Hox-4.2 is a member of the vertebrate Hox genes. These homeobox genes encode sequence-specific DNA-binding proteins that play a role in vertebrate pattern formation. Their complex spatiotemporal expression patterns during embryogenesis are established in response to positional cues. To characterize regulatory regions involved in Hox-4.2 expression, transient expression assays in P19 murine embryonal carcinoma cells with luciferase reporter gene constructs, under the control of 5$ sp prime$ flanking sequences of Hox-4.2, were performed. Cotransfections of such luciferase reporter constructs with an expression vector for the Hox-4.2 product, resulted in the identification of a 217 bp fragment that functioned as an autoregulatory enhancer. Two binding sites for the Hox-4.2 product within this fragment were characterized by electrophoretic mobility shift assays and their role for autoregulation was demonstrated by site-directed mutagenesis. These findings suggest an autoregulatory mechanism in the control of Hox-4.2 expression and established the Hox-4.2 product as a transcriptional activator in a mammalian system. The use of luciferase reporter gene constructs in P19 embryonal carcinoma cells also led to the identification of a retinoic acid response element (RARE) in Hox-4.2 5$ sp prime$ flanking sequences which was critical for retinoic acid-mediated activation of reporter gene expression in cultured cells. The involvement of retinoic acid receptors in this response was demonstrated by mutation of the Hox-4.2 RARE and cotransfection of an expression vector for a dominant negative form of retinoic acid receptor $ alpha$, both of which inhibited the response to retinoic acid. Furthermore, retinoic acid receptors bound to the RARE as demonstrated in electrophoretic mobility shift assays. The characterization of the Hox-4.2 RARE represented one of the first direct indications for a retinoic acid receptor-mediated regulation of Hox genes. In addition, lacZ reporter gen
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.41282 |
| Date | January 1993 |
| Creators | Püpperl, Heike |
| Contributors | Featherstone, Mark S. (advisor) |
| 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 | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001351902, proquestno: NN91820, Theses scanned by UMI/ProQuest. |
Page generated in 0.0014 seconds