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Identifier uch.biology.phd//2004hatzis
Title Μελέτη της μεταγραφικής ρύθμισης του ηπατικού πυρηνικού παράγοντα 4α
Alternative Title Study of the transcriptional regulation of hepatpcyte nuclear factor 4α
Author Χατζής, Παντελής
Thesis advisor Παπαματθαιάκης, Ιωσήφ
Abstract The work presented in the following pages focused on the elucidation of the mechanisms involved in the transcriptional regulation of the gene of hepatic nuclear factor 4α (HNF4α), a transcription factor which is expressed in a variety of tissues, such as the liver, the pancreas, the intestine and the kidney and which plays a significant role in the establishment and maintenance of the differentiated phenotype, in the organization of the specialized functions of those tissues. This is achieved through its participation in a regulatory network of transcription factors, the interplay and the interaction with relevant target genes of which determines the balanced expression of those gene products that are responsible for the function of the tissue. This work aimed at the understanding of the factors and mechanisms through which the expression of human HNF4α is regulated. To this end, as is presented in the first part of the work, 12.1 kb of upstream sequences of the gene were cloned and analyzed. Major sites of DNaseI hypersensitivity were identified both in the proximal promoter region and the first intron of the gene and in the upstream region, -6.6, -8.0 and -8.8 kb 5’ of the transcription start site. Transient transfection experiments using reporter genes under the control of progressive 5’ deletions of the regulatory region demonstrated that the region of the proximal promoter was sufficient for the induction of high levels of transcription in hepatic cells. DNaseI footprinting analysis and electrophoretic mobility shift assays revealed binding sites for the transcription factors HNF1α and β, Sp1, GATA6 and HNF6 in this region. Synergy between HNF1α and HNF6 and HNF1β and GATA6 produced high levels of transcription from the promoter, indicating that there are at least two alternative mechanisms for the transcriptional activation of the HNF4α gene. Chromatin immunoprecipitation experiments demonstrated that in differentiated hepatic cells it is the transcription factos HNF1α, HNF6, Sp1 and COUPTFII that interact with the promoter. The latter represses the promoter through a hormone response element that is not conserved in the homologous mouse region. This element was shown to respond to retinoic acid signaling, as incubation of cells with RA leads to the recruitment of the respective receptors to the promoter and to the upregulation of HNF4α expression levels. The second part of this work investigates the order of factor recruitment and chromatin modifications to the proximal promoter and an upstream enhancer of the human HNF4α gene, located -6.6 kb upstream of the transcription start site, around the initial activation of the gene during the differentiation of human enterocytes. These experiments demonstrated that two independent protein complexes formed in the two regulatory regions, which included transcription factors, acetyltransferases and chromatin remodelers at the enhancer and transcription factors, general transcription factors and RNA polymerase-II at the promoter. These events were followed by a unidirectional movement of the enhancer complex along the intermediate regions until it encountered the proximal promoter. This movement was accompanied by a spreading of histone hyperacetylation from the enhancer towards the promoter. The initiation of transcription from the gene coincided with the formation of a stable enhancer-promoter complex and the remodeling of the nucleosomes covering the transcription start site. These results gave a more complete picture of the events that take place in the regulatory regions of human HNF4α around the initial activation of the gene during a cellular differentiation model. They also provided experimental validation for a mechanism by which the communication between topologically remote regulatory regions is achieved through their physical contact and thus gave a mote complete picture of the complexity of the process of the transcriptional of at least one tissue-specific gene.
Language Greek
Subject Ήπαρ; Μεταγραφικές ρυθμίσεις; Ενισχυτές; Υποκινητής; Έντερα
Issue date 2004-03-30
Collection   School/Department--School of Sciences and Engineering--Department of Biology--Doctoral theses
  Type of Work--Doctoral theses
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