Lees, Jacqueline A.; (1990) A functional analysis of the mouse oestrogen receptor. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Steroid receptors represent a class of ligand inducible transcription factors that regulate the rate of transcription of hormone responsive genes by interaction with defined enhancer elements. This thesis examines the mechanisms of receptor-DNA interaction and transcriptional activation using the mouse oestrogen receptor as a model system. cDNA clones encoding the mouse oestrogen receptor have been isolated and characterised. Subsequently, these clones have been used to generate a series of receptor deletion mutants, which have been expressed both in vitro and in transient transfection assays in order to map sequences responsible for DNA binding, ligand binding and transcriptional activation. This study has shown that, like many other transcription factors, the sequence specific DNA binding activity of the receptor is mediated by a discrete domain (amino acids 121-339), encompassing two zinc finger motifs, that is required, but not sufficient, for transcriptional activation. However, high affinity DNA binding requires additional residues, contained within the C-terminus of the receptor, that are essential for receptor dimerisation. By generating receptor fusion proteins it has been possible to demonstrate that residues 501-522 of the mouse oestrogen receptor are sufficient to restore the DNA binding and dimerisation activity of C-termlnal deletion mutants, suggesting that they encode a major portion of the mouse oestrogen receptor dimer interface. This 22 amino acid domain is well conserved throughout the steroid receptor superfamily, but does not resemble the dimerisation domains of other eukaryotic transcription factors, suggesting that it may represent a novel dimerisation motif. Furthermore, site-directed mutagenesis of the wild-type mouse oestrogen receptor has allowed the identification of residues within this region that are required for either dimerisation (Arg-507, Leu-511 and lle-518) or ligand binding (lle-518 and Gly-525) suggesting that these two functional domains overlap and that the steroid binding may take place at the dimer interface. In addition to the ligand binding and dimerisation domains, the C-terminus of the mouse oestrogen receptor also contains an oestradiol inducible transactivation domain that remains active when fused to a heterologous (Lex A) DNA binding domain. Whilst these three functions clearly overlap, they are not entirely coincident since deletion of receptor residues between 552 and 538 almost completely abolishes the activity of this oestradiol inducible transactivation domain without the loss of either dimerisation or ligand binding activity. It is not clear how ligand binding activates this transactivation domain, but oestradiol binding alters the mobility of the receptor-DNA complex in a band shift assay, suggesting that it may induce a conformational change in the receptor that could bring together dispersed C-terminal residues to form this motif. The activity of the receptor deletion mutants has shown that the receptor also contains an N-terminal transactivation domain that is able to function in the absence of oestradiol. Furthermore, a number of these mutants differentially stimulated different target promoters, suggesting that the activity of these two transactivation domains is modulated by the test promoter environment, presumably because of their differing abilities to cooperate with heterologous transcription factors.

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