Opioid use in pregnancy is the latest in a long list of social issues in the United States that is being addressed with punitive policies that disproportionately affect women of color and their families and do not focus on environmental factors contributing to increasing drug use in pregnancy. Evidence is emerging demonstrating that these punitive policies are not achieving their intended goals. Additionally, these policies continue a trend of pitting maternal and fetal interests against each other. Historically, bioethics has divided conversations on biomedical and environmental ethics. However, recent merging of these fields may facilitate the biopolitical translation of emerging epigenetics research of substance use in pregnancy. This paper reviews the ineffectiveness of punitive policies in reducing drug use in pregnancy and related negative health outcomes for both mothers and infants. It demonstrates how these policies perpetuate racial injustice through existing systemic oppressive structures. Leveraging epigenetics to draw a link between the environment and the maternal-fetal dyad can help members of the scientific community advocate for policies that promote equitable and justice-based public health interventions as well as relieve the maternal fetal conflict. / Urban Bioethics

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No / When cells change functions or activities (such as during differentiation, response to extracellular stimuli, or migration), gene expression undergoes large-scale reprogramming, in cell type- and function-specific manners. Large changes in gene regulation require changes in chromatin architecture, which involve recruitment of chromatin remodeling enzymes and epigenomic modification enzymes to specific genomic loci. Transcription factors must also be accurately assembled at these loci. SATB1 is a genome organizer protein that facilitates these processes, providing a nuclear architectural platform that anchors hundreds of genes, through its interaction with specific genomic sequences; this activity allows expression of all these genes to be regulated in parallel, and enables cells to thereby alter their function. We review and describe future perspectives on SATB1 function in higher-order chromatin structure and gene regulation, and its role in metastasis of breast cancer and other tumor types.

Regulation of chromatin by epigenetic modifications is a fundamental step during the control of gene expression in eukaryotic cells. The participation of different factors including histone chaperones, chromatin remodeling complexes and histone-modifying complexes regulate chromatin dynamics and ensure the correct metabolism of transcripts that need to be exported to the cytoplasm. In these lines, post-translational modifications including monoubiquitination of histone H2B (H2Bub1) and methylation of histone H3 represent a well-studied histone cross-talk which is required for chromatin integrity and transcription. Additionally, the transition from H2Bub1 to its deubiquitinated form by Ubp8, the DUB enzyme from SAGA (Spt-Ada-Gcn5-acetyltranferase) co-activator complex, is fundamental to obtain a correct gene expression. In this work, we demonstrate that the histone chaperone Asf1 and the Ran-binding protein Mog1, participate in maintaining correct levels of H2Bub1. We show that Mog1 is required for the trimethylation of histone H3 at lysine 4 (H3K4me3), hence, acting as a modulator of histone cross-talk. Mog1 role into gene expression is also demonstrated by its physical and genetically interaction with transcription factors including SAGA and COMPASS complexes. Indeed, we demonstrate that Mog1 interacts genetically with TREX-2 subunits and affects mRNA export. During this work, we have also focused in understanding the molecular mechanisms surrounding Spinocerebellar Ataxia Type 7 (SCA7) which is a rare disease caused by amino acid glutamine (Q) repeats within the DUBm component, ATXN7. Therefore, our interest has been directed towards the study of new mechanisms that trigger SCA7 such as the DUB activity from SAGA complex, protein-protein interaction networks and metabolic profiles. / La regulación de la cromatina a través de modificaciones epigenéticas es un paso fundamental durante el control de la expresión génica en células eucariotas. La participación de diferentes factores tales como chaperonas de histonas, complejos de remodelación de la cromatina y complejos modificadores de histonas, regulan la dinámica de la cromatina y garantizan el correcto metabolismo de los transcritos que necesitan ser exportados al citoplasma. De esta forma, las modificaciones postraduccionales que incluyen la monoubicuitinación de la histona H2B (H2Bub1) y la metilación de la histona H3 representan un "cross-talk" de histonas la cual es requerida para la integridad de la cromatina y la transcripción. Además, la transición de H2Bub1 a su forma desubicuitinada por Ubp8, la enzima DUB del complejo co-activador SAGA (Spt-Ada-Gcn5-acetiltranferasa), es necesaria para obtener una expresión génica correcta. En este trabajo, se demuestra que la chaperona de histona Asf1 y la proteína de unión a Ran, Mog1, participan en el mantenimiento de los niveles de H2Bub1. Se demuestra que Mog1 es necesaria para la trimetilación de la histona H3 en la lisina 4 (H3K4me3), actuando como un modulador del "cross-talk" de histonas. El papel de Mog1 en la expresión génica también se demuestra por sus interacciones físicas y genéticas con factores de transcripción, incluyendo los complejos SAGA y COMPASS. Además, demostramos que Mog1 interactúa genéticamente con subunidades de TREX-2 y afecta a la exportación de mRNAs. Durante este trabajo, también nos hemos centrado en la comprensión de los mecanismos moleculares que envuelven a la Ataxia Espinocerebelosa Tipo 7 (SCA7), que es una enfermedad rara causada por una repetición de aminoácidos glutamina (Q) dentro del componente del DUBm, ATXN7. Por lo tanto, nuestro interés se ha dirigido hacia el estudio de nuevos mecanismos que desencadenan SCA7, como la actividad DUB del complejo SAGA, las interacciones proteína-proteína y los perfiles metabólicos. / La regulació de la cromatina a través de modificacions epigenètiques és un pas fonamental durant el control de l'expressió gènica en cèl·lules eucariotes. La participació de diferents factors tals com chaperones d'histones, complexos de remodelació de la cromatina i complexos modificadors d'histones, regulen la dinàmica de la cromatina i garanteixen el correcte metabolisme dels transcrits que necessiten ser exportats al citoplasma. D'aquesta forma, les modificacions postraduccionals que inclouen la monoubicuitinació de la histona H2B (H2Bub1) i la metilació de la histona H3 representen un "cross-talk" d'histones la qual és requerida per a la integritat de la cromatina i la transcripció. A més, la transició d'H2Bub1 a la seua forma desubicuitinada per Ubp8, l'enzim DUB del complex co-activador SAGA (Spt-Ada-Gcn5-acetiltranferasa), és necessària per a obtenir una expressió gènica correcta. En aquest treball, es demostra que la chaperona de histona Asf1 i la proteïna d'unió a Ran, Mog1, participen en el manteniment dels nivells d'H2Bub1. Es demostra que Mog1 és necessària per a la trimetilació de la histona H3 en la lisina 4 (H3K4me3), actuant com un modulador del "cross-talk" d'histones. El paper de Mog1 en l'expressió gènica també es demostra per les seues interaccions físiques i genètiques amb factors de transcripció, incloent els complexos SAGA i COMPASS. A més, vam demostrar que Mog1 interactua genèticament amb subunitats de TREX-2 i afecta a l'exportació de mRNA. Durant aquest treball, també ens hem centrat en la comprensió dels mecanismes moleculars que envolten a l'Atàxia Espinocerebelosa Tipus 7 (SCA7), que és una malaltia rara causada per una repetició d'aminoàcids glutamina (Q) dins del component del DUBm, ATXN7. Per tant, el nostre interès s'ha dirigit cap a l'estudi de nous mecanismes que desencadenen SCA7, com l'activitat DUB del complex SAGA, les interacciones proteïna-proteïna i els perfils metabòlics. / Oliete Calvo, P. (2017). Study of the SAGA deubiquitination module: identification of new modulators and its implication on Spinocerebellar Ataxia Type 7 [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/86155

The current findings indicate that activated protein C (aPC) can induce FOXP3 generation and regulatory T cells (Tregs) through epigenetic modulation and metabolic reprogramming. The current findings show that preincubation of T cells with aPC increased the frequency of Treg markers, CD4+FOXP3+ T cell frequency and suppressive function of T-cells, suggesting an increase of Tregs. The emergence of FOXP3 expression and Treg-like characteristics is linked to alterations in the epigenetic profile of T cells, characterized by a decline in overall DNA methylation, a decrease in the repressive histone marks H3K27me3 , and a reduced methylation of the FOXP3 promoter region. In addition, the induction of Tregs by aPC is accompanied by changes in mitochondrial metabolism. T lymphocytes that were preincubated with aPC exhibit a decline in mitochondrial respiration, a decrease in mitochondrial membrane potential, and a transition towards metabolic quiescence. The metabolic alterations are associated with a reduction of crucial metabolites, specifically α-ketoglutarate (αKG) and glutamine, that are known to regulate T cell function and epigenetic regulation. Reversal of the aPC-induced Treg-like phenotype and associated altered mitochondrial metabolism can be achieved through the supplementation of exogenous αKG or glutamine, supporting a functional interaction of these reduced metabolites and altered T-cell function. The current results show that chronically elevated levels of aPC in transgenic APChigh mice lead to a higher incidence of CD4+FOXP3+ Tregs in the spleen without alterations in thymic Tregs (tTregs), thereby indicating that aPC facilitates the development of Tregs in peripheral lymphoid organs but does not influence primary T-cell development. T cells derived from APChigh mice display a decrease in mitochondrial metabolism, which is consistent with the findings observed in vitro. Supplementation of αKG in cultured T cells derived from APChigh mice restores mitochondrial function and decreases the frequency of CD4+FOXP3+ Tregs. These findings support a model in which aPC alters T-cell metabolism, possibly by reducing expression of glucose (GLUT1) and glutamine (ASCT2 and SNAT1) transporters and thus the availability of metabolic substrates in T-cells. The reduced availability of these substates alters epigentic gene-expression and favors Treg development. In summary, the findings indicate that activated protein C (aPC) promotes the induction of regulatory T cells (Tregs) via metabolic modulation and altered epigenetic gene-expression. The results provide new insights into the long-lasting effects of aPC and indicate the possibility of aPC as a therapeutic target for regulating immune responses and fostering immune tolerance in diverse pathologies. Additional research is necessary to investigate the potential therapeutic applications.

<p dir="ltr">Across diverse organisms and tissues, aging cells undergo extensive rewiring on an epigenetic and transcriptomic level, leading to widespread changes in rhythmic gene expression. Rhythmic gene expression is dictated by the circadian rhythm, which is synchronized to the external environment through the detection of light by the eye. The photosensitive tissue of the eye, the retina, exhibits considerable age-dependent transcriptomic and epigenetic alterations. During aging, the prevalence of ocular disease increases along with a decline in visual function overall, predisposing older individuals to circadian rhythm desynchronization, which is associated with a host of pathologies including neurodegenerative disease and cancer. Despite links between the health of the eye during aging and circadian rhythm dysfunction, a cohesive understanding of the molecular underpinnings that tie these together has not been reached.</p><p dir="ltr">To first understand the transcriptional mechanisms that are necessary to maintain photoreceptor viability and function during aging, we performed a targeted photoreceptor-specific RNAi screen in <i>Drosophila </i>to identify transcriptional regulators necessary for protection against premature, age-dependent retinal degeneration. Using RNAi lines targeting transcription factors, chromatin remodelers, and histone modifiers, we identified 18 targets necessary for protection against premature and progressive retinal degeneration. These targets were enriched for factors involved in the regulation of RNA polymerase II (Pol II) initiation, pausing, and elongation, suggesting regulation of the transcription cycle is critical for photoreceptor health during aging. Transcriptome profiling of photoreceptors from select RNAi lines revealed that knockdown of the pausing factor <i>Spt5 </i>or the chromatin remodeler <i>domino </i>resulted in similar transcriptome-wide changes to those observed in aged photoreceptors. Together, these data showed that transcriptional regulators are key in maintaining photoreceptor viability during aging, and age-dependent changes in gene expression not only correlate with but may also contribute to an increased risk of retinal degeneration. This research is presented in Chapter 2: “Targeted RNAi screen identifies transcriptional mechanisms that prevent premature degeneration of adult photoreceptors”.</p><p dir="ltr">To determine how the chromatin landscape dictates changes in the photoreceptor transcriptome with aging, we profiled chromatin marks associated with active transcription in young and old <i>Drosophila </i>photoreceptors using ChIP-seq. Both H3K4me3 and H3K36me3 decrease globally across actively-expressed genes during aging independent of differential gene expression. Knockdown of the H3K36me3 methyltransferase Set2 in young photoreceptors led to substantial changes in splicing events similar to those observed in aging photoreceptors, impacting genes involved in phototransduction and neuronal function. Because proper splicing is essential for visual behavior, and <i>Drosophila </i>visual function decreases with age, H3K36me3 may play a role in maintaining visual function in the eye through the regulation of alternative splicing. This research is presented in Chapter 3: “Establishing the contribution of active histone methylation marks to the aging transcriptional landscape of Drosophila photoreceptors”.</p><p dir="ltr">Since the molecular circadian clock is necessary for light-dependent photoreceptor survival in <i>Drosophila</i>, we sought to characterize the rhythmic gene expression changes during aging by performing nuclear RNA-seq on young and old <i>Drosophila </i>photoreceptors across the circadian day. RNA-seq revealed that over 50% of the photoreceptor transcriptome is rhythmic, and one-third of expressed genes showed altered rhythmicity with age. CUT&RUN of the core molecular clock transcriptional activators CLOCK and CYCLE in young and old photoreceptors identified relatively few target genes, and that CLOCK and CYCLE occupancy on chromatin does not substantially change with age, suggesting other epigenetic factors underly the drastic shifts in the photoreceptor rhythmic transcriptome during aging. Profiling of H3K4me2/3, H3K36me3, H3K9me3, and H3K27me3 at a single time-point using CUT&RUN showed distinct patterns of distribution across genes in young and old photoreceptors, in addition to a genome-wide loss in levels of all marks. We performed ATAC-seq and CUT&RUN of Pol II, H3K4me1, H3K4me2, and H3K4me3 across the circadian day in young and old photoreceptors to observe their daily patterns. Chromatin accessibility and Pol II occupancy oscillate throughout the day at all expressed genes, and the phase of this oscillation shifts in old photoreceptors. This oscillating pattern occurred at all expressed genes regardless of the timing of rhythmic gene expression. H3K4me1, me2, and me3 showed different oscillating patterns at all expressed genes that were nearly abolished in old photoreceptors. Though the overall oscillating patterns of H3K4 methylation also did not correlate with the timing of rhythmic gene expression, levels of H3K4 methylation do correlate with when the gene is most highly expressed. CUT&RUN of H3K4 methylation in young photoreceptors with a knockdown of either of the H3K4 methyltransferases Trr or Trx determined that they have overlapping yet non-redundant roles in maintaining H3K4 methylation genome-wide. Transcriptome profiling of young photoreceptors with Trr or Trx knockdown showed that they are necessary for the majority of rhythmic gene expression in young photoreceptors. Additionally, Trr or Trx knockdown led to loss and modulation of rhythmic gene expression similar to the changes observed in aging photoreceptors. Together, these data suggest that the genome-wide decreases in histone methylation in aging photoreceptors are the driving force behind shaping the rhythmic transcriptome. This research is presented in Chapter 4: “Histone methylation loss underlies the rewiring of the rhythmic transcriptome in aging photoreceptors”.</p>

Cell phenotypic transitions, or cell fate decision making processes, are regulated by complex regulatory networks composed of genes, RNAs, proteins and metabolites. The regulation can take place at the epigenetic, transcriptional, translational, and post-translational levels to name a few. Epigenetic histone modification plays an important role in cell phenotype maintenance and transitions. However, the underlying mechanism relating dynamical histone modifications to stable epigenetic cell memory remains elusive. Incorporating key pieces of molecular level experimental information, we built a statistical mechanics model for the inheritance of epigenetic histone modifications. The model reveals that enzyme selectivity of different histone substrates and cooperativity between neighboring nucleosomes are essential to generate bistability of the epigenetic memory. We then applied the epigenetic modeling framework to the differentiation process of olfactory sensory neurons (OSNs), where the observed 'one-neuron-one-allele' phenomenon has remained as a long-standing puzzle. Our model successfully explains this singular behavior in terms of epigenetic competition and enhancer cooperativity during the differentiation process. Epigenetic level events and transcriptional level events cooperate synergistically in the OSN differentiation process. The model also makes a list of testable experimental predictions. In general, the epigenetic modeling framework can be used to study phenotypic transitions when histone modification is a major regulatory element in the system. Post-transcriptional level regulation plays important roles in cell phenotype maintenance. Our integrated experimental and computational studies revealed such a motif regulating the differentiation of definitive endoderm. We identified two RNA binding proteins, hnRNPA1 and KSRP, which repress each other through microRNAs miR-375 and miR-135a. The motif can generate switch behavior and serve as a noise filter in the stem cell differentiation process. Manipulating the motif could enhance the differentiation efficiency toward a specific lineage one desires. Last we performed mathematical modeling on an epithelial-to-mesenchymal transition (EMT) process, which could be used by tumor cells for their migration. Our model predicts that the IL-6 induced EMT is a stepwise process with multiple intermediate states. In summary, our theoretical and computational analyses about cell phenotypic transitions provide novel insights on the underlying mechanism of cell fate decision. The modeling studies revealed general physical principles underlying complex regulatory networks. / Ph. D.

Yes / Background: miRNAs (microRNAs) play a key role in triple-negative breast cancer (TNBC) progression, and its heterogeneity at the expression, pathological and clinical levels. Stratification of breast cancer subtypes on the basis of genomics and transcriptomics profiling, along with the known biomarkers’ receptor status, has revealed the existence of subgroups known to have diverse clinical outcomes. Recently, several studies have analysed expression profiles of matched mRNA and miRNA to investigate the underlying heterogeneity of TNBC and the potential role of miRNA as a biomarker within cancers. However, the miRNA-mRNA regulatory network within TNBC has yet to be understood. Results and Findings: We performed model-based integrated analysis of miRNA and mRNA expression profiles on breast cancer, primarily focusing on triple-negative, to identify subtype-specific signatures involved in oncogenic pathways and their potential role in patient survival outcome. Using univariate and multivariate Cox analysis, we identified 25 unique miRNAs associated with the prognosis of overall survival (OS) and distant metastases-free survival (DMFS) with “risky” and “protective” outcomes. The association of these prognostic miRNAs with subtype-specific mRNA genes was established to investigate their potential regulatory role in the canonical pathways using anti-correlation analysis. The analysis showed that miRNAs contribute to the positive regulation of known breast cancer driver genes as well as the activation of respective oncogenic pathway during disease formation. Further analysis on the “risk associated” miRNAs group revealed significant regulation of critical pathways such as cell growth, voltage-gated ion channel function, ion transport and cell-to-cell signalling. Conclusion: The study findings provide new insights into the potential role of miRNAs in TNBC disease progression through the activation of key oncogenic pathways. The results showed previously unreported subtype-specific prognostic miRNAs associated with clinical outcome that may be used for further clinical evaluation. / EPSRC (EP/R043787/1).

Regulation of cancer cell phenotype by the tumor microenvironment has motivated further investigation into how microenvironmental factors could contribute to tumor initiation, development, and therapeutic resistance. Analyzing how the microenvironment drives tumor development and cancer cell heterogeneity is particularly important in cancers such as glioblastoma multiforme (GBM) that have no known risk factors and are characterized by a high degree of heterogeneity. GBM patients have a median survival of 15 months and therefore are in great need of more effective therapeutic options. The goal of this research is to generate in vitro models of the heterogeneous brain tumor microenvironment, with a focus on vascular dynamics, to probe the impact of microenvironmental cues on tumor progression and to integrate the tumor models with highly sensitive analytical tools to characterize the epigenome of discrete phenotypic subpopulations that contribute to intratumoral cellular heterogeneity. As GBM tumors are characterized by a dense vasculature, we delved into microenvironmental factors that may be promoting angiogenesis. The correlations emerging between inflammation and cancer led to analysis of the inflammatory molecule lipopolysaccharide (LPS). We utilized 3D micro-tissue models to simulate vascular exposure to ultra-low chronic inflammatory levels of LPS and observed an increase in vascular formation when brain endothelial cells were exposed to ultra-low doses of LPS. We also utilized our micro-tissue models to analyze histone methylation changes across the epigenome in response to microenvironmental cues, namely culture dimensionality and oxygen status. The H3K4me3 modification we analyzed is associated with increased gene transcription, therefore the alterations we observed in H3K4me3 binding across the genome could be a mechanism by which the tumor microenvironment is regulating cancer cell phenotype. Lastly, we developed a microfluidic platform in which vascular dynamics along with microenvironmental heterogeneities can be modeled in a more physiologically relevant context. We believe the studies presented in this dissertation provide insight into how vasculature primed by chronic inflammation and epigenetic alterations in tumor cells could both contribute to enhanced tumor development. Modeling these biological processes in our advanced microfluidic platform further enables us to better understand microenvironmental regulation of tumor progression, uncovering new potential therapeutic targets. / PHD / Regulation of cancer cell behavior by the tumor microenvironment, which includes the surrounding extracellular matrix, native healthy cells, and signaling molecules, has motivated further investigation into how microenvironmental factors could contribute to tumor initiation, development, and therapeutic resistance. Analyzing how the microenvironment drives tumor development and heterogeneity in cancer cell behavior is particularly important in cancers such as glioblastoma multiforme (GBM) that have no known risk factors and are characterized by a high degree of heterogeneity. GBM patients have a median survival of 15 months and therefore are in great need of more effective therapeutic options. The goal of this research is to generate models of the heterogeneous brain tumor microenvironment with a focus on how microenvironmental cues impact blood vessel development, which facilitates tumor progression. We will also use these tumor models, along with sensitive analytical tools, to characterize epigenetic modifications that potentially contribute to tumor cell heterogeneity. As GBM tumors are characterized by a dense vasculature, we delved into microenvironmental factors that may promote blood vessel growth. The correlations emerging between inflammation and cancer led to analysis of the inflammatory molecule lipopolysaccharide (LPS). We utilized 3D tumor models to simulate blood vessel exposure to ultra-low chronic inflammatory levels of LPS and observed an increase in blood vessel formation when brain endothelial cells were exposed to ultra-low doses of LPS. We also utilized our tissue models to analyze histone methylation changes across the epigenome in response to microenvironmental cues, namely culture dimensionality and oxygen status. The histone methylation changes we observed across the genome could be a mechanism by which the tumor microenvironment is regulating cancer cell v behavior. Lastly, we developed a microfluidic platform in which blood vessel development along with microenvironmental heterogeneities can be modeled in a more physiologically relevant context. We believe the studies presented in this dissertation provide insight into how blood vessel exposure to chronic inflammatory factors and epigenetic alterations in tumor cells could both contribute to enhanced tumor development. Modeling these biological processes in our advanced microfluidic platform further enables us to better understand microenvironmental regulation of tumor progression, uncovering new potential therapeutic targets.

No / We organized the first International Symposium on Skin Epigenetics at the Centre for Skin Sciences at the University of Bradford (West Yorkshire, UK) on 2nd and 3rd April 2012. The goal of the Symposium was to bring together two research communities—skin and chromatin biologists—and discuss the most important aspects of epigenetic regulatory mechanisms that control skin development and regeneration. The symposium was attended by more than 80 participants from countries across Europe, Australia, Japan, Singapore, and USA, and representing academic institutions and industry. Epigenetic regulation of gene expression programs in the skin is a novel trend in research in cutaneous biology, and several landmark papers arising in the field were published recently (reviewed in Botchkarev et al., 2012; Botchkareva, 2012; Frye and Benitah, 2012; Yi and Fuchs, 2012; Zhang et al., 2012). The Symposium program included six Keynote lectures, the inaugural John M. Wood Memorial Lecture, and six sessions that covered major levels of epigenetic regulation.