Browsing by Subject "Transcription, Genetic"
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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 Roles of Class II Histone Deacetylases in the Cardiovascular System(2005-12-19) Chang, Shurong; Olson, Eric N.Histone acetylation/deacetylation, which is orchestrated by two opposing families of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs), represents one of the fundamental mechanisms to control gene transcription. Class II histone deacetylases regulate developmental and physiological processes through interaction with and repression of a variety of transcription factors, including myocyte enhancer factor 2 (MEF2). Using gene targeting combined with biochemical assays, the function and regulation of class II HDACs are being elucidated. Here I show that in the absence of HDAC5, the heart becomes profoundly enlarged in response to calcineurin signaling and pressure overload. The cardiac phenotype of HDAC5 mutant mice is remarkably similar to that of HDAC9 mutant mice, strongly suggesting that these two HDACs play comparable roles in the control of cardiac growth. HDAC 5 and 9 also appear to play overlapping roles during heart development, as evidenced by cardiac malformations that occur in mice lacking both genes. Histone deacetylase 7 (HDAC7) is specifically expressed in the endothelium during early embryogenesis. Disruption of the HDAC7 gene in mice results in embryonic lethality due to a failure in endothelial cell-cell adhesion and consequent dilatation and rupture of blood vessels. HDAC7 represses MMP10 gene transcription by associating with MEF2, a direct activator of MMP10 transcription and essential regulator of blood vessel development. By in vitro kinase assays, I showed that class II HDACs are substrates for a novel stressresponsive kinase(s) specific for conserved serines that regulate MEF2-HDAC interactions. A eukaryotic expression screen revealed a remarkable variety of signaling pathways that converge on the signal-responsive phosphorylation sites in HDAC5, thereby enabling HDAC5 to connect extracellular signals to the genome. Microarray analysis was performed to provide a genome-wide molecular description of the target genes of the HDAC5/MEF2 complex in the muscle differentiation pathway. This approach was validated by characterizing the transcriptional regulatory element of a novel gene identified in the microarray analysis, which was confirmed as a direct target of MEF2. Taken together, this study provided mechanistic insights into the regulatory pathways for class II HDACs and the biological functions of these histone modifying enzymes.Item Transcriptional Gene Silencing in Mammalian Cells by MicroRNAs That Target Gene Promoters(2011-08-26T17:35:48Z) Younger, Scott Thomas; Corey, David R.A rich history exists for RNA-based regulation of gene transcription. It was reported more than a decade ago that RNA is capable of inducing DNA methylation and transcriptional gene silencing in plants. It was subsequently shown that small RNAs are involved in the establishment of heterochromatic regions of the yeast genome. More recently it has been demonstrated that small duplex RNAs designed to be complementary to gene promoters are potent regulators of gene transcription in mammalian cells. Potent and robust transcriptional regulation by designed small RNAs suggests the existence of endogenous mechanisms that facilitate recognition of gene promoters by small RNAs in mammalian cells. microRNAs (miRNAs) are endogenous small RNAs that regulate gene expression post transcriptionally through base complementarity to target sequences within 3’ UTRs of mRNA transcripts. In this body of work I test the hypothesis that miRNAs can also recognize sequences within gene promoters using two alternative approaches. In the first approach I computationally evaluate the potential for miRNAs to recognize gene promoters by performing a genome-wide survey of putative miRNA target sites within promoter sequences. In the second approach I use the well characterized human progesterone receptor (PR) gene as a model to experimentally validate that miRNAs possess the ability to regulate transcription in a cell culture system. Upon completion of this work I found that gene promoters are significantly enriched for miRNA target sites. Furthermore, the frequency of miRNA target sites within promoter sequences is comparable to their frequency within 3’ UTRs. I experimentally screened multiple miRNAs predicted to target the PR gene promoter, identified several that were capable of inhibiting transcription of the PR gene, and characterize the mechanism of transcriptional silencing. miRNAs have been understood to regulate gene expression at the post transcriptional level through recognition of 3’ UTRs within mRNA transcripts. My study extends miRNA function to recognition of sequences within gene promoters. Sequence specific recognition of gene promoters by miRNAs may complement protein transcription factors. In addition, the ability of small RNAs to rapidly evolve specificity for new sequences would have evolutionary advantages.Item Transcriptional Regulation of Dehydroepiandrosterone Sulfotransferase (SULT2A1) By Estrogen-Related Receptor-alpha (ERR-alpha)(2007-12-03) Seely, Jeremiah Brent; Rainey, WilliamThe estrogen-related receptors (ERR alpha , beta and gamma ) are a subfamily of orphan nuclear receptors (designated NR3B1, NR3B2 and NR3B3) that are structurally and functionally related to estrogen receptors alpha and beta . Herein we test the hypothesis that ERRalpha regulates transcription of the genes encoding the enzymes involved in adrenal steroid production. Real-time RT-PCR was first used to determine the levels of ERRalpha mRNA in various human tissues. Adult adrenal levels of ERRalpha transcript were similar to that seen in heart, which is known to highly express ERRalpha . Expression of ERRalpha in the adult adrenal was then confirmed using western blotting and immunohistochemistry. To examine the effects of ERRalpha on steroidogenic capacity we used reporter constructs with the 5′-flanking regions of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage (CYP11A), 3beta hydroxysteroid dehydrogenase type II (HSD3B2), 17alpha hydroxylase, 17,20 lyase (CYP17), and DHEA sulfotransferase (SULT2A1). Cotransfection of these reporter constructs with wild-type ERRalpha or VP16-ERRalpha expression vectors demonstrated ERRalpha enhanced reporter activity driven by flanking DNA from CYP17 and SULT2A1. SULT2A1 promoter activity was most responsive to the ERRalpha and VP16-ERRalpha , increasing activity 2.6- and 79.5-fold respectively. ERRalpha effects on SULT2A1 were greater than the stimulation seen in response to steroidogenic factor 1 (SF1). Transfection of serial deletions of the 5′-flanking DNA of the SULT2A1 gene and EMSA experiments indicated the presence of three functional regulatory cis-elements which shared sequence similarity to binding sites for SF1. Taken together, the expression of ERRalpha in the adrenal and its regulation of SULT2A1 suggest an important role for this orphan receptor in the regulation of adrenal steroid production.Item Transcriptional Regulation of Neural Crest-derived Pharyngeal Arch Artery Development(2004-12-15) Ivey, Kathryn Nicole; Mendelson, Carole R.The heart is the first organ to form and is required for growth and development of mammalian embryos. As the heart matures, formation of the outflow tract is vital to establish appropriate connections with the vasculature. This process requires contribution from specialized neural crest cells, which originate in the neural folds and migrate to give rise to specific segments of the great vessels as well as particular facial structures. Many syndromic birth defects in humans affecting the heart and face arise as a result of inappropriate development of neural crest cells and can be modeled in animals through ablation of premigratory neural crest cells or targeted deletion of genes required for their proliferation, migration or survival. However, the transcription factors and signaling molecules that specify unique subsets of neural crest cells are still being detailed. This thesis represents efforts to understand those particulars. Endothelin-1 (Et-1), a small signaling peptide, is important for development of neural crest-derived structures and targeted deletion of the gene encoding Et-1 or its receptor, Endothelin-A (EtA), results in craniofacial and outflow tract anomalies along with downregulation of particular neural crest-derived pharyngeal arch mesenchyme markers. Mice deficient for both Gaq and Ga 11 are phenotypically similar to EtA or Et-1-null mice. My analysis of expression patterns of Et-1 dependent and independent transcription factors in Gaq /G a11-deficient embryos revealed that expression of genes encoding Et-1 dependent transcription factors was specifically downregulated in the pharyngeal arches of Gaq /G a11-deficient mice indicating that Gaq and Ga11 proteins serve as intracellular mediators of Et-1 signaling in the pharyngeal arch mesenchyme. Et-1 is also important for development of the neural crest-derived fetal vessel, the ductus arteriosus, which bridges the pulmonary and systemic circulations during gestation and must close at birth for extrauterine survival. The ductus arteriosus is composed of highly differentiated, contractile smooth muscle. I found that Et-1 is expressed specifically in the smooth muscle of the ductus arteriosus during development along with Hif2a and Ap2ᠡnd that, through epistatic relationships and negative feedback regulation, these three factors cooperatively regulate development of the specialized, neural crest-derived smooth muscle of this vessel.