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Successful human adenovirus (HAdV) replication relies on multiple protein-protein interactions between viral and host proteins. HAdV type 5 (HAdV-5) pVI is a multifaceted protein necessary for viral endosomal escape, activation of viral protease, as well as nuclear shuttling of certain viral proteins. Preliminary mass spectrometry experiments indicated that pVI can bind cellular importins and histone chaperones, of which many are considered novel pVI targets. Here, the binding of the pVI protein to cellular importins was validated, and preliminary studies were done to characterize whether HAdV-5 infection changes importin levels in the infected cells. The validation studies were inconclusive, but it was observed that the accumulation of the importin proteins was not altered in during HAdV-5 infection. In addition, the role of NAP1L1 and NAP1L4, two ubiquitously expressed histone chaperones, was examined during HAdV-5 infection and their effect on HAdV-5 genome structure. Here, it is shown that NAP1L1 knockdown affected viral mRNA and protein as well as hindered viral histone-like protein pVII deposition onto viral DNA during the late stage of infection. In contrast, the NAP1L4 protein was shown to co-localize to viral replication centers (VRCs), and its elimination promoted the pVII protein deposition on virus DNA. These results suggest that NAP1L1 is involved in viral transcription and chromatin assembly, whereas NAP1L4 has anti-viral properties during the assembly process.

Chez S.cerevisiae, NuA4 est un complexe Histone acetyltransferase (HAT) de 1,3 MDa contenant 13 sous unités. Esal, seule HAT essentielle chez la levure, est la sous-unité catalytique qui acétyle les histones H4 et H2A et une des six protéines essentielles du complexe. De plus, six protéines sont présentes dans d'autres complexes de modification (SAGA, Sin3/Rpd3) et de remodelage ATP- dépendant de la chromatine (Ino80 et Swrl) et deux sous-groupes ont été identifiés comme ayant une activité cellulaire indépendante et distincte de NuA4 (PiccoloNuA4 et le trimère Eaf5/7/3). Cette organisation modulaire de NuA4 correspond aux multiples besoins de recrutements et de régulation d'Esal par la cellule lors des événements de réparation, de transcription et de replication de la chromatine. Chez l'humain, le complexe Tip60 est l'orthologue de NuA4 et regroupe les activités de modification de la chromatine de NuA4 et de Swrl. L'organisation spatiale de NuA4 présente donc beaucoup d'intérêt. Au sein du laboratoire du professeur Jacques Côté, je concentre mon travail sur la production et la purification du complexe NuA4. Avec nos échantillons hautement purifiés, les techniques d'analyse par microscopie électronique (EM) et de reconstitution informatique, réalisées par nos collaborateurs Johnathan Chittuluru et Francisco Asturias, permettent, avec une forte résolution, de visualiser le complexe en 3D. Les atouts majeurs associés à cette technique sont aussi de visualiser les interactions du complexe avec un nucléosome et de localiser les sous-unités dans le complexe (par deletion ou étiquetage). Dès lors, ces résultats apportent des indications pertinentes sur NuA4 et ces différents modules qui par recrutement ou interaction directe avec les histones participent à la régulation dynamique de la chromatine.

The epigenome carries dynamic information that controls gene expression and maintains cell identity during both disease and normal development. The inherent plasticity of the epigenome paves new avenues for developing diagnostic and therapeutic tools for human diseases. Microfluidic technology has improved the sensitivity and resolution of epigenomic analysis due to its outstanding ability to manipulate nanoliter-scale liquid volumes. In this thesis, I report three projects focusing on low-input, cell-type-specific and spatially resolved histone modification profiling on microfluidic platforms. First, I applied Microfluidic Oscillatory Washing-based Chromatin Immunoprecipitation followed by sequencing (MOWChIP-seq) to study the effect of culture dimensionality, hypoxia stress and bacterium infection on histone modification landscapes of brain tumor cells. I identified differentially marked regions between different culture conditions. Second, I adapted indexed ChIPmentation and introduced mu-CM, a low-input microfluidic device capable of performing 8 assays in parallel on different histone marks using as few as 20 cells in less than 7 hours. Last, I investigated the spatially resolved epigenome and transcriptome of neuronal and glial cells from coronal sections of adult mouse neocortex. I applied unsupervised clustering to identify distinct spatial patterns in neocortex epigenome and transcriptome that were associated with central nervous system development. I demonstrated that our method is well suited for scarce samples, such as biopsy samples from patients in the context of precision medicine. / Doctor of Philosophy / Epigenetic is the study of alternations in organisms not caused by alternation of the genetic codes. Epigenetic information plays pivotal role during growth, aging and disease. Epigenetic information is dynamic and modifiable, and thus serves as an ideal target for various diagnostic and therapeutic strategies of human diseases. Microfluidics is a technology that manipulates liquids with extremely small volumes in miniaturized devices. Microfluidics has improved the sensitivity and resolution of epigenetic analysis. In this thesis, I report three projects focusing on low-input, cell-type-specific and spatially resolved histone modification profiling on microfluidic platforms. Histone modification is one type of epigenetic information and regulates gene expression. First, we studied the influence of culture condition and bacterium infection on histone modification profile of brain tumor cells. Second, we introduced mu-CM, combining a low-input microfluidic device with indexed ChIPmentation and is capable of performing 8 assays in parallel using as few as 20 cells. Last, we investigated spatial variations in the epigenome and transcriptome across adult mouse neocortex, the outer layer of brain involving in higher-order function, such as cognition. I identified distinct spatial patterns responsible for central nervous system development using machine learning algorithm. Our method is well suited for studying scarce samples, such as cells populations isolated from patients in the context of precision medicine.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by abnormalities in multiple components of the immune system resulting in progressive damage to multiple organs. Current treatments for SLE are often intensive and result in side effects and the potential for continued flares and progression of disease. Histone deacetylase (HDAC) enzymes control multiple cellular functions by removing acetyl groups from lysine residues in various proteins. HDAC inhibitors have been investigated as a potential treatment for SLE with promising results, however selective HDAC6 inhibition (HDAC6i) has become a leading candidate for pharmacologic inhibition to reduce the potential for side effects. We hypothesize that HDAC6i will decrease SLE disease by targeting substrates of HDAC6 in multiple components of immunity and organ systems. NZB/W mice were treated with ACY-738 or ACY-1083, followed by evaluation of multiple disease parameters and mechanisms involved in disease pathogenesis within the kidney, bone marrow, and spleen. Within the kidney, HDAC6i decreased glomerular pathology scores, proteinuria, and IgG and C3 deposition. Within glomerular cells, HDAC6i increased alpha-tubulin acetylation and decreased nuclear NF-κB. Within the spleen, there was a dose-dependent decrease in the frequency of Th17 cells and a mild decrease in the frequency of Treg cells. Concurrently, there were decreased levels of IL-12/IL-23 and minimal decreases in TGF-β in the serum. Within the bone marrow, B cell development through Hardy fractions exhibited accelerated progression through later stages as NZB/W mice aged. This accelerated progression may allow B cells to bypass important regulatory checkpoints in maintaining immune tolerance and contribute to autoimmunity. Treatment with an HDAC6i corrected the aberrant B cell development in the bone marrow and RNAseq analysis unveiled six genes (Cebpb, Ccr9, Spib, Nfil3, Lgals1, and Pou2af1) that may play a role in the aforementioned abnormalities. Overall, these findings show that HDAC6i decreased disease in NZB/W mice by targeting multiple components of the immune response, including glomerular cells, T cell subsets in the spleen, and bone marrow B cells. In conclusion, selective HDAC6i is an excellent candidate for pharmacologic therapy for SLE because it targets multiple immune abnormalities involved in SLE pathogenesis while remaining selective and safe. / Ph. D.

This dissertation presents applications of machine learning and statistical approaches to infer protein-DNA bindings in the presence of epigenetic modifications. Epigenetic modifications are alterations to the DNA resulting in gene expression regulation where the structure of the DNA remains unaltered. It is a heritable and reversible modification and often involves addition or deletion of certain chemical compounds to the DNA. Histone modification is an epigenetic change that involves alteration of the histone proteins – thus changing the chromatin (DNA wound around histone proteins) structure – or addition of methyl-groups to the Cytosine base adjacent to a Guanine base. Epigenetic factors often interfere in gene expression regulation by promoting or inhibiting protein-DNA bindings. Such proteins are known as transcription factors. Transcription is the first step of gene expression where a particular segment of DNA is copied into the messenger-RNA (mRNA). Transcription factors orchestrate gene activity and are crucial for normal cell function in any organism. For example, deletion/mutation of certain transcription factors such as MEF2 have been associated with neurological disorders such as autism and schizophrenia. In this dissertation, different computational pipelines are described that use mathematical models to explain how the protein-DNA bindings are mediated by histone modifications and DNA-methylation affecting different regions of the brain at different stages of development. Multi-layer Markov models, Inhomogeneous Poisson analyses are used on data from brain to show the impact of epigenetic factors on protein-DNA bindings. Such data driven approaches reinforce the importance of epigenetic factors in governing brain cell differentiation into different neuron types, regulation of memory and promotion of normal brain development at the early stages of life. / Doctor of Philosophy / A cell is the basic unit of any living organism. Cells contain nucleus that contains DNA, self replicating material often called the blueprint of life. For sustenance of life, cells must respond to changes in our environment. Gene expression regulation, a process where specific regions of the DNA (genes) are copied into messenger RNA (mRNA) molecules and then translated into proteins, determines the fate of a cell. It is known that various environmental (such as diet, stress, social interaction) and biological factors often indirectly affect gene expression regulation. In this dissertation, we use machine learning approaches to predict how certain biological factors interfere indirectly with gene expression by changing specific properties of DNA. We expect our findings will help in understanding the interplay of these factors on gene expression.

Blast-induced neurotrauma (BINT) is a prevalent brain injury within both military and civilian populations due to current engagement in overseas conflict and ongoing terrorist events worldwide. In the early 2000s, 78% of injuries were attributable to an explosive mechanism during overseas conflicts, which has led to increased incidences of BINT [1a]. Clinical manifestations of BINT include long-term psychological impairments, which are driven by the underlying cellular and molecular sequelae of the injury. Development of effective treatment strategies is limited by the lack of understanding on the cellular and molecular level [2a]. The overall hypothesis of this work is that epigenetic regulatory mechanisms contribute to the progression of the BINT pathology and neurological impairments. Epigenetic mechanisms, including DNA methylation and histone acetylation, are important processes by which cells coordinate neurological and cellular response to environmental stimuli. To date, the role of epigenetics in BINT remains largely unknown. To test this hypothesis, an established rodent model of BINT was employed [3a]. Analysis of DNA methylation, which is involved in memory processes, showed decreased levels one week following injury, which was accompanied by decreased expression of the enzyme responsible for facilitating the addition of methyl groups to DNA. The one week time point also showed dramatic decreases in histone acetylation which correlated to decline in memory. This change was observed in astrocytes and may provide a mechanistic understanding for a hallmark characteristic of the injury. Treatment with a specific enzyme inhibitor was able to mitigate some of the histone acetylation changes. This corresponded with reduced astrocyte activation and an altered behavioral phenotype, which was characterized by high response to novelty. The diagnostic efficacy of epigenetic changes following blast was elucidated by the accumulation of cell-free nucleic acids in cerebrospinal fluid one month after injury. Concentrations of these molecules shows promise in discriminating between injured and non-injured individuals. To date, the diagnostic and therapeutic efforts of BINT have been limited by the lack of a mechanistic understanding of the injury. This work provides novel diagnostic and therapeutic targets. The clinical potential impact on diagnosis and therapeutic intervention has been demonstrated. / Ph. D.

Le génome est organisé sous forme de chromatine afin de contourner la problématique d’espace limité dans le noyau. De plus, cette structure hautement condensé est une barrière physique aux processus cellulaires qui nécessite l’accès à l’information génétique. Les dernières années d'études ont dévoilé des complexes modificateurs de la chromatine comme des acteurs clés dans plusieurs mécanismes de modulation de la chromatine. L'un de ces modificateurs est NuA4, un complexe conservé au cours de l’évolution qui acétyle les histones H2A, H2A.Z et H4. Dans cette thèse, en utilisant Saccharomyces cerevisiae comme organisme modèle, nous avons identifié l'implication de NuA4 dans l'incorporation de H2A.Z et la biosynthèse des voies purines. Dans une seconde partie, nous étudions la participation de NuA4 dans la réponse aux dommages de l'ADN. Plus précisément, nous avons caractérisé la phosphorylation des sous-unités NuA4 dépendante de Mec1/Tel1. L’ensemble de ces travaux, comment NuA4 coordonne différentes activités cellulaires. / Cell genome is packaged into chromatin in order to compensate the limited space within the nucleus. However, this highly condensed structure also presents strong physical barriers for cellular processes using DNA as templates. Recent years of studies have unveiled chromatin modifying complexes as key players in several mechanisms of chromatin modulation. One of these modifiers is NuA4, an evolutionary conserved large multi-subunit histone acetyltransferase complex that acetylates histone H2A, H2A.Z and H4. In this thesis, using Saccharomyces cerevisiae as model system, we identified the implication of NuA4 in global histone variant H2A.Z incorporation and purine biosynthesis pathways. Moreover, we also show previously uncharacterized involvement of NuA4 in DNA damage response pathways through Mec1/ Tel1-dependent phosphorylation events on NuA4 subunits. Further analysis will shed light on detailed mechanisms about how NuA4, as a multifunctional complex, coordinates various cellular activities.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease requiring a genetic predisposition coupled with an environmental trigger in order for initiation of disease. While the exact pathoaetiology has yet to be determined, both B and T cell dysregulation are thought to contribute to disease. Histone deacetylases (HDACs) are a class of enzymes that hydrolyze the lysine bound acetyl group in both histone and non-histone proteins thereby altering protein structure and function. While the use of pan-HDAC inhibitors has proven to be effective for the treatment of a number of acute diseases, they may not be viable as therapeutics for chronic disease due to cytotoxicity and adverse side effects following long term treatment. We sought to determine whether treatment with a class I and II HDAC inhibitor (HDACi) or a specific HDAC6i would be able to ameliorate disease in lupus-prone NZB/W mice. We found that both the class I and II HDACi (ITF2357) and the HDAC6i (ACY-738) were able to decrease SLE markers of disease including splenomegaly, proteinuria, and anti-dsDNA and IgG production in the sera. Treatment with ITF2357 resulted in an increase in the number of immunosuppressive regulatory T (Treg) cells and a decrease in the pro-inflammatory Th17 phenotype. Furthermore, ITF2357 was found to increase Foxp3 acetylation leading to increased Foxp3 stability allowing for differentiation into the Treg phenotype. ACY-738 treatment was able to correct aberrant bone marrow B cell differentiation while also increasing the number of splenic Treg cells in NZB/W mice. These results suggest that HDAC inhibition is able to ameliorate SLE in NZB/W mice by altering aberrant T and B cell differentiation. Additional studies were performed to further examine the expression and function of different HDAC isoforms in immune cells. Due to the ability of HDAC inhibition to decrease markers of SLE disease as well as alter B and T cell development and differentiation, we sought to determine if specific HDAC isoforms are altered in lupus vs non lupus mice in early and late disease states. We determined the level of class IIb HDAC (HDACs 6, 9, and 10) expression in bone marrow B cells, splenic B and T cells, and glomerular cells from early- and late-disease MRL/lpr lupus-prone mice compared to healthy, age-matched C57BL/6 control mice. Expression of HDAC6 and HDAC9 were significantly increased in all of the tissues tested from MRL/lpr mice. Furthermore, both cytoplasmic and nuclear HDAC activity was increased in diseased MRL/lpr mice, and HDAC activity and expression continued to increase as disease progressed. In vitro treatment with ACY-738, a selective HDAC6i, was able to decrease cytoplasmic HDAC activity and inhibit iNOS production. Furthermore, ACY-738 was able to alter apoptosis through increased Bax expression in B cells. Treatment with ACY-738 was also able to inhibit Hsp90 expression and decrease NF-κB nuclear translocation, which are both upregulated during active SLE. Our studies indicate that HDAC activity contributes to SLE pathogenesis and that the use of isoform-selective HDAC inhibitors may be a viable treatment for SLE. / Ph. D.