Browsing by Subject "Epigenetics"
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Item Alcohol promotes mammary tumor development through regulation of estrogen signaling(2012-05) Wong, Amy W.; Nuñez, Nomelí P.Breast cancer is the most common malignancy affecting women and the second leading cause of death among women in the United States. Alcohol consumption is one of the few modifiable risk factors for breast cancer development but the mechanism by which it contributes to mammary cancer development and progression remains unclear, although it has been suggested that estrogen is critical for this process. To determine if alcohol promotes mammary tumor development via the estrogen pathway, estrogen receptor alpha-negative (ER[alpha]-negative) MMTV-neu mice were treated with various doses of ethanol and activation of estrogen signaling was measured. Our results showed that alcohol consumption increased estrogen signaling activation, serum estrogen levels and, most interestingly, expression of ER[alpha] in tumor tissue in the ER[alpha]-negative mice. Several lines of evidence in literature suggest that ER[alpha] expression in ER[alpha]-negative cancer cells is inhibited through epigenetic regulation. Epigenetics is the study of heritable changes in gene expression caused by mechanisms other than DNA sequence changes. Thus, to determine whether alcohol may regulate ER[alpha] re-expression in ER[alpha]-negative breast cancer cells through epigenetic mechanisms, we examined the effects of ethanol on CpG methylation and histone modifications (acetylation and methylation) of two ER[alpha]-negative breast cancer cell lines, MDA-MB-231 (human) and MMTV-neu (mouse). We also examined whether the epigenetic modifications subsequently affect the recruitment of transcriptional regulation complexes to the ER[alpha] promoter to regulate ER[alpha] transcription. Results showed that alcohol promotes ER[alpha] re-expression in these ER[alpha]-negative cell lines and that this effect was associated with decreased CpG methylation, an overall increase of histone acetylation and decrease of histone methylation, and an alteration in the enrichment of the ER[alpha] transcriptional regulation complexes (pRb2/p130-E2F4/5-HDAC1-SUV39H1-p300 and pRb2/p130-E2F4/5-HDAC1-SUV39H1-DNMT1) at the ER[alpha] promoter, which may contribute to cancer cell progression. In addition, we found that the inhibition of ER[alpha] by tamoxifen specifically blocks the effects of alcohol on ER[alpha] reactivation. To determine how alcohol promotes cell invasive ability, a critical process for cancer progression, we examined the role of two genes, metastasis suppressor Nm23 and integrin alpha-5 ITGA5, which we identified to be important for alcohol-induced breast cancer cell invasion. It has previously been shown that estrogen may regulate Nm23 expression and that estrogen regulation may be important for ITGA5-mediated cancer progression. Our results showed that alcohol promotes cancer cell invasion through the down-regulation of Nm23, which led to the subsequent increase of ITGA5 and increase of cell invasion. Collectively, data from my research strongly supports and provides evidence that alcohol promotes breast cancer development and progression through the regulation of estrogen signaling.Item Assessing the preservation of cytosine methylation in ancient DNA from five prehistoric Native American populations(2013-05) Smith, Ricky Wayne Aldon; Bolnick, Deborah Ann; DiFiore, AnthonyCytosine methylation of CpG dinucleotides is an important epigenetic mark that regulates gene expression in humans. While methylation patterns in extant populations have been widely studied, few studies have attempted to analyze methylation in ancient DNA. Indeed, it was only recently shown that methyl groups can be preserved in ancient DNA. However, it is unknown how often methylation patterns can be recovered from ancient samples with preserved nuclear DNA. If they are frequently preserved, it may ultimately be possible to infer patterns of gene activity at the population level in ancient times. In this study, I assessed the preservation of cytosine methylation in ancient DNA from the remains of 30 prehistoric Native Americans from California, Illinois, Kentucky, and Mexico. These samples were previously shown to contain endogenous mitochondrial and nuclear DNA. I analyzed the cytosine methylation states of CpG-rich retrotransposons, which are epigenetically inactivated by cytosine methylation in humans. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite. Bisulfite products were pyrosequenced, and C-to-T conversions at potentially methylated CpG dinucleotides were quantified. I found that cytosine methylation is readily recoverable from human remains with preserved nuclear DNA from various localities over the time depth tested (~6000 years). This study presents the first direct evidence of cytosine methylation in ancient human remains, and suggests that it may be possible to analyze patterns of gene activity in ancient populations.Item Colon Cancer Chemoprotection through Epigenetic Effects of a Fish Oil/Pectin Diet(2012-10-19) Cho, Young MiAccumulated genetic and epigenetic abnormalities contribute to the development of colon cancer. We have shown that a combination of fish oil (containing decosahexaenoic acid, DHA, 22:6 n-3) and pectin (fermented to butyrate by colonic microflora) is protective against colon carcinogenesis in part by regulating the expression of genes involved in apoptosis, leading to apoptosis induction. To determine how FO/P enhances apoptosis, we measured the expression of genes involved in apoptosis. We performed a pathway analysis on differentially expressed genes identified at three times during colon tumorigenesis: initiation, aberrant crypt foci (ACF) formation, and tumor stage, and compared these results with phenotypic observations at those times. At initiation, FO/P down-regulated the expression of genes involved with cell adhesion and enhanced apoptosis compared with corn oil/cellulose (CO/C). At the ACF stage, expression of genes involved in cell cycle regulation was modulated by FO/P and proliferation was reduced in FO/P rats compared with CO/C rats. FO/P increased apoptosis and the expression of genes that promote apoptosis at the tumor endpoint compared with CO/C. We next determined if changes in expression of genes involved in apoptosis by FO/P are associated with changes in promoter methylation of a key apoptosis regulator, Bcl-2. Genomic DNA was isolated from carcinogen-induced colon tumors and non-involved tissues. FO/P increased Bcl-2 promoter methylation in tumor tissues and colonocyte apoptosis relative to those observed with CO/C. A negative correlation between Bcl-2 DNA methylation and Bcl-2 mRNA levels was observed in the tumors. Additionally, we examined gene specific promoter methylation of 24 apoptosis-related genes using human colon cancer cells. Cells were treated with DHA or linoleic acid (18:2 n-6), and select cultures were also treated with butyrate. The combination of DHA and butyrate led to a significant reduction in the methylation of pro-apoptotic genes and an increase in apoptosis. These data suggest that part of the mechanisms involved in the induction of apoptosis by FO/P may be through epigenetic regulation of genes involved in apoptosis throughout colon carcinogenesis.Item Expression of the bovine DNA (cytosine 5) methyltransferase family during preimplantation development and aberrations induced by somatic cell nuclear transfer(Texas A&M University, 2005-02-17) Golding, Michael CameronBovine preimplantation embryos derived from nuclear transfer experiments exhibit a global state of genomic hypermethylation that likely account for the large number of developmental abnormalities observed to date. The central hypotheses of this work is that the genomic hypermethylation and improper epigenetic reprogramming reported in studies of bovine nuclear transfer, are in large part due to abnormal expression and regulation of the DNA methyltransferase proteins. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2γ and Dnmt3a4 represent previously unreported sequence combinations. Investigation of bovine DNA methyltransferase expression in the bovine oocyte and early preimplantation development has revealed an intricate system divergent from observations previously reported in the mouse. Specifically, the somatic version of Dnmt1 along with Dnmt2, 3a and 3b are all expressed during these initial stages of bovine development. Further, real time analyses of the Dnmt transcripts in cloned and in vitro produced embryos reveal significant differences in the mRNA expression levels of Dnmt1 and 2 but not Dnmt3a and 3b suggesting that the de novo methyltransferases may be functioning normally while Dnmt1 and Dnmt2 are aberrantly methylating the genome during a critical time when methylation levels should be receding. Real time PCR analysis of the Dnmt transcripts in fetal and adult tissues has revealed a developmental and tissue specific expression pattern suggesting that proper expression and function of these enzymes is a key element in the process of differentiation. These results are further supported by studies of Dnmt expression in aging bovine fibroblast cultures, which suggest that the Dnmts may play some as yet unidentified role in cellular senescence. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos. Bovine preimplantation embryos derived from nuclear transfer experiments exhibit a global state of genomic hypermethylation that likely account for the large number of developmental abnormalities observed to date. The central hypotheses of this work is that the genomic hypermethylation and improper epigenetic reprogramming reported in studies of bovine nuclear transfer, are in large part due to abnormal expression and regulation of the DNA methyltransferase proteins. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2γ and Dnmt3a4 represent previously unreported sequence combinations. Investigation of bovine DNA methyltransferase expression in the bovine oocyte and early preimplantation development has revealed an intricate system divergent from observations previously reported in the mouse. Specifically, the somatic version of Dnmt1 along with Dnmt2, 3a and 3b are all expressed during these initial stages of bovine development. Further, real time analyses of the Dnmt transcripts in cloned and in vitro produced embryos reveal significant differences in the mRNA expression levels of Dnmt1 and 2 but not Dnmt3a and 3b suggesting that the de novo methyltransferases may be functioning normally while Dnmt1 and Dnmt2 are aberrantly methylating the genome during a critical time when methylation levels should be receding. Real time PCR analysis of the Dnmt transcripts in fetal and adult tissues has revealed a developmental and tissue specific expression pattern suggesting that proper expression and function of these enzymes is a key element in the process of differentiation. These results are further supported by studies of Dnmt expression in aging bovine fibroblast cultures, which suggest that the Dnmts may play some as yet unidentified role in cellular senescence. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos.Item Genetic networks and epigenetic mechanisms of temperature-dependent sex determination in the red-eared slider turtle, Trachemys scripta(2014-05) Matsumoto, Yuiko; Crews, David; Atkinson, Nigel; Hofmann, Johann; Vokes, Steve; Zakon, HaroldIn the red-eared slider turtle, Trachemys scripta, gonadal sex is determined by the incubation temperature during the mid-trimester of development; temperature effects can be overridden by exogenous ligands if they are administered during the temperature-sensitive period of development. How the physical signal of temperature is transduced into a biological signal that ultimately results in determining gonad sex is not known. My thesis research focuses on five candidate sex determining genes: cyp19a1 (aromatase), Forkhead box protein L2, R-spondin1, Doublesex mab3-related transcription factor 1, and Sex-determining Region on Y chromosome-box 9. The first three genes are markers of ovarian differentiation while the latter two genes are markers of testicular differentiation. Both in ovo (egg) and in vitro (gonadal explants) studies were conducted. Chapters 1 and 2 examine how exogenous steroid ligands interact with candidate genes as the gonads differentiate into testes or ovaries. Topical application of testosterone with aromatase inhibitor to eggs incubating at the female-producing temperature (31 ºC; FPT) suppresses expression of ovarian markers while increasing expression of testicular markers. Administration of 17β-estradiol (E2) to eggs incubating at a male-producing temperature (26 ºC; MPT) increases expression of ovarian markers while testicular markers are suppressed. This suggests that exogenous ligands modify gonadal trajectory by re-directing (suppression and activation) the expression of candidate genes. Chapter 3 identifies the gonad-specific promoter and the temperature-dependent DNA methylation signatures of the aromatase gene during gonadal differentiation. DNA methylation of the aromatase promoter is lowest at FPT relative to MPT. Exogenous E2 and certain polychlorinated biphenyls retain typical methylation patterns observed at MPT (Chapter 4). This suggests that despite the ability of exogenous ligands to alter the transcriptional profiles and gonad phenotypes, the MPT set the temperature typical epigenetic marks first at the beginning of TSP. Recruitment of modified histone proteins, H3K4me3 and H3K27me3, at the aromatase promoter is FPT-specific during gonad determination. Temperature shift experiments suggest a lack of histone enrichment is due to MPT cue, but is not reversible by FPT. Preliminary analysis of modified histones by Next-generation sequencing shows high duplication levels across samples, leaving room for technical improvement in future study.Item Investigation of the proteomic interaction profile of uncoupling protein 3 and its effect on epigenetics(2014-08) Yan, Xiwei; Mills, Edward MichaelUncoupling proteins (UCPs) are localized on the inner mitochondrial membrane (IMM) and “uncouple” the electrochemical proton gradient formed by the electron transport chain (ETC) from ATP production. Though the prototypical uncoupling protein 1 (UCP1) is known to mediate the cold-induced thermogenesis in rodents and human neonates, the physiological and biochemical functions of the homologs UCP2-5 are still under debate. Our research focuses on UCP3, the homolog prevalently expressed in skeletal muscle (SKM), the most important metabolic organs. UCP3 has long been speculated to have a pivotal role in maintaining the mitochondrial metabolism. Several biochemical roles have been attributed to UCP3, including the regulation of fatty-acid transport and oxidation, reactive oxygen species (ROS) scavenging and calcium uptake. And several proteins have been identified to directly bind with UCP3 and facilitate its function. But to further understand how UCP3 relates to different aspects of mitochondrial functions, a more comprehensive profile of the UCP3 interaction partners is needed. We performed a mass spectrometry-based experiment and successfully identified a list of over 170 potential proteins that may directly or indirectly interact with UCP3, and several novel functions of UCP3 are implied by these protein-protein interactions. Additionally, researches have shown that the metabolic defects are important contributing factors to the epigenetic changes. Considering the roles of UCP3 in sustaining the normal mitochondrial metabolism, we hypothesized that UCP3 has a novel function in regulating the genomic DNA methylation processes. The data we obtained from the pilot study confirms that loss of UCP3 will lead to aberrant DNA methylation changes. But further experiment is still needed to investigate the regulatory pathway between UCP3 and DNA methylation. The physiological role of UCP3 in defending against cancer, diabetes and obesity has been investigated, but the mechanisms how UCP3 protect the organism from these diseases have not been elucidated. Our research sheds light on the understanding of UCP3 functions and may be of significant therapeutic benefit in the prevention and treatment of these diseases.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 Roles for polyploidy, circadian rhythms, and stress responses in hybrid vigor(2014-05) Miller, Marisa Elena; Chen, Z. JeffreyHybrid plants and animals, like corn and the domestic dog, grow larger and more vigorously than their parents, a common phenomenon known as hybrid vigor or heterosis. In hybrids between Arabidopsis ecotypes or species (in allotetraploids), altered expression of circadian clock genes leads to increased starch and chlorophyll content and greater biomass. In plants and animals, circadian clock regulation plays a key role in optimizing metabolic pathways, increasing fitness, and controlling responses to biotic and abiotic stresses. In the allotetraploids, the increased level of heterosis is likely caused by interspecific hybridization as well as genome doubling. However, it is unknown how genome dosage and allelic effects influence heterosis, and whether additional clock output traits, such as stress responses, are altered in hybrids. In three related projects, the effects of genomic hybridization (including parent-of-origin effects) and genome dosage on heterosis were elucidated. In my first project, I found that although ploidy influenced many traits, including seed and cell size, biomass and circadian clock gene expression were most strongly influenced by hybridization. Additionally, parent-of-origin effects between reciprocal hybrids were frequently observed for many traits. In my second project, I described a unique role for RNA-directed DNA methylation (mainly CHH methylation) in mediating the parent-of-origin effect on expression of the circadian clock gene CCA1 in reciprocal hybrids. Altered CCA1 expression peaks were associated with heterosis of biomass accumulation in the reciprocal hybrids. Lastly, I used transcriptome sequencing in hybrids at different times of day to examine changes in downstream clock-regulated pathways. In the hybrids, many genes in photosynthetic pathways were upregulated, while many genes involved in biotic and abiotic stresses were repressed during the morning and afternoon, respectively. Additionally, natural variation between parents in stress-responsive gene expression was found to be crucial for producing vigorous hybrids. These conceptual advances increase the mechanistic understanding of heterosis, and may guide selection of parents for making better hybrids.