Browsing by Subject "epigenetics"
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Item Arabidopsis thaliana histone deacetylase 1 (AtHD1) and epigenetic regulation(Texas A&M University, 2004-09-30) Tian, LuEpigenetic regulation is a mechanism by which heritable changes in gene expression are controlled by chromatin status rather than primary DNA sequence. Changes in chromatin structure affect accessibility of DNA elements to the transcriptional machinery and thus affect transcription activity of the gene. A key event in this process is reversible modification of core histones, which is catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDs, HDAs, or HDACs). In general, histone deacetylation is related to transcriptional gene silencing, whereas acetylation is associated with gene activation.To study the role of histone deacetylase in plant gene regulation and development, we generated constitutive antisense histone deacetylase 1 (CASH) transgenic plants. AtHD1 is a homolog of RPD3 protein, a global transcriptional regulator in yeast. Expression of the antisense AtHD1 caused dramatic reduction in endogenous AtHD1 transcription, resulting in accumulation of acetylated histones. Down-regulation of histone deacetylation caused a variety of growth and developmental abnormalities and ectopic expression of tissue-specific genes. However, changes in genomic DNA methylation were not detected in repetitive DNA sequences in the transgenic plants.We also identified a T-DNA insertion line in exon 2 of AtHD1 gene (athd1-t1), resulting in a null allele at the locus. The complete inhibition of the AtHD1 expression induced growth and developmental defects similar to those of CASH transgenic plants. The phenotypic abnormalities were heritable across the generations in the mutants. When the athd1-t1/athd1-t1 plants were crossed to wild-type plants, the mutant phenotype was corrected in the F1 hybrids, which correlated with the AtHD1 expression and reduction of histone H4 Lys12 acetylation. Microarray analysis was applied to determine genome-wide changes in transcriptional profiles in the athd1-t1 mutant. Approximately 6.7% (1,753) of the genes were differentially expressed in leaves between the wild-type (Ws) and the athd1-t1 mutant, whereas 4.8% (1,263) of the genes were up- or down-regulated in flower buds of the mutant. These affected genes were randomly distributed across five chromosomes of Arabidopsis and represented a wide range of biological functions. Chromatin immunoprecipitation assays indicated that the activation for a subset of genes was directly associated with changes in acetylation profiles.Item Characterization and Analysis of the Bovine Epigenome during Preimplantation Embryo Development In Vitro(2012-10-19) Williamson, Gayle LingerDuring early mammalian embryogenesis, the embryonic genome undergoes critical reprogramming events that include changes in both DNA methylation and histone modifications necessary to control chromatin structure and thus, gene expression. Improper reprogramming of the epigenome during this window of development can lead to a vast number of imprinting anomalies, which are increased in children and livestock conceived in vitro. In the bovine, which closely resembles human preimplantation development, epigenetic changes occur from fertilization through the blastocyst stages. In particular, and concurrent with embryonic genome activation (EGA), de novo DNA methylation begins at the 8-cell stage. In order to explore the roles of histone-modifying enzymes during this crucial period of development, we characterized the transcript expression of several enzymes key enzymes across in vitro bovine preimplantation development using quantitative real-time PCR. Two of the 7 genes analyzed (Suz12 and Lsh) exhibited notable increases at the 8-16 cell stages, with basal levels observed both before and after this. These increases coincided with both EGA and de novo DNA methylation. We further explored their roles in bovine preimplantation embryos by knocking down expression via the use of gene-specific targeting siRNAs. Independent suppression of either Suz12 or Lsh via cytoplasmic microinjection of targeting siRNAs resulted in lower development rates (p < 0.0001), and poorer embryo quality of the morulas and blastocysts that survived. In addition, Suz12 suppression led to reductions in both H3K27 (p < 0.0001) and H3K9 (p = 0.07) trimethylation, and an increase in DNA methylation levels (p < 0.0001), as compared to the null-injected controls. Lsh suppression did not change H3K27, but led to a reduction in H3K9 trimethylation (p = 0.006) and an increase in DNA methylation (p < 0.0001). Clearly our data demonstrate that these epigenetic modifiers play a critical role in formation of the embryonic epigenome, but further research would be necessary in order to fully characterize gene activities during this developmental window.Item Generating a Consistent Framework for Evaluating Cell Response to External Stimuli through Epigenetic Assessors(2011-08-08) Wang, BoMesenchymal stem cells are more and more widely used in tissue engineering due to their pluripotency and no relative ethical problems. Traditional characterization techniques to detect mesenchymal stem cell states include flow cytometry, gene expressing profiling and immunohistochemistry. However, these methods can only provide transient and low level information from current RNA or protein levels about mesenchymal stem cells, which may cause problems when predicting the possible downstream lineages they will commit into. We have developed chromatin immunoprecipitation (ChIP)-based epigenetic technique to detect mesenchymal stem cell states. For the systems we tested, this epigenetic assessor successfully characterized cell state changes and gave similar results obtained from gene expression profiling or protein expression assay. This epigenetic technique can provide information about mesenchymal stem cells states from a more fundamental chromatin level, which is promising for predicting future lineages from current states.Item Nutritional Programming of Neuroendocrine Pathways Controlling the Onset of Puberty in Heifers(2014-05-01) Alves, Bruna R. C.Accelerated body weight gain during the juvenile period leads to early maturation of the reproductive neuroendocrine system and puberty by advancing the onset of high-frequency release of gonadotropin-releasing hormone (GnRH). In a series of studies conducted as part of this dissertation, it was hypothesized that nutritional regimens that accelerate pubertal development alter the function of kisspeptin and neuropeptide Y (NPY) neurons, known modulators of the release of GnRH. In addition, alterations in the pattern of methylation of target genome sequences were investigated in DNA collected from the arcuate nucleus, a hypothalamic region well-characterized for its role in mediating the metabolic control of reproductive function. Early-weaned crossbred heifers were fed diets to promote an average daily gain of 0.5 Kg (Low Gain; n=12) or 1.0 Kg (High Gain; n=12) from approximately 4 to 8 mo of age. This nutritional regimen has been demonstrated to facilitate early onset of puberty in heifers. At eight mo of age, heifers were slaughtered and a block of tissue containing the preoptic area and hypothalamus was collected and processed for examining the expression of NPY and KISS1, investigating the innervation of GnRH and kisspeptin neurons by NPY fibers, and characterizing the methylation profile of target genomic DNA sequences isolated from the arcuate nucleus. Mean body weight and circulating concentrations of leptin were greater in High-gain than in Low-gain heifers. Elevated rate of body weight gain was associated with reduced expression of NPY in the arcuate nucleus, reduced proportion of GnRH neurons in apposition to NPY-containing varicosities, particularly in GnRH neurons located in the mediobasal hypothalamus, and a reduced number of KISS1-expressing cells in the mid portion of the arcuate nucleus. The rate of body weight gain during the juvenile period did not affect NPY innervation of kisspeptin neurons. Differential methylation of target genomic DNA sequences isolated from the arcuate nucleus was observed in association with rate of body weight gain in juvenile heifers, and genes affected included GHR, IGF2, IGF1R, LEPR, PEG3, LIN28B and HMGA2. Overall, results from these studies support the hypothesis that accelerated body weight gain during the juvenile period promotes alterations in cellular functions within the hypothalamus that facilitate early onset of puberty in heifers.Item The development of a bovine interspecies model for the analysis of genomic imprinting in normal and nuclear transfer derived fetuses(Texas A&M University, 2004-11-15) Dindot, Scott VictorThe advent of somatic cell nuclear transfer in cattle has provided the opportunity for researchers to generate genetically identical animals as well as animals that possess precise genetic modifications for agriculture and biomedical purposes. However, in spite of the revolutionary impact this technology presents, problems remain which hinder the production of healthy animals on a consistent basis. Research on cloned mice implicates improper reprogramming of epigenetic modifications and genomic imprinting for the low pregnancy rates and high incidence of abnormalities that are manifested in cloned animals; however, a systematic and comprehensive analysis of nuclear reprogramming in cloned cattle remains undone. The purpose of this research is to assess and characterize the patterns of genomic imprinting in normal and nuclear transfer derived bovine fetuses. To facilitate the identification of imprinted genes in the bovine, a Bos gaurus/Bos taurus interspecies model has been incorporated to maximize the genetic heterozygosity that exists between the alleles of putative imprinted genes for allelic discrimination and parental inheritance. The sequence of twenty-six genes, previously reported as imprinted in mice and humans, was analyzed in Bos gaurus (Gaur) and Bos taurus (Angus) cattle for the presence of single nucleotide polymorphisms (SNP). SNPs were detected in the Gene trap locus 2 (GTL2), Insulin like growth factor 2 (IGF2), Wilms tumor 1 (WT1) and the X chromosome inactivation specific transcript (XIST). Allelic expression analysis in interspecies hybrids indicated maternal genomic imprinting at the IGF2 and XIST loci, paternal genomic imprinting at the GTL2 locus and no imprinting at the WT1 locus. Analysis in cloned hybrids indicated fidelity of allelic expression at the IGF2 and GTL2 loci, however disruption of imprinting was observed at the XIST locus in the placenta of clones. These results are the largest identification of imprinted genes in the bovine and the first identification of the disruption of an imprinted gene in an animal derived from somatic cell nuclear transfer.Item The Identification and Characterization of Copy Number Variants in the Bovine Genome(2013-06-26) Doan, RyanSeparate domestication events and strong selective pressures have created diverse phenotypes among existing cattle populations; however, the genetic determinants underlying most phenotypes are currently unknown. Bos taurus taurus (Bos taurus) and Bos taurus indicus (Bos indicus) cattle are subspecies of domesticated cattle that are characterized by unique morphological and metabolic traits. Because of their divergence, they are ideal model systems to understand the genetic basis of phenotypic variation. Here, we developed DNA and structural variant maps of cattle genomes representing the Bos taurus and Bos indicus breeds. Using this data, we identified genes under selection and biological processes enriched with functional coding variants between the two subspecies. Furthermore, we examined genetic variation at functional non-coding regions, which were identified through epigenetic profiling of indicative histone- and DNA-methylation modifications. Copy number variants, which were frequently not imputed by flanking or tagged SNPs, represented the largest source of genetic divergence between the subspecies, with almost half of the variants present at coding regions. We identified a number of divergent genes and biological processes between Bos taurus and Bos indicus cattle; however, the extent of functional coding variation was relatively small compared to that of functional non-coding variation. Collectively, our findings suggest that copy number and functional non-coding variants may play an important role in regulating phenotypic variation among cattle breeds and subspecies.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.Item Transcriptional Regulation of Pregnane X Receptor by Protein Arginine Methyltransferase(2011-08-08) Xie, YingPregnane X receptor (PXR) is a ligand-dependent transcription factor that plays an important role in xenobiotic/drug metabolism. The ligand-receptor interaction transcriptionally activates phase I and phase II enzymes, and membrane-bound transporters in a coordinated manner and ultimately leads to detoxification and excretion of the ligands. One of the direct target genes is cytochrome P450 3A4 (CYP3A4) which is responsible for metabolism of over 50% of clinically used drugs. Understanding the regulation of PXR is important for treatment of disease and avoidance of untoward drug-drug interactions. In this research, we have used various biochemical and molecular approaches to investigate factors that regulate the transcriptional activity of PXR. We have stably transfected PXR into HepG2 human liver hepatoma cells. Using these PXR-HepG2 cells, we surveyed the histone methyltransferases that interact with PXR. Based on results from co-immunoprecipitation/methyltransferase, N-terminal peptide sequencing, GST-pulldown assays, we found that protein arginine methyltransferase 1 (PRMT1) is a predominant histone methyltransferase in HepG2 cells. Evidence from other laboratories suggests that histone methylation by PRMT1 sets the stage for subsequent histone modifications such as the acetylation of histone H4. These modifications are believed to be important for transcriptional and epigenetic regulation of gene expression. We hypothesize that PRMT1 plays a role in the epigenetic changes regulated by PXR. PRMT1-dependent histone methylation changes may be involved in epigenetic cell memory where prior exposure to certain agents may alter the chromatin (or priming the chromatin) with a "primed" state which alters the subsequent magnitude or duration of gene expression. In our study, we have found that pretreatment of PXR-HepG2 cells with DMSO greatly enhanced PXR-mediated activation of CYP3A4 upon rifampicin treatment. DMSO pretreatment altered histone modifications association with the promoter of the PXR-regulated gene (CYP3A4). Inhibition of histone methylation by PRMT1 either through RNAi or the methyltransferase inhibitor (Adox) abolished the priming effects. My research results strongly indicate that PRMT1 is involved in transcriptional regulation of PXR and may be involved in epigenetic memory of liver cells where prior exposure to agents changes the subsequent detoxification responses.