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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.

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.

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.

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.

Numerous memory studies have demonstrated that epigenetic-mediated transcriptional regulation, such as post-translational histone modifications, is essential to memory formation and maintenance. Moreover, many studies on the mechanisms of memory have focused on fear memories underlying traumatic events, which helps to understand post-traumatic stress disorder (PTSD). However, these mainly focus on individuals directly experiencing the event, while different species have shown the ability to learn fear indirectly by observing a conspecific experiencing a trauma. Thus, our understanding of indirect fear learning (IFL)'s characteristics is very limited. The trimethylation of histone 3 lysine 4 (H3K4me3) is an essential regulator of active gene transcription in cells and has been shown to be critical for memory formation in the hippocampus, a major site of memory storage. However, it is unknown how H3K4me3 is coordinated to target genes during memory formation. Monoubiquitination of histone H2B (H2Bubi) is critical for recruiting H3K4me3 to DNA in a gene-specific manner during memory formation in the hippocampus. Furthermore, there is a great overlap between H3K4me3 and phosphorylation of histone H2A.X at serine 139 (H2A.XpS139), a marker to study DNA double-strand break (DSB) loci. DSB is a critical mechanism for solving DNA-related topological issues during transcription and replication, which could be triggered in some immediate early genes (IEGs) by neuronal activity, such as memory consolidation.Here, we used rat fear conditioning paradigms in combination with quantitative molecular assays, such as chromatin immunoprecipitation (ChIP), and gene editing techniques, like siRNAs and CRISPR-dCas9 manipulations, to study the role of hippocampal 1) H2Bubi and 2) DSBs in contextual fear memory consolidation and reconsolidation, respectively. Additionally, we behaviorally and molecularly characterized IFL and compared it to directly acquired fear subjects. We found that contextual fear conditioning changed the expression of 86 genes in the hippocampus one hour after training. Remarkably, siRNA knockdown of the H2Bubi ligase, Rnf20, abolished changes in all but one of these genes, Per1. Additionally, we report that the loss of Rnf20 in neurons, but not astrocytes, of the hippocampus impaired long-term memory formation. We next found an increase in H2A.XpS139 and H3K4me3 levels in the Npas4, an IEG important for contextual fear memory, promoter region 5 minutes after retrieval. In vivo siRNAmediated knockdown of the enzyme responsible for DSB, topoisomerase II β, prior to retrieval, decreased Npas4 promoter-specific H3K4me3 and H2A.XpS139 levels and impaired long-term memory. Lastly, our data show that both sexes can indirectly acquire fear from either sex using the auditory-cued IFL model. Moreover, our data show that molecular profiles in the amygdala are largely unique to direct or indirect fear learning and vary by sex. Collectively, this data reveals novel roles for histone phosphorylation and ubiquitination in regulating H3K4me3 and memory formation and shows behavioral and molecular differences in each sex based on the way they acquire fear. / Doctor of Philosophy / Changes in epigenetic mechanisms, processes that control the expression of genes without changing the original sequences, play a crucial role in the formation and maintenance of memory. Moreover, many studies on the mechanisms of memory have focused on fear memories underlying traumatic events, helping to understand post-traumatic stress disorder (PTSD). However, these majorly focus on individuals directly experiencing the event, while different species have shown the ability to learn fear indirectly by observing a conspecific experiencing a trauma. Thus, our understanding of indirect fear learning (IFL)'s characteristics is very limited. In the present study, we investigated some of these epigenetic mechanisms called histone modifications. In the brain, histone 3 lysine 4 trimethylation (H3K4me3), a histone modification, is critical for memory formation in the hippocampus, a key area for memory storage. However, it is still not fully understood how H3K4me3 is coordinated during memory formation. Another histone modification called H2B monoubiquitination (H2Bubi) helps recruit H3K4me3 to DNA and so is also crucial for memory formation. Here, using rat models, we found that the expression of 86 genes is changed during memory formation in the hippocampus and that this result is almost entirely dependent on the presence of H2Bubi. We also discovered that H2Bubi is critical for longterm memory formation only in neurons of the hippocampus, and not astrocytes (another type of brain cells). Additionally, there is a connection between H3K4me3 and the phosphorylation of histone H2A.X, another epigenetic mechanism that co-occurs with DNA breaks and may serve as a markerfor studying these breaks. DNA breaks play a vital role during gene expression and could be triggered by neuronal activity during memory formation. We observed an increase in H2A.X phosphorylation and H3K4me3 levels in a memory-permissive gene five minutes after memory retrieval. Inhibition of DSBs, prior to retrieval abolished these changes, and impaired long-term memory. This suggests a critical role for DSBs in memory maintenance and that H2A.X phosphorylation is necessary for the recruitment of H3K4me3 to DNA. Lastly, our data demonstrated that both males and females could learn fear indirectly from either sex by observing them undergoing auditory-cued fear conditioning. Additionally, we found distinct molecular patterns in the amygdala, a brain region involved in fear processing, depending on whether fear was directly or indirectly acquired, and it varied between sexes. Collectively, data from this dissertation reveals novel roles for histone modifications in memory formation and shows behavioral and molecular differences in each sex based on the way they acquire fear.

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. / 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. To date, the development of effective treatment strategies has been unsuccessful. The work described herein seeks to evaluate the specific cellular mechanisms that contribute to the progression of the BINT pathology and neurological impairments. Epigenetic mechanisms are regulatory mechanisms that coordinate DNA modifications and DNA storage to facilitate altered cellular phenotypes. DNA modifications often involves DNA methylation, which is the addition of methyl groups to the DNA backbone. DNA storage is regulated by specific modifications to histone proteins. Histone acetylation is a well-studied modification process that is capable inciting either chromatin relaxation or compaction. Both 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. 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 one of the enzymes 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 memory impairment. This change was observed in astrocytes which are support cells in the brain and are particularly vulnerable to blast-induced aberrations. Drug administration, targeting the histone acetylation equilibrium, successfully mitigated astrocyte activation and altered the behavioral phenotype. Diagnosis of BINT remains clinically challenging. An accumulation of cell-free nucleic acids was observed the in cerebrospinal fluid one month after injury. Concentrations of these molecules shows promise in discriminating between injured and non-injured individuals. These nucleic acids are susceptible to DNA methylation and may provide a platform for studying epigenetic biomarkers. 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 potential clinical impact on diagnosis and therapeutic intervention has been demonstrated.

Tableau d'honneur de la Faculté des études supérieures et postdorales, 2014-2015 / La chromatine est une structure nucléaire composée des histones, autour desquelles l’ADN s’enroule pour être empaqueté dans le noyau. La dynamique de cette structure permet de réguler plusieurs procédés nucléaires, tels que la transcription, la réplication et la réparation de l’ADN. Il existe, entre autre, des complexes de modifications de la chromatine qui collaborent à la régulation de ces différentes fonctions nucléaires. Les acétyltransférases de la famille MYST participent à l’acétylation des queues N-term des histones. Très conservées de la levure à l’humain, elles possèdent des rôles importants dans plusieurs processus cellulaires. Deux des complexes MYST ont été au cœur de mes études doctorales, soit le complexe HBO1 et MOZ/MORF. Mon projet de doctorat avait comme premier objectif de disséquer les différents domaines protéiques présents au sein de ces deux complexes et de caractériser leurs interactions soit avec les autres sous-unités, soit avec la chromatine. Par des analyses biochimiques, nous avons déterminé le mode d’assemblage des complexes MYST. Nous avons également caractérisé leurs différents domaines de reconnaissance de modifications post-traductionnelles des histones, afin de déterminer leur mode de recrutement. Des analyses à l’échelle du génome entier nous ont aussi permis de localiser ces protéines à des loci bien précis. De plus, il nous a été possible de constater l’importance de l’association des protéines INGs sur la fonction suppresseur de tumeur du complexe HBO1-JADE. Suite à une purification de la protéine BRPF1, j’ai pu constater l’association de HBO1 avec cette protéine. Comme deuxième objectif de thèse, j’ai donc eu à caractériser le nouveau complexe HBO1-BRPF1 et à démontrer sa spécificité d’acétylation. En utilisant des essais d’acétylation in vitro combinés à des expériences d’immunoprécipitation de la chromatine, j’ai pu établir un nouveau mode de régulation de l’activité acétyltransférase de la protéine HBO1. Ce mécanisme étonnant démontre un changement de spécificité de l’activité catalytique des MYST en fonction de leur association aux protéines d’échafaudage. Tous ces résultats démontrent donc qu’il est important de considérer l’ensemble des sous-unités des complexes MYST, car elles sont toutes aussi importantes que l’enzyme pour la reconnaissance, la spécificité d’acétylation ainsi que les fonctions cellulaires de ces complexes. / Chromatin is a nuclear structure formed by DNA that is wrapped around histone octamers, allowing for its compaction in the nucleus. This structure is dynamic and regulates many nuclear processes, such as transcription, replication and DNA repair. Among other factors, complexes that modify chromatin collaborate for the regulation of these nuclear functions. The MYST acetyltransferase family participate in the acetylation of histone N-term tails. Highly conserved from yeast to human, they play various roles in many cellular pathways. During my PhD, I have focused on two of these MYST acetyltransferases, HBO1 and MOZ/MORF. The first objective of my project was to dissect the different protein domains comprised within these complexes and define their interactions either with other subunits or with chromatin. Using biochemical experiments, we brought to the forefront the assembly mechanism of the MYST complexes. Additionally, we characterized their chromatin recognition domains, which helped us determine their recruitment mechanism. Genome-wide analysis also gave us the precise localisation of these proteins on many loci. Moreover, we could determine that the association with ING subunits is essential for the tumor suppressor function of these complexes. Following purification of the BRPF1 protein, we could detect binding of the HBO1 protein. Thus, the second objective of my PhD project was to characterize the newly identified HBO1-BRPF1 complex and determine its acetylation specificity. Using in vitro acetylation assays combined with chromatin immunoprecipitation experiments, we unravelled a new regulation mechanism of the HBO1 acetyltransferase activity. This surprising mechanism shows a switch of histone tail acetylation specificity depending of the associated scaffold proteins, an activity previously thought to be intrinsic to the catalytic subunit. These data highlight a new role of the associated scaffold subunits within MYST-ING acetyltransferase complexes in directing the acetylation of specific histone tails. Altogether, these results demonstrate that it is important to consider MYST acetyltransferases as complexes, since their different subunits contribute to chromatin recognition, acetylation specificity and cellular functions.

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. / Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by multiple abnormalities in the immune system resulting in progressive immune-mediated damage to multiple organs. Current treatment regimens are often intensive, result in side effects, and may only provide temporary relief of disease. Histone deacetylase (HDAC) inhibition is currently being investigated as a new treatment modality for SLE with aims for improved efficacy and decreased potential for unwanted side effects. HDAC enzymes remove acetyl groups from multiple proteins (substrates) and subsequently regulate their function. HDAC6 is a specific HDAC enzyme that is of particular interest and are the subject of the following studies. These studies hypothesize that HDAC6 inhibition will decrease SLE by targeting multiple protein targets involved in the immune-mediated pathway of disease initiation and progression. NZB/W mice were utilized as a model of the human disease, and were treated by HDAC6 inhibitors during various stages of disease progression. Long-term treatment initiated early in disease decreases disease as evidenced by decreased renal pathology scores, immune complex deposition in the kidneys, decreased T cell subtypes in the spleen, and decreased inflammatory cytokines. HDAC6 inhibition corrects abnormal B cell development within the bone marrow of NZB/W mice, which is otherwise altered during disease progression. Furthermore, HDAC6 inhibition altered gene expression within the bone marrow, and deep sequencing analysis revealed multiple genes that may be involved in the pathway of disease progression. Overall, HDAC6 inhibition targets multiple pathways involved in SLE disease initiation and progression in various organs including the bone marrow, spleen, and kidneys. Because SLE is a disease that is multi-factorial and effects multiple organs, it would be ideal that a potential drug therapy also targets multiple targets and organ systems while remaining safe to use. Based on these studies, HDAC6 inhibitors are excellent candidates for the treatment of SLE.