GH acts through its specific receptor, GHR. In the human liver, more than twelve hGHR mRNAs are transcribed from unique TUTR exons, seven of which are found in two small clusters: module A (V2,V9,V3) and module B (V7,V1,V4,V8). While module A mRNAs are ubiquitously expressed, module B transcripts are restricted to normal postnatal liver, suggesting developmental- and liver-specific regulation of the hGHR gene. To begin characterising the elements regulating module B mRNA expression, I studied the promoter region of the V1 exon, the most abundantly expressing variant in liver. A 1.8 kb region upstream of the V1 transcriptional start site (TSS) was actively repressed in transient transfection assays (TTAs). However, T or 3' deletions relieved the suppression, suggesting the presence of multiple negative and positive regulatory elements. Two sites for growth factor independence-1 (Gfi-1) and Gfi-1b and a GAGA element were identified in the most 3' 300 by regulatory region. Gfi-1 was detected by western blot in human foetal and postnatal liver. Gfi-1 and Gfi-lb strongly repressed while the Drosophila GAGA factor (GAF) stimulated V1 transcription through their specific sites, as determined by TTAs and site-directed mutagenesis (SDM). Six putative sites for hepatic nuclear factor-4 (HNF-4) were also identified in the 1.8 kb region. HNF-4alpha is developmentally regulated in human liver: HNF-4alpha,2 and alpha8 proteins are expressed in foetal hepatocytes but only HNF-4alpha2 is detected postnatally. TTAs and SDM demonstrated that HNF-4alpha2 and HNF-4alpha8 have a similar dual effect on V1 transcription: activation via site #1 in the proximal promoter and repression through site #6, ~1.7 kb upstream of the TSS. Results from EMSA/EMSSA/ChIP analyses suggest these sites are bound by HNF-4alpha. / Thus, V1 transcriptional activity is repressed by Gfi-1/Gfi-1b, stimulated through a GAGA element by GAF, and repressed or stimulated by HNF-4alpha. (2+8) depending on the site. Similar sites are present in homologous regions of the bovine, ovine and mouse GHR genes suggesting that their regulatory roles are conserved. However, none of these factors individually appear to be responsible for the postnatal hepatic-specific expression of V1 mRNA. To define the specific regulatory mechanisms, future studies should examine their interactions with additional liverenriched factors (e.g. C/EBPalpha).
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.102688 |
| Date | January 2006 |
| Creators | Osafo, Joy Kwakyewaa. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | © Joy Kwakyewaa Osafo, 2006 |
| Relation | alephsysno: 002565523, proquestno: AAINR27825, Theses scanned by UMI/ProQuest. |
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