However, changes in genomic DNA methylation were not detected in repetitive DNA sequences in the transgenic plants.We also identified a T-DNA insertion line in exon 2 of AtHD1 gene (athd1-t1), resulting in a null allele at the locus. The complete inhibition of the AtHD1 expression induced growth and developmental defects similar to those of CASH transgenic plants. The phenotypic abnormalities were heritable across the generations in the mutants. When the athd1-t1/athd1-t1 plants were crossed to wild-type plants, the mutant phenotype was corrected in the F1 hybrids, which correlated with the AtHD1 expression and reduction of histone H4 Lys12 acetylation. Microarray analysis was applied to determine genome-wide changes in transcriptional profiles in the athd1-t1 mutant. Approximately 6.7% (1,753) of the genes were differentially expressed in leaves between the wild-type (Ws) and the athd1-t1 mutant, whereas 4.8% (1,263) of the genes were up- or down-regulated in flower buds of the mutant. These affected genes were randomly distributed across five chromosomes of Arabidopsis and represented a wide range of biological functions. Chromatin immunoprecipitation assays indicated that the activation for a subset of genes was directly associated with changes in acetylation profiles.

DNA methylation-dependent gene silencing, catalyzed by DNA methyltransferases (DNMTs) and mediated by methyl binding domain proteins (MBDs) and histone deacetylases (HDACs), is essential for mammalian development, with the nervous system demonstrating particular sensitivity to perturbations. Little is known, however, about the role of DNA methylation in the stage-specific differentiation of neurons. In the olfactory epithelium (OE), where neurogenesis is continuous and the cells demonstrate a laminar organization with a developmental hierarchy, we identified sequential, transitional stages of differentiation likely mediated by different DNMT, MBD and HDAC family members. Biochemically, HDAC1 and HDAC2 associate with repressor complexes recruited by both MBD2 and MeCP2. HDAC1 and HDAC2, however, are divergently expressed in the OE, a pattern that is recapitulated in the brain. Rather than simultaneous inclusion in a complex, therefore, the individual association of HDAC1 or HDAC2 may provide specificity to a repressor complex in different cell types. Furthermore, distinct transitional stages of differentiation are perturbed in the absence of MBD2 or MeCP2. MeCP2 is expressed in the most apical immature olfactory receptor neurons (ORNs), and is up-regulated with neuronal maturation. In the MeCP2 null OE there is a transient delay in ORN maturation and an increase in neurons of an intermediate developmental stage. Two protein variants of MBD2 are expressed in the OE, with MBD2b expressed in cycling progenitor cells and MBD2a in the maturing ORNs. MBD2 null ORNs undergo increased apoptotic cell death. There is also a significant increase in proliferating progenitors in the MBD2 null OE, likely due, at least in part, to feedback from the dying ORNs, acting to up-regulate neurogenesis. Increased cell cycling in the MBD2 null is also observed post-lesion, however, in the absence of feedback back from the ORNs, a phenotype that is recapitulated by an acute inhibition of HDACs with valproic acid. Therefore, disruptions at both transitional stages of ORN differentiation are likely in the MBD2 null mouse. Together, these results provide the first evidence for a sequential recruitment of different MBD proteins and repressor complexes at distinct transitional stages of neuronal differentiation.

During cellular differentiation, gene expression is globally regulated through changes in the epigenome. How a single genome can give rise to a diversity of cell and tissue types remains a complex area of investigation, and here we sought to explore the molecular regulation of gene expression during the differentiation of skeletal muscle cells from committed myogenic progenitors. Using a systematic and integrated analysis of global transcriptional and epigenetic data, we characterized the regulation of gene expression in differentiating myoblasts and found that muscle-specific gene expression is regulated through differential activation of tissue-specific regulatory DNA elements by the myogenic transcription factor MyoD. In addition, the genome-wide localization of MyoD, and the mechanisms underlying its function in transcriptional regulation, varies between myogenic progenitors and differentiating myoblasts. Our study explores the recruitment and function of MyoD at regulatory elements of target genes and additionally describes a novel role for ligand-inducible signaling in the regulation of MyoD function and ultimately in myogenic differentiation.