Browsing by Subject "Epigenetics"

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Adaptive lymphocyte responses in tolerance and autoimmunity
(2022-09) Titcombe, Philip
T cells that encounter self-antigens after exiting the thymus avert autoimmunity through peripheral tolerance. Pathways for this include an unresponsive state known as anergy, clonal deletion, and T regulatory (Treg) cell induction. The transcription factor cues and kinetics that guide distinct peripheral tolerance outcomes remain unclear. In the first part of this dissertation, we report that anergic T cells are epigenetically primed for regulation by the non-classical AP-1 family member BATF. Tolerized BATF-deficient CD4+ T cells were resistant to anergy induction and instead underwent clonal deletion due to pro-apoptotic BIM (Bcl2l11) upregulation. During prolonged antigen exposure, BIM de-repression resulted in fewer PD-1+ conventional T cells as well as loss of peripherally-induced FOXP3+ Treg cells. Simultaneous Batf and Bcl2l11 knockdown meanwhile restored anergic T cell survival and Treg cell maintenance. The data identify the AP-1 nuclear factor BATF as a dominant driver of sustained T cell anergy and illustrate a mechanism for divergent peripheral tolerance fates. When T cell tolerance breaks down, self-reactive T cell clones may provide aberrant help signals to B cells and trigger an autoimmune response. The specificity of an individual B cell is governed by the antibody molecules that are expressed on the cell surface as the B cell receptor (BCR). Despite processes to select against dangerous BCRs with self-reactivity, rare B cell clones still sometimes manage to escape self-tolerance and mediate autoimmune damage. The precise targets and cellular events that initiate B cell autoimmunity are still not known. Currently, our understanding of autoimmune disease pathogenesis relies on inference from the antibody reactivities that can be readily observed in fully established disease. While these general autoantibody profiles have been useful in diagnosis, they provide limited information about actual pathogenesis. This is because the diversity of self-antigen targets and accompanying autoantibody reactivities greatly expand by the time that an autoimmune disease can be detected. In the second part of this dissertation, we used a novel enrichment strategy to investigate self-reactive B cells in rheumatoid arthritis (RA) – an autoimmune disease with well-defined late-stage autoantibody targeting of citrullinated self-antigens. We identified biased immunoglobulin gene usage in RA subject BCRs. Clustering of related immunoglobulin reads revealed that clonal expansion of rare individual B cell lineages occurs in parallel with divergent sequence mutations. Correspondingly, recombinant monoclonal antibodies (mAbs) generated from such BCR lineages demonstrated citrulline–dependent cross-reactivity extending beyond the citrullinated peptides used for B cell capture. A pair of citrullinated autoantigen–specific mAbs with cross-reactive binding profiles also promoted arthritis in mice. Our findings suggest that broad autoantibody specificities in RA arise from a restricted repertoire of evolving citrulline–multispecific B cell clades with pathogenic potential.
Connecting variation in genome structure and chromatin composition in Zea mays
(2021-02) Noshay, Jaclyn
In many crop species there is tremendous intraspecific variation for genome structure due to highly variable transposon insertions. The goal of my thesis research is to provide insight on genetic and epigenetic dynamics and their relationship with functional variation which has the potential to influence variation in regulation and gene expression. ‘Epigenetics’ describes heritable information not solely due to the DNA sequence, whereas ‘genetics’ is heritable information directly related to the DNA sequence. The central question of the chapters presented is to ask, what are the relative contributions of genetic (transposable element insertions) and epigenetic (localized chromatin changes) factors to variation in DNA methylation and gene expression? In order to address this topic, I first present background information on both DNA methylation and epigenetic influence in maize. It is pertinent to understand the many roles and mechanisms of DNA methylation in plant species in order to decipher the contributions to variation. While this DNA methylation has previously been assessed, the ability to tease apart epigenetics from genetics through polymorphism detection on a genome-wide scale is possible only with new technology. These advancements have helped us to understand information found within the maize epigenome which may not be captured by genetic variation and therefore provide additional data for predicting traits and improving the efficiency of plant improvement strategies. I have conducted several research studies to address the question of epigenetic stability in maize. To first address the dynamic between DNA methylation and transposable elements across the genome, I sought to characterize epigenetic patterns associated with TE families and the cause or effect of TE insertion on DNA methylation architecture.Chapter III presents the assessment of natural variation of transposon insertions and the impact on epiallele state. After identifying how these TEs interact with their flanking sequence I further questioned the genomic influence of the TE body in chapter IV. Accessible chromatin data has allowed identification of putative regulatory regions genome-wide and I pursued the question of how novel TE sequence can contribute to the regulatory dynamics of an organism. Through polymorphic TE insertions we were able to assess the influence of these enhancers on nearby gene expression. The final chapter of my thesis seeks to question the stability of the maize methylome. Now focusing on the shared and nonshared sequence between maize genotypes, I was able to analyze epigenetic variation in the presence or absence of sequence variation. A pan-genome study allows for identification of both core (present across all genotypes) and dispensable (variable between genotypes) epigenetic regions. Presence of variable methylation state is indicative of epigenetic patterns not predictable by sequence. The work presented below describes these broad inquiries in further detail working to answer essential questions regarding genetic and epigenetic contributions to maize phenotype.
Development and Cellular Evaluation of Selective N-Terminal BET Bromodomain Inhibitors
(2021-08) Divakaran, Anand
As regulators of transcription, proteins that interpret post-translational modifications to N-terminal histone tails are essential for maintaining cellular homeostasis. When dysregulated, these ‘reader’ proteins become drivers of disease. In the case of bromodomains, which recognize N-acetylated-lysine, developing domain selective inhibitors has been a significant challenge to medicinal chemists. However, recent development of inhibitors with domain-selectivity within the Bromodomain and Extra Terminal (BET) family of bromodomains suggest the tandem BET bromodomains, BD1 and BD2, play differential roles in regulating gene expression. We identified tri-substituted imidazole-based inhibitors that are > 50-fold selective for the N-terminal bromodomains of BET proteins (BET-BD1) and display an unusual mode of domain selectivity via the displacement of conserved structured waters. We subsequently described a structure-based design approach to eliminate off-target p38a kinase affinity, while improving BET selectivity and affinity for BD1 of the bromodomain-containing protein 4 (BRD4). Using these molecules, we observe differential transcriptional effects relative to pan-BD1 inhibitors. Whereas BRD4-BD1 inhibition can reduce the expression of inflammatory cytokines, a weak effect on MYC super-enhancer regulation is only observed at concentrations when both BD1 and BD2 bromodomains are inhibited. Using our structural insights, new chemical-biology strategies were developed to study the role of BRD4 functional modulation through selective bromodomain targeting. Existing strategies to selectively target BRD4 rely on the use of pan-BET inhibitors. In the case of selective BRD4 degraders, pan-BET inhibitors are optimized for BRD4:protein-ubiquitin ligase (E3) ternary complex formation. Here, we present and validate a strategy for selectively targeting BRD4 through bivalent inhibition of both bromodomains, as well as a strategy to degrade BRD4 through its N-terminal bromodomain. Based on our novel BRD4-BD1 selective inhibitors, our unoptimized degrader dBRD4-BD1 induces BRD4 degradation at a half-maximal degradation concentration (DC50) of 0.28 μM, and results in the upregulation of BRD2 and 3. The design of selectivity upfront enables the study of BRD4 biology in the absence of wider BET-inhibition and simplifies design of future BRD4-selective degraders as new E3 recruiting ligands are discovered. Together, these approaches highlight the value of chemical probes that selectively target the unique functions of epigenetic reader domains.
The Effect of Developmental Iron Deficiency on Gene Expression, Tet Proteins, and Dna Hydroxymethylation In the Rodent Brain
(2020-06) Barks, Amanda
Fetal-neonatal iron deficiency (ID) has a lasting negative impact on neurodevelopment, resulting in significant cognitive, socio-emotional, and learning and memory deficits in adulthood, as well as increased risk for neuropsychiatric disease. Given that ID is the most common micronutrient deficiency worldwide, and that pregnant women and young children are disproportionately affected, it presents a significant public health concern. Preclinical models have demonstrated that the developing central nervous system (CNS) is particularly affected by ID, and that the deleterious neurodevelopmental effects and neuropsychiatric risks that follow are associated with dysregulation of CNS gene expression. Dysregulated genes map to signaling pathways and networks critical for neurodevelopment and neuronal function, suggesting that these critical functions are compromised by ID. If developmental ID is corrected by iron repletion within a critical period, correction of neurodevelopmental deficits is possible. However, if iron repletion occurs outside of the critical period, the phenotypic and gene expression changes persist into adulthood despite correction of the deficiency. While changes in gene expression can be understood as the proximate cause of the ID neurocognitive phenotype, it is still unclear what the ultimate cause is. As such, there is a gap in our understanding of how developmental ID establishes and maintains gene expression changes in the CNS. A potential mechanism by which iron could enact these changes is through Ten-Eleven Translocation (TET) enzymes, a family of iron-dependent hydroxylases that generate the epigenetic modification 5-hydroxymethylcytosine (5hmC), or DNA hydroxymethylation. Epigenetic modifications such as DNA hydroxymethylation have the ability to stably influence gene expression throughout the lifespan, and are known to be labile to environmental influences. Of particular relevance, 5hmC is more abundant in the brain than any other tissue, and it increases in enrichment as neurodevelopment progresses, particularly in genes critical for neuronal development and function. The central hypothesis of my thesis research is that dysregulation of TET enzymatic activity and 5hmC by fetal-neonatal ID drives gene expression changes in brain that contribute to the long-term neurocognitive phenotype of developmental ID. To test this hypothesis, the following aims were proposed: 1) Determine the effect of fetal-neonatal ID on TET activity and 5hmC in two regions of the developing rat brain, the hippocampus and the cerebellum, and 2) Determine whether treatment of developmental ID with dietary iron repletion can reverse the changes to this epigenetic system. Completion of these aims contributes to the long-term goal of understanding the cellular and molecular underpinnings of CNS dysfunction and increased neuropsychiatric disease risk following developmental ID. Because the standard therapy of iron repletion incompletely rescues the neurodevelopmental phenotype of ID, there is a need for better therapeutic options. By better understanding the underlying mechanisms of ID-related hippocampal dysfunction, it may be possible to identify new therapeutic targets for more effective treatment of iron deficiency.
Epigenetic regulation of killer immunoglobulin-like receptor Gene expression in developing human natural killer cells.
(2010-05) Cichocki, Frank M.
The immune system is our primary defense against infection and disease. Immune cells need to recognize and efficiently destroy invasive pathogens while, at the same time, exercising tolerance towards normal cells and tissues within the body. Because pathogenic organisms are constantly evolving to evade detection, the immune system must employ multiple recognition strategies to keep pace. Natural killer (NK) cells have evolved a self versus non-self recognition strategy known as “missing self” that is based upon the recognition of self major histocompatibility complex (MHC) molecules by stochastically expressed inhibitory receptors on the surface of NK cells. When MHC expression is downregulated by a virus or cellular transformation event, the dampening signals that balance against NK cell activation are lost due to a lack of inhibitory receptor engagement. This lack of inhibitory signaling, along with the engagement of activating receptors, leads to the elimination of the distressed cell through targeted NK cell-mediated cytotoxicity. The work presented in this manuscript focuses on the transcriptional regulation of a critically important family of human NK cell inhibitory receptors known as killer immunoglobulin-like receptors (KIR). The KIR genes are present within the leukocyte receptor complex on chromosome 19 and are expressed in a variegated, clonally restricted pattern on fully differentiated NK cells. How this pattern of gene expression is regulated during NK cell development is not well understood despite the demonstrated clinical relevance of KIR during hematopoietic cell transplantation to treat patients with leukemia, the influence of the KIR repertoire on the progression of HIV to AIDS, and the importance of KIR during pregnancy. Progress in the elucidation of how KIR genes are regulated has been slow due to the complexity of the KIR locus and the lack of KIR genes in mice, which are much more amenable to genetic manipulation. We have shown that the 5’ upstream regulatory region of each KIR gene contains a previously uncharacterized distal promoter with a functional c-Myc binding site. Stimulation of primary peripheral blood NK cells with IL-15 induces c-Myc binding at the distal promoter, which acts to promote KIR transcription. We also found that the overexpression of c-Myc protein in the NK92 cell line, which lacks surface KIR due to dense methylation of CpG dinucleotides proximal to the transcriptional start site, causes de novo surface KIR expression. Taken together, these results suggest that IL-15 directly promotes KIR transcription by inducing the binding of c-Myc to the distal promoter. We hypothesize that the recruitment of c-Myc and the initiation of active transcription from the distal promoter may also be key steps in the removal of repressive epigenetic marks within KIR promoters during human NK cell development to allow for stable gene expression. In addition to identifying a novel distal promoter, our group has found that the conventional proximal KIR promoter exhibits bi-directional transcriptional activity, meaning that transcription can initiate in either the sense or antisense orientation. We observed a strong inverse correlation between the expression of KIR antisense transcripts and receptor expression on the cell surface, leading to the hypothesis that antisense transcripts directly participate in RNA-mediated transcriptional repression of individual KIR genes. We found that over-expressing full-length antisense transcripts during NK cell development led to an approximately 70% reduction in KIR expression compared to controls. Furthermore, we determined that full-length antisense transcripts are processed into a 28 base RNA with biochemical properties similar to those attributed to members of the PIWI family of small RNAs. We also demonstrate that the 28 base sequence is necessary for antisense transcript-mediated repression of KIR gene expression. This work establishes a direct association between KIR antisense transcription and the initiation of DNA methylation within the KIR promoter. Further elucidation of the mechanisms that regulate KIR expression during NK cell development may provide a basis for new strategies in the design of NK cell-based therapies
Exposures and the Impacts on the Epigenome that Persist from Youth to Adulthood and Intergenerationally
(2022-01) Colwell, Mathia
Environmental exposures have an impact on the epigenome that persists throughout a lifetime and can be transmitted intergenerationally. While a plethora of exposures exist, the mechanisms of epigenetic destruction are unique with each exposure. My research explores two exposures, arsenic and the chemotherapeutic drug 5-aza-2'-deoxycytidine (Decitabine), both of which disrupt different elements of the DNA methylation pathway resulting in persistent epigenetic changes. I first share my findings on the impacts of maternal arsenic exposure, which disrupts the 1-carbon metabolism pathway. I provide evidence that in utero arsenic exposure in mice disrupts epigenetic reprogramming in the developing embryo and primordial germ cells, causing dysregulated methylation and the onset of diseases into adulthood. This work is the first of its kind to show the intergenerational effects of in utero arsenic exposure on adulthood disease phenotypes and the inherited DNA methylation damage in offspring. In my second study I examine the epigenetic damage caused by decitabine exposure, which disrupts the maintenance of DNA methylation in replicating cells. I identify site specific and global methylation damage within the reproductive tracts of mice as a response to a curated chemotherapeutic exposure paradigm. This data highlights the persistent changes of the epigenome in healthy non-target tissues. My findings promote the cautionary application of decitabine as a hypomethylating agent, as it paradoxically alters DNA methylation where the long-term effects on non-target tissues remain unknown. Together my research contributes to the long-term goal of understanding how environmental exposures disrupt the methylation pathway and the lasting consequences of disrupted DNA methylation.
Identification and characterization of DNA methylation variation within maize
(2013-05) Eichten, Steven Richard
DNA methylation is a genetic modification known to repress the activity of transposable elements, repetitive sequences, and in some cases genes. Although DNA methylation is often found in common locations across different individuals, evidence has shown that DNA methylation can vary between individuals at certain loci and can therefore have the opportunity to create a unique regulatory environment for the surrounding sequence. Beyond this, the relationship between DNA methylation state and the genetic content of an individual is still unclear. DNA methylation may act as a downstream effect of certain genetic signals, or it may act independently of genetic state as an epigenetic modification. The goal of this thesis is to profile the DNA methylation landscape across maize (Zea mays) and identify the genomic regions that display differential DNA methylation patterns. These regions of differential methylation are then further studied to understand their stability across generations, their influences on gene expression, as well as their connection to the genetic context they are found. The chapters describe the identification of thousands of differentially methylated regions (DMRs) between maize lines. These DMRs are shown to occur throughout the genome and have high stability across generations. In contrast, few DMRs are found across different tissues within the same genotype. DMRs are shown to often be associated with the local genetic variation. This genetic relationship is highlighted, along with the discovery of a mechanism of genetic control by the spreading of DNA methylation from certain retrotransposable elements. These results indicate that DMRs are present in maize and are created through both epigenetic and genetic means.
Infertility and infertility treatment: childhood cancer and epigenetic risks.
(2010-05) Puumala, Susan Elizabeth
Although childhood cancer is rare, it can have a devastating impact on the children who develop it and their families. Just as in adults, cancer in children is comprised of many disease types. Since the window for exposure is quite limited in childhood cancer, exposures encountered in utero are likely to contribute to carcinogenesis. Thus, etiologic studies have focused on exposures that occur during pregnancy for cancers that arise early in life. Parental infertility and infertility treatment have been hypothesized as possible risk factors for childhood cancer. The link is suspected since many infertile couples may have specific genetic or epigenetic anomalies that could be passed on to their children, which in turn could lead to carcinogenesis. Infertility treatment occurs at or near conception and may persist for several days after conception, making it plausible for treatment to affect early embryo development. In addition, infertility or infertility treatment may alter epigenetic patterns in the developing embryo resulting in an increase risk of childhood cancer. All of these reasons suggest a need to examine infertility and its treatment further. Three related studies are combined in this thesis to examine the potential influence of infertility and infertility treatment on childhood cancer. The first two studies examined the potential link between infertility and its treatment and two specific childhood cancer diagnoses: infant leukemia and germ cell tumors (GCT). As these two diagnoses are quite rare, few previous studies have been performed to examine infertility or its treatment as a possible risk factor. Overall, no significant associations between infertility or its treatment and infant leukemia or childhood GCT were found. However, some notable subgroup associations were found in both studies. In infant leukemia, there was an increased risk of the rare MLL- subtype in children born to women not trying to conceive compared to those trying to conceive for less than one year for all types combined and for acute lymphoblastic leukemia (ALL). In contrast, there was a decreased risk of acute myeloid leukemia (AML) for children born to women who reported use of medication to help them become pregnant. In GCT, there was an increased risk for non-gonadal tumors in females born to women with at least two fetal losses. The final study examined DNA methylation as a potential mechanism by which assisted reproductive technology (ART) might influence the risk of childhood cancer. The data were consistent with no difference in methylation between groups at all loci for lymphocyte samples. Possible differences were found in buccal cell samples for two loci; IGF2 DMR0 and IGF2R. Subgroup analysis indicated potential lower methylation in those whose parents used ART for unexplained infertility. Correlation between lymphocyte and buccal cell samples was low for all loci. The combined results of all three studies indicate no increased risk of infant leukemia, pediatric germ cell tumors, or epigenetic disruptions in specific loci associated with Beckwith-Wiedemann syndrome and certain types of childhood cancer. While an association may still exist for different types of childhood cancer or methylation levels in other loci or tissues, these studies should reassure parents of children conceived through infertility treatment at least somewhat.
Modulating Protein-Protein Interactions Through Peptidomimetics Utilizing Thioether Oxidation States, Noncovalent Interactions & Epigenetic Regulation
(2017-09) Perell, Gabriella
Interactions among proteins play a fundamental role in regulating all biological processes. Such interactions that dictate disease progression are viable regions to target with chemical probes and inhibitors. With an array of interacting sites that vary in size, polarity, and structure, these intransigent chemical targets have been termed undruggable due to the difficulties to target; however, advancements in the area of chemical biology now allow for exploration and progress of this formidable territory. With an estimated 650,000 protein-protein interactions (PPIs) found in the human interactome, probes that can target such interactions are highly sought after due to their significance in disease suppression. In Chapter 2, we explore the importance of a new non-covalent interaction between aromatic π-systems and sulfoxide moieties that contribute to binding energy. With an enrichment of aromatic amino acids at protein interfaces, the utility of this interaction can be exploited at protein interaction sites. We further probe protein interfaces in Chapter 3 by incorporating oxidizable thioether-bridged -helical peptidomimetics. These probes modulate the polarity, structure, stability, affinity, and permeability, thereby allowing the mimetics to disrupt native PPIs. We utilized a specific Protein Observed 19F (PrOF) NMR technique to monitor crosslinked peptide affinity, as well as circular dichroism, tryptic digests, HPLC, fluorescence microscopy, fluorescence polarization, and computational calculations to evaluate biophysical characteristics. We further probe perfluorinated sulfone crosslinks in collaboration with Ratimir Derda in Chapter 3 for interactions with human serum albumin. By using two different methods to incorporate thioether crosslinks, either with an SN2 dibromoalkylation or a thiol-ene reaction as discussed in Chapter 4, we can alter the oxidation states of either one or two sulfur groups within the macrocycle. Chapter 5 is an assessment of a histone variant, H2A.Z for specific acetylation patterns to interact with certain bromodomains, BPTF and PfGCN5. Using PrOF NMR, a ligand observed NMR technique CPMG, and photo-crosslinking, we identify from of a series of various acetylation patterns, that H2A.Z Ack4,11 shows the highest affinity towards the bromodomain BPTF. We further investigate a different bromodomain, CBP in collaboration with Stuart Conway for interactions with five previously identified small molecules. Using PrOF NMR, and fluorescence polarization, we identify affinity towards three bromodomains: CBP, Brd4 and BrdT.
Oxytocin and epigenetic mechanisms in mouse models of empathy and autism
(2016-08) Pisansky, Marc
Empathy, the capacity to infer the emotional state of another, represents a normal process of social cognition that is impaired in several psychiatric diseases. This dissertation research investigates (1) behavioral indices and features of empathy in mice, (2) the role of the neuropeptide oxytocin in mouse empathy, and (3) empathic and other endophenotypes in an epigenetic mouse model of autism spectrum disorders (ASDs). Empathy was modeled using a novel set of behavioral paradigms that measure fear transmitted from a distressed “demonstrator” mouse to an “observer” conspecific. Socially transmitted fear was influenced by the sex of the observer, familiarity to the demonstrator, and distress vocalizations emitted from the demonstrator. Repeated observation of a distressed conspecific elicited a switch from freezing to escape fear behaviors that was specific to familiar conspecifics. Oxytocin – whether exogenously applied or released via chemogenetic stimulation – enhanced socially transmitted fear in unfamiliar mice, whereas oxytocin receptor antagonism reduced this empathic behavior in familiar mice. Genetically-modified mice lacking chromodomain helicase DNA-binding 5 (Chd5), a chromatin remodeler that regulates neurodevelopmental processes, exhibited specific impairments in these empathic behaviors. Chd5-/- mice further displayed characteristic socio-communicative and neophobic behaviors reminiscent of ASD symptomology. Cortical tissue from Chd5-/- mice revealed altered transcriptional expression and neuron-level morphological variations characteristic of ASD etiology and pathophysiology. Thus, this research furthers our understanding of neural and epigenetic contributions to neurodevelopment and social cognition, and provides clues to understanding empathy and treating psychiatric disease.
Pre-clinical Strategies to Overcome Drug-Resistant Multiple Myeloma: Predictive Transcriptomics and Targeting the Myeloma Epigenome
(2018-04) Harding, Taylor
Multiple myeloma remains an incurable hematological malignancy due to the failure of standard-of-care therapies to broadly target a genetically heterogeneous disease and an inability overcome inevitable drug-resistant relapse. This dissertation will address this outstanding problem through two approaches: transcriptomic profiling to predict resistance to proteasome inhibitors and pre-clinical evaluation of epigenetic-targeting therapies to broadly target the myeloma epigenome. First, our goal was to develop a gene expression signature that predicts response specific to proteasome inhibitor (PI) treatment in MM. Using a well-characterized panel of human myeloma cell lines (HMCLs) representing the biological and genetic heterogeneity of MM, we created an in vitro chemosensitivity profile in response to treatment with the four PIs as single-agents. Through gene expression profiling and machine learning-based computational approaches we identified a 42-gene expression signature that could not only distinguish good and poor PI-response in the HMCL panel, but could also be successfully applied to four different clinical datasets on MM patients undergoing PI-based chemotherapy to distinguish between extraordinary (good and poor) outcomes. Our results demonstrate the use of in vitro modeling and machine learning-based approaches to establish predictive biomarkers of response and resistance to drugs that may serve to better direct myeloma patient treatment options. Epigenetic abnormalities are abundantly present in multiple myeloma and accumulating evidence suggests that the histone methyltransferase EZH2 is aberrantly active in MM. We tested the efficacy of EZH2 specific inhibitors in a large panel of human MM cell lines (HMCLs) and found that only a subset of HMCLs demonstrate single agent sensitivity despite ubiquitous global H3K27 demethylation. Pre-treatment with EZH2 inhibitors greatly enhanced the sensitivity of HMCLs to the pan-HDAC inhibitor panobinostat in nearly all cases regardless of single agent EZH2 inhibitor sensitivity. Transcriptomic profiling revealed large-scale transcriptomic alteration by EZH2 inhibition highly enriched for cancer-related pathways. Further analysis demonstrated that combination treatment further perturbed oncogenic pathways and signaling nodes consistent with an antiproliferative/pro-apoptotic state. We conclude that combined inhibition of HDAC and EZH2 inhibitors is a promising therapeutic strategy to broadly target the epigenetic landscape of aggressive MM.
Repeated morphine exposure activates synaptogenesis and other neuroplasticity-related gene networks in the dorsomedial prefrontal cortex of male and female rats
(2023-04) Liu, Shirelle
Opioid abuse is a chronic disorder likely involving stable neuroplastic modifications. While a number of molecules contributing to these changes have been identified, the broader spectrum of genes and gene networks that are affected by repeated opioid administration remain understudied.In this study, Next-Generation RNA-sequencing (RNA-seq) was employed followed by quantitative chromatin immunoprecipitation to investigate changes in gene expression and their regulation in adult male and female rats’ dorsomedial prefrontal cortex (dmPFC) after a regimen of daily injection of morphine (5.0 mg/kg; 10 days). Ingenuity Pathway Analysis (IPA) was used to analyze affected molecular pathways, gene networks, and associated regulatory factors. A complementary behavioral study evaluated the effects of the same morphine injection regimen on locomotor activity, pain sensitivity, and somatic withdrawal signs. Behaviorally, repeated morphine injection induced locomotor hyperactivity and hyperalgesia in both sexes. 90% of differentially expressed genes (DEGs) in morphine-treated rats were upregulated in both males and females, with a 35% overlap between sexes. A substantial number of DEGs play roles in synaptic signaling and neuroplasticity. Chromatin immunoprecipitation revealed enrichment of H3 acetylation, a transcriptionally activating chromatin mark. Although broadly similar, some differences were revealed in the gene ontology networks enriched in females and males. The results cohere with findings from previous studies based on a priori gene selection. This study also reveals novel genes and molecular pathways that are upregulated by repeated morphine exposure, with some common to males and females and others that are sex-specific.
The Role of Anergy in Peripheral Regulatory T cell Generation
(2016-06) Kalekar, Lokesh
The role that anergy, an acquired state of T cell functional unresponsiveness, plays in natural peripheral tolerance remains unclear. In this study, we demonstrate that anergy is selectively induced in fetal antigen-specific maternal CD4+ T cells during pregnancy. A naturally occurring subpopulation of anergic polyclonal CD4+ T cells, enriched in self antigen-specific T cell receptors, is also observed in healthy hosts. Neuropilin-1 expression in anergic conventional CD4+ T cells is associated with thymic regulatory T cell (Treg cell)-related gene hypomethylation, and this correlates with their capacity to differentiate into Foxp3+ Treg cells that suppress immunopathology. Thus, our data suggest that not only is anergy induction important in preventing autoimmunity, but it also generates the precursors for peripheral Treg cell differentiation
The role of the transcriptional corepressor Bcor in embryonic stem cells and mouse development.
(2009-06) Wamstad, Joseph Alan
Mutations in the transcriptional corepressor BCOR results in X-linked MCOPS2 (microphthalmia syndromic 2) disorders, including Oculofaciocardiodental syndrome (OFCD) and MAA2-associated Lenz microphthalmia. BCOR regulates gene expression in association with a Polycomb group like complex of proteins capable of epigenetic modification of chromatin, including the H2A E3 ubiquitin ligases RING1A/B and the histone demethylase FBXL10. To evaluate the role of Bcor in mouse development, we determined the expression pattern of Bcor during embryogenesis and generated multiple mutant alleles of Bcor in mice and ES cells. Bcor is strongly expressed in extraembryonic tissue during mid-gestation and in embryonic structures that correlate with tissues affected in OFCD patients. Bcor loss-of-function in mice results in a strong parent-of-origin effect, most likely indicating a requirement for Bcor in extraembryonic development. In vitro differentiation of embryonic stem (ES) cells harboring Bcor loss-of-function alleles demonstrates a role for Bcor in the regulation of gene expression very early in the differentiation of ES cells into ectoderm, mesoderm and downstream hematopoietic lineages. Chimeric mice generated with these same Bcor loss-of-function ES cells, display reduced contribution to hematopoietic lineages, ocular abnormalities and tail malformations. To circumvent the early embryonic requirement of Bcor in development, we created a conditional allele of Bcor in ES cells and mice that mimics a mutation found in OFCD patients. Ubiquitous inactivation of Bcor in mice results in male lethality prior to embryonic turning and female lethality late in gestation. Additionally, ablation of Bcor in ES cells results in the up regulation of important developmental regulators. In total, these results indicate that Bcor plays an essential role in the differentiation of multiple tissue lineages during early embryonic and extraembryonic development.

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