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Fibroblast to myofibroblast differentiation is essential for the initial healing response, but excessive myofibroblast activation leads to pathological fibrosis. Upon injury, quiescent fibroblasts differentiate into contractile, synthetic myofibroblasts. Initially fibrosis is reparative, but when chronic it contributes to organ dysfunction and failure. Cytosolic calcium (cCa2+) signaling is necessary for myofibroblast differentiation yet the role of mitochondrial calcium (mCa2+) has not been explored. cCa2+ signaling is rapidly integrated into the mitochondrial matrix via the mitochondrial calcium uniporter channel (mtCU), a mechanism theorized to integrate cellular energetic demand with metabolism and respiration. This is intriguing, as it is now appreciated that metabolic reprogramming is required for numerous cellular differentiation programs. The Mcu gene encodes the channel-forming subunit of the mtCU and is required for acute mCa2+ uptake. To examine the contribution of mCa2+ signaling to myofibroblast differentiation, we isolated mouse embryonic fibroblasts (MEFs) from Mcufl/fl mice and deleted Mcu with adenovirus-expressing Cre recombinase. Mcu-/- MEFs exhibited decreased mCa2+ uptake and enhanced cCa2+ transient amplitude when treated with ATP (purinergic, IP3-mediated Ca2+ release). Loss of Mcu promoted myofibroblast differentiation: increased alpha-smooth muscle actin (α-SMA) expression and contractile function (gel retraction), increased myofibroblast gene expression, and decreased proliferation. Further, we found that treatment of wild-type fibroblasts with fibrotic agonists – transforming growth factor beta (TGFβ) and Angiotensin II (AngII) – increased expression of the mtCU gatekeeper, MICU1, to modulate mtCU activity and down-regulate mCa2+ uptake. This suggests that fibrotic agonists signal to acutely inhibit mCa2+ uptake to initiate myofibroblast differentiation. Next, we evaluated the relationship between mCa2+ uptake, metabolism, and myofibroblast differentiation. Fibrotic stimuli increased glycolysis and loss of MCU augmented this phenotype. In addition, genetic activation of glycolysis promoted myofibroblast differentiation, while genetic inhibition of glycolysis ablated the increased differentiation observed in Mcu-/- MEFs. We hypothesize that loss of mCa2+ uptake promoted aerobic glycolysis by reducing the activity of key Ca2+-dependent enzymes such as pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogase (αKGDH). Metabolomic analysis revealed a multitude of changes induced by both TGFβ and the loss of MCU, including increased levels of pyruvate, consistent with inactive PDH. In addition, metabolite quantification showed TGFβ increased alpha-ketoglutarate (αKG) levels ~2-fold and this increase was augmented by loss of Mcu. This is interesting because αKG promotes histone and DNA demethylation by modulating αKG-dependent dioxygenases. Indeed we observed that TGFβ and loss of MCU induced demethylation of histone lysine residues. Further, using ChIP-qPCR we found that TGFβ decreased H3K27me2 marks at the periostin and platelet-derived growth factor receptor alpha loci, which are early and robust indicators fibroblast activation. Finally, to examine the contribution of mCa2+ in cardiac fibrosis, we generated conditional, fibroblast-specific knockout mice by crossbreeding Mcufl/fl mice with Col1a2-CreERT mice (Mcufl/fl x Col1a2-Cre), permitting tamoxifen-inducible gene deletion in adult mice. Loss of Mcu (Mcufl/fl x Col1a2-Cre) increased myofibroblast differentiation and exacerbated fibrosis following myocardial infarction or chronic AngII infusion. In summary, our data linked changes in mCa2+ uptake with metabolic alterations necessary for chromatin modifications and activation of the myofibroblast gene program. While mCa2+ signaling is most well known and studied for its role in cell death, we have demonstrated a previously unrecognized role for modulation of mCa2+ uptake as a key regulator of myofibroblast differentiation. / Biomedical Sciences

Epidemiological data indicate that children conceived in vitro have a greater relative risk of low birth-weight, major and minor birth defects, and rare disorders involving imprinted genes, suggesting that epigenetic changes may be associated with assisted reproduction. DNA methylation and gene expression differences have been found in cord blood and placenta comparing children conceived in vitro using assisted reproductive technology (ART) and children conceived in vivo. The source of these differences (the effect of ART versus underlying infertility) has never been identified in humans. In order to determine what fraction of the DNA methylation and gene expression difference is attributable to the ART procedure and what fraction is attributable to underlying infertility, quasi-transcriptome-wide DNA methylation profiles were compared between (1) in vitro ART children of mothers who are infertile as a result of a physical impediment to fertilization (tubal blockage) or children conceived with the aid of donor oocytes as a group (tubal and donor egg group), and (2) children of parent(s) who have idiopathic infertility (infertility group). Both groups were compared to children conceived in vivo. Our data suggest strongly that many of the DNA methylation and gene expression differences observed between the in vitro and in vivo conceptions are associated with some aspect of ART procedure, rather than underlying infertility. / Molecular Biology and Genetics

DNA hypermethylation is known to contribute to the formation of cancer and this process has been widely studied. However, DNA hypomethylation has received far less attention and the factors controlling the balance between hypo and hypermethylation and its impact on tumorigenesis remains unclear. TET1 is a DNA demethylase that regulates DNA methylation, hydroxymethylation and gene expression. Full length TET1 (TET1FL) has a CXXC domain that binds to un-methylated CG islands (CGIs). This CXXC domain allows TET1 to protect CGIs from aberrant methylation but it also limits its ability to regulate genes outside of CGIs. This dissertation reports a novel isoform of TET1 (TET1ALT) that has a unique transcription start site from an alternate promoter in intron 2, yielding a protein with a unique translation start site. Importantly, TET1ALT lacks the CXXC domain but retains the catalytic domain. TET1ALT is repressed in ESCs but becomes activated in embryonic and adult tissues while TET1FL is ex / Biomedical Sciences

Quaternary ammonium compounds (QACs) are antimicrobial disinfectants commonly used in commercial and household settings. While these compounds have been used for decades, reproductive toxicity has not been thoroughly evaluated. Extensive use of QACs results in ubiquitous human exposure to potentially toxic compounds. Reproductive toxicity of two common QACs, alkyl dimethyl benzyl ammonium chloride (ADBAC) and didecyl dimethyl ammonium chloride (DDAC), was investigated to determine gender-specific toxicity with an emphasis on male reproductive function. Breeding pairs of mice exposed for six months to ADBAC+DDAC exhibited decreases in fertility and fecundity, with fewer pregnancies and decreased numbers of pups over a six month period. Females proceeded through significantly fewer estrus cycles, and both ovulation and implantation rates were reduced. Males exhibited declines in both sperm concentration and motility. Male reproductive toxicity was further assessed in a series of in-vitro and in-vivo experiments. ADBAC+DDAC were cytotoxic to testicular Sertoli cells in culture at concentrations greater than or equal to 0.0005%. Changes in blood-testis-barrier integrity (BTB) were observed at 0.01% ADBAC+DDAC using a two-compartment culture system that measures transepithelial electrical resistance (TER). Sertoli cell cytotoxicity correlated with decreased TER at ADBAC+DDAC concentrations above 0.001%. In-vitro fertilization capacity of epididymal sperm was reduced in males given a 10-day rest period following ADBAC+DDAC exposure. Multigenerational changes in sperm parameters and in mRNA expression of enzymes involved with epigenetic modifications were evaluated across three generations. Sperm concentration and motility were reduced in F0 males exposed directly to ADBAC+DDAC. In F1 males, sperm concentration was increased and motility decreased, while there was no change in the F2 progeny. Genes involved in epigenetic modifications were altered in the exposed F0, with upregulation of two histone acetyltransferases (Hat1 and Kat2b) and downregulation of one lysine-specific demethylase (Kdm6b). F1 and F2 generations were not different from controls except for downregulation of the methyltransferase Dnmt1 in F1 progeny. The reproductive toxicity of ADBAC+DDAC identified in these studies, particularly to the male, compels further investigation into the potential effects that these compounds may have on human reproduction. / 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. 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.

Epigenetics involves heritable changes in an individual’s traits resulting from variations in gene expression without alterations to the DNA sequence. In eukaryotes, genomic DNA is usually folded with histones into chromatin. Post-translational modifications (PTMs) on histones not only play crucial roles in regulating various biological processes, including gene expression, but also store the majority of epigenetic information. A fundamental question in this field is how cells transmit these PTMs to their progeny. Before I began my thesis research, a well-established dogma in the field was that parental histones containing PTMs are symmetrically distributed to daughter DNA strands during DNA replication. These modified histones serve as templates for PTM duplication, thereby restoring the original chromatin states on both daughter strands. Several histone chaperones have been identified as regulators of parental histone segregation. However, their impact on epigenetic inheritance is controversial, which I reasoned is due to the lack of proper systems to examine epigenetic inheritance. This prompted me to use the unique characteristics of fission yeast heterochromatin as a model of epigenetic inheritance. In this organism, heterochromatin formation involves two distinct steps: establishment and inheritance. Reporter systems have been established to allow precise examination of heterochromatin inheritance. However, parental histone segregation pathways have not been characterized in this organism, and their impact on heterochromatin inheritance is unknown. My thesis work investigates the role of parental histone chaperones in regulating parental histone segregation and epigenetic inheritance in fission yeast. It comprises 5 chapters: Chapter 1 introduces epigenetics, with a focus on chromatin-based epigenetic inheritance. It also highlights the unique features of fission yeast heterochromatin that make it an excellent model for studying epigenetic inheritance.Chapter 2 is the focus of my thesis work. I employed inheritance-specific reporters in fission yeast to investigate the roles of three parental histone chaperones on epigenetic inheritance. In addition, in collaboration with Dr. Zhiguo Zhang’s lab, I adapted the Enrichment and Sequencing of Protein-Associated Nascent DNA (eSPAN) method, a recently developed technique designed to quantify the bias of specific proteins at replication forks, to examine parental histone segregation in fission yeast. My analyses demonstrated a critical role for parental histone segregation in epigenetic inheritance. Moreover, I discovered that both the symmetric segregation of parental histones and their density on daughter strands are critical for this process. Chapter 3 uncovers a novel function of a DNA replication protein Mrc1 in regulating epigenetic inheritance, distinct from its established roles in DNA replication checkpoint activation and replication speed control. I demonstrated the critical role of Mrc1 in regulating the symmetrical transfer of parental histone and the proper inheritance of heterochromatin. These results provide essential mechanistic insights into the function of Mrc1. Chapter 4 explores the function of an additional DNA replication protein and histone chaperone, Swi7 (Pol alpha). I have found that mutations in Swi7 lead to defects in parental histone segregation and heterochromatin inheritance, laying a strong foundation to further investigate its mechanism of action. Chapter 5 discusses potential future research directions that can build upon my thesis work.In conclusion, my thesis represents a thorough examination of parental histone chaperones in regulating epigenetic inheritance in fission yeast. By combining innovative genetic assays and advanced methodologies such as eSPAN, I have provided critical insights into the molecular mechanisms of epigenetic inheritance. In addition, the assays that I have developed during my thesis work also pave the way for future studies aimed at elucidating the mechanism of epigenetic inheritance in this important model organism.

No / Spatial genome organization in the cell nucleus plays a crucial role in the control of genome functions. Our knowledge about spatial genome organization is relying on the advances in gene imaging technologies and the biochemical approaches based on the spatial dependent ligation of the genomic regions. Fluorescent in situ hybridization using specific fluorescent DNA and RNA probes in cells and tissues with the spatially preserved nuclear and genome architecture (3D-FISH) provides a powerful tool for the further advancement of our knowledge about genome structure and functions. Here we describe the 3D-FISH protocols allowing for such an analysis in mammalian tissue in situ including in the skin. These protocols include DNA probe amplification and labeling; tissue fixation; preservation and preparation for hybridization; hybridization of the DNA probes with genomic DNA in the tissue; and post-hybridization tissue sample processing.

<p>Mammalian development is orchestrated by global transcriptional changes, which drive cellular differentiation, giving rise to diverse cell types. The mechanisms that mediate the temporal control of early differentiation can be studied using embryonic stem cell (ESCs) and embryonal carcinoma cells (ECCs) as model systems. In these stem cells, differentiation signals induce transcriptional repression of genes that maintain pluripotency (PpG) and activation of genes required for lineage specification. Expression of PpGs is controlled by these genes’ proximal and distal regulatory elements, promoters and enhancers, respectively. Previously published work from our laboratory showed that during differentiation of ESCs, the repression of PpGs is accompanied by enhancer silencing mediated by the Lsd1/Mi2-NuRD-Dnmt3a complex. The enzymes in this complex catalyze histone H3K27Ac deacetylation and H3K4me1/2 demethylation followed by a gain of DNA methylation mediated by the DNA methyltransferase, Dnmt3a. The absence of these chromatin changes at PpG enhancers during ESC differentiation leads to their incomplete repression. In cancer, abnormal expression of PpG is commonly observed. Our studies show that in differentiating F9 embryonal carcinoma cells (F9 ECCs), PpG maintain substantial expression concomitant with an absence of Lsd1-mediated H3K4me1 demethylation at their respective enhancers. The continued presence of H3K4me1 blocks the downstream activity of Dnmt3a, leading to the absence of DNA methylation at these sites. The absence of Lsd1 activity at PpG enhancers establishes a “primed” chromatin state distinguished by the absence of DNA methylation and the presence of H3K4me1. We further established that the activity of Lsd1 in these cells was inhibited by Oct3/4, which was partially repressed post-differentiation. Our data reveal that sustained expression of the pioneer pluripotency factor Oct3/4 disrupts the enhancer silencing mechanism. This generates an aberrant “primed” enhancer state, which is susceptible to activation and supports tumorigenicity. </p> <p>As differentiation proceeds and multiple layers of cells are produced in the early embryo, the inner cells are depleted of O₂, which triggers endothelial cell differentiation. These cells form vascular structures that allow transport of O₂ and nutrients to cells. Using ESC differentiation to endothelial cells as a model system, studies covered in this thesis work elucidated a mechanism by which the transcription factor Vascular endothelial zinc finger 1 (Vezf1) regulates endothelial differentiation and formation of vascular structures. Our data show that Vezf1-deficient ESCs fail to upregulate the expression of pro-angiogenic genes in response to endothelial differentiation induction. This defect was shown to be the result of the elevated expression of the stemness factor Cbp/p300-interacting transactivator 2 (Cited2) at the onset of differentiation. The improper expression of Cited2 sequesters histone acetyltransferase p300 from depositing active histone modifications at the regulatory elements of angiogenesis-specific genes that, in turn, impedes their activation. </p> <p>Besides the discovery of epigenetic mechanisms that regulate gene expression during differentiation, our studies also include development of a sensitive method to identify activities of a specific DNA methyltransferase at genomic regions. In mammals, DNA methylation occurs at the C5 position of cytosine bases. The addition of this chemical modification is catalyzed by a family of enzymes called DNA methyltransferases (Dnmts). Current methodologies, which determine the distribution of Dnmts or DNA methylation levels in genomes, show the combined activity of multiple Dnmts at their target sites. To determine the activity of a particular Dnmt in response to an external stimulus, we developed a method, Transition State Covalent Crosslinking DNA Immunoprecipitation (TSCC-DIP), which traps catalytically active Dnmts at their transition state with the DNA substrate. Our goal is to produce a strategy that would enable the determination of the direct genomic targets of specific Dnmts, creating a valuable tool for studying the dynamic changes in DNA methylation in any biological process.</p>