Browsing by Subject "Chromatin"
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Item The Analysis of the MCF7 Cancer Model System and the Effects of 5-AZA-2'-Deoxycytidine Treatment on the Chromantin State Using a Novel Microarray-Based Technology for High Resolution Global Chromatin State Measurement(2006-07-10) Weil, Michael Ryan; Garner, Harold Ray (Skip)A microarray method to measure the global chromatin state of the human genome was developed in order to provide a novel view of gene regulation. The 'chromatin array' employs traditional methods of chromatin isolation, microarray technology, and advanced data analysis, and was applied to a cancer model system. Chromatin is first separated by its condensation state using chromatin fractionation. By probing with a comparative genomic hybridization-style microarray, the chromatin condensation state of thousands of individual loci in an MCF7 tumor model cell line was determined and correlated with transcriptional activity. The chromatin array showed a significant portion (>3,000) of the genes were in a condensation state that was neither condensed or relaxed as a result of heterogeneity in the condensation states in the population. The utility of the chromatin array in deciphering gene regulation was demonstrated in a MCF7 cell line treated with 5 Aza dC, which disrupts genome methylation, and as a result causes global relaxation of chromatin structure. 5 Aza dC treatment results in strong changes in expression, and a normalized global chromatin relaxation of two-fold. A significant subset of 378 genes was condensed by 5 Aza dC treatment, indicating that a mechanism of chromatin regulation exists that can resist the effects of 5 Aza dC treatment. The genes with the largest changes in response to 5 Aza dC treatment showed a strong correlation with CpG island-based regulation (p < 0.0001), and a restoration of transcription patterns associated with normal mammary tissue. Analysis using splice-form specific microarray probes demonstrated that the chromatin state was not uniform across a gene. These findings indicate that certain gene regions exhibit differential sensitivity to 5 Aza dC treatment, and therefore may be regulated independently. Using functional annotation, expression microarray, and comparative genomic hybridization data, this work should provide a framework through which the biological implications of the relationship between chromatin accessibility and expression may be deciphered.Item Epigenetic Mechanisms in Drug Addiction(2010-11-02T18:19:06Z) Renthal, William Russell; Nestler, Eric J.Changes in gene expression in brain reward regions are thought to contribute to the pathogenesis and persistence of drug addiction. Recent studies have begun to focus on the molecular mechanisms by which drugs of abuse, and related environmental stimuli, such as drug-associated associated cues or stress, converge on the genome to alter specific geneprograms. Increasing evidence suggests that these stable gene expression changes in neurons are mediatedin part by epigenetic mechanisms that alter chromatin structure on specific gene promoters. Indeed, genome-wide analysis using chromatin immunoprecipitation coupled with promoter microarrays in vivo, identified on which genes chronic cocaine exposure alters histone acetylation and methylation in the nucleus accumbens, a key brain reward region. In addition to providing novel insight into basic transcriptional mechanisms co-opted by cocaine, these data revealed a new class of cocaine-regulated genes, the sirtuins, which potently regulate reward behavior. In order to further understand the mechanisms by which cocaine regulates chromatin structure, I investigated enzymes which control levels of histone acetylation, histone deacetylases (HDACs). Chronic, but not acute, exposure to cocaine decreased the function of a class II HDAC, HDAC5, in the NAc, which allows for increased histone acetylation and transcription of HDAC5 target genes. This regulation is behaviorally important, as loss of HDAC5 causes hypersensitive responses to chronic, but not acute, cocaine. I have also identified a key roleof the class I HDAC, HDAC1, which interacts with the drug-induced transcription factor, ΔFosB, to repress c-fos gene induction in striatum after chronic psychostimulant exposure. Taken together, these findings suggest that proper balance of histone acetylation in the NAc is a crucial factor in the saliency of cocaine action, and that disruption of this balance may be involved in the transition from acuteadaptive responses to chronic psychiatric illness.Item Functional characterization of smyd1, a methyltransferase essential for heart and skeletal muscle development(2006) Zhu, Li; Tucker, Philip W.Item Mechanisms underlying vernalization-mediated VERNALIZATION INSENSITIVE 3 (VIN3) induction in Arabidopsis thaliana(2013-05) Zografos, Brett Robert; Sung, SibumVernalization is defined as the response to prolonged cold exposure required for acquiring the molecular competence necessary to undergo floral transition. FLOWERING LOCUS C (FLC), a potent floral repressor in Arabidopsis, is highly expressed before vernalizing cold treatment but is repressed during prolonged vernalization. VERNALIZATION INSENSITIVE 3 (VIN3) is a Plant HomeoDomein (PHD)- containing protein that is required for establishing vernalization-mediated repression of FLC. The induction of VIN3 is one of the earliest molecular events in vernalization response and its expression is intimately linked to prolonged cold exposure. However, mechanisms underlying VIN3 induction remain poorly understood. The constitutive repression of VIN3 in the absence of cold is due to multiple repressive components, including a transposable element-derived sequence, LIKE-HETEROCHROMA TIN PROTEIN 1 (LHP1), and POLYCOMB REPRESSION COMPLEX 2 (PRC2). Furthermore, the full extent of VIN3 induction by vernalization requires activating complex components, including EARLY FLOWERING 7 (ELF7) and EARLY FLOWERING IN SHORT DAYS (EFS). Dynamic changes in the histone modifications present at VIN3 chromatin during vernalization were also observed, indicating that chromatin changes play a critical role in regulating VIN3 induction. However, VIN3 induction by vernalization still occurs in the absence of activation complexes and de- repression of VIN3 in the absence of the repressive complexes is not sufficient for achieving complete induction. Thus, unknown cold-influenced regulators responsible for achieving maximum VIN3 induction during vernalization must exist. Therefore, forward genetic screening was undertaken to elucidate upstream regulators of VIN3. Molecular characterization of T-DNA mutant populations elucidated two interesting mutants: a mutant that ectopically expressed VIN3 before cold (ectopic VIN3 induction, evi1) and mutants that failed to induce VIN3 during vernalization (defects in VIN3 induction, dvi1). FLC is over-expressed in dvi1 despite its failure to induce VIN3 expression during vernalization, suggesting that this mutant may regulate both VIN3 and FLC. In evi1, FLC is hyper-repressed after 40 days of vernalization, leading to an acceleration of flowering time. These results indicate that regulators of VIN3 in the vernalization pathway exist and that these regulators may use different mechanisms in order to influence VIN3 expression.Item The Role of Chromatin Remodeling in Hippocampus in Depression and Antidepressant Action(2008-05-13) Tsankova, Nadeja Mincheva; Nestler, Eric J.This thesis presents a novel level by which neuroplastic changes in the brain may be disrupted with depression and reversed by treatment with antidepressants: regulation at the level of chromatin remodeling. The technique of brain chromatin immunoprecipitation was pioneered to directly measure the in vivo modifications of histones, a form of chromatin remodeling, at gene promoter regions in the hippocampus after chronic defeat stress, a model of depression, and chronic treatment with the antidepressants imipramine and electroconvulsive seizure (ECS). Chromatin modifications and transcriptional changes were assayed in one gene in particular, the brain-derived neurotrophic factor (BDNF). BDNF is alternatively spliced to generate several mRNA transcripts, driven by unique promoters. I measured the expression levels of each BDNF transcript (I-IV) in rat after ECS, as well as each BDNF transcript (I-V) in mice after chronic stress and imipramine treatments, and found that these chronic treatments induce lasting changes in the expression of specific BDNF splice variants. These changes correlated with sustained modifications in histones at the exact promoter regions, driving the differential changes in BDNF expression. Chronic defeat stress induced robust enrichment of H3-K27 methylation at BDNF P3 and P4 promoters (modifications expected to repress promoter activity), while chronic imipramine in defeated animals lead to lasting upregulation in the levels of H3 acetylation and H3-K4 methylation at P3 and P4 (modifications expected to stimulate promoter activity). Finally, I discovered a novel role for the histone deacetylase HDAC5 in the therapeutic efficacy of chronic imipramine after defeat stress. I found that chronic imipramine downregulates HDAC5 after stress, that HDAC5 overexpression in the hippocampus blocks the behavioral effects of imipramine in defeated mice, that HDAC5 inhibition exerts a subtle antidepressant-like effect, and that HDAC5 deficiency reduces the pathological response to stress. This unexpected role for HDAC5 provides an important mechanistic link between the adaptive chromatin remodeling changes at genes and the ability of chronic antidepressants to exert therapeutic efficacy after chronic stress. These experiments provide one of the first endeavors to understand the role of chromatin remodeling in modulating long-term adaptive changes in brain associated with complex psychiatric conditions, such as depression.Item The Ribosomal DNA Genes Influence Genome-Wide Gene Expression in Drosophila melanogaster(2012-07-16) Paredes Martinez, Lida SilvanaChromatin structure is a fundamental determinant of eukaryotic gene expression and it is composed of two chromatin environments, euchromatin and heterochromatin. Euchromatin provides an accessible platform for transcription factors; hence it is permissive for gene expression. Heterochromatin on the other hand is highly compacted and inaccessible, which in most cases leads to transcriptional repression. A locus that is composed of both of these environments is the ribosomal DNA (rDNA). In eukaryotes the rDNA is composed of hundreds to thousands of tandemly repeated genes where maintaining both silent and active copies is fundamental for the stability of the genome. The aim of this research was to investigate the role of the rDNA in gene expression in Drosophila melanogaster. In D. melanogaster the rDNA loci are present on the X and Y chromosomes. This research used the Y-linked rDNA array to investigate the role of this locus on gene expression. A genetic and molecular strategy was designed to create and quantify specific, graded and isogenic Y- linked rDNA deletions. Then the deletions were used to address the effect of rDNA deletions on gene expression using reporter genes sensitive to Position Effect Variegation (PEV). In addition, the effect of the deletions in nucleolus size and structure as well as the effect of spontaneous rDNA deletions on gene expression were tested in this study. This research found that changes in rDNA size change the chromatin balance, which resulted in increased expression of the reporter genes, decreased nucleolus volume, and altered nucleolus structure. These findings prompted a further research question on whether this effect on gene expression occured globally in the genome. This was addressed by performing microarray analysis where the results showed that rDNA deletions affect about half of the genes on the genome. Presented in this dissertation is evidence that suggest a novel role for the rDNA is a global modulator of gene expression and also is a contributor to the gene expression variance observed in natural populations.