1 To identify amino acid residues that define ligand binding specificity in the receptors for androgen, glucocorticoid and progesterone.
2 To define the DNA binding site repertoire for retinoid and thyroid hormone receptors and elucidate the role of receptor dimerisation in DNA binding activity.
3 To identify proteins that mediate the transcriptional activity of nuclear receptors.
4 To develop defined cell-free transcription systems consisting of purified/recombinant proteins so that the individual steps in transcriptional activation by nuclear receptors can be elucidated
5 To characterise the function of oestrogens as mitogens by analysing their effects on cell cycle related gene expression.
6 To define the molecular mechanism by which nuclear receptors regulate the transcription of the growth hormone and apolipoprotein genes.
7 To develop reporter systems in mammalian cells for research and high throughput drug screening programs.
1 The ligand binding domains of AR, GR and PR have been divided into five segments and 20/30 AR/PR and 22/30 GR/PR chimeras have been expressed. No individual segment was responsible for ligand specificity but the third segment of the AR and PR was important in ligand binding and/or transcriptional activation. Furthermore, natural androgens were found to bind to the AR at sites partially distinct from those of non-steroidal antiandrogens.
2 The retinoid and thyroid hormone receptors bind to direct repeats (DR) as heterodimers with RXR. When the repeats are spaced by 6 base pairs, or less, dimerisation is mediated by both the DNA binding and ligand binding domains but when the repeats are spaced by more than 6 base pairs the dimer interface between the DNA binding domains is not formed. RAR/RXR heterodimers bind in either orientation to such widely spaced repeats; in contrast, RAR binds to the upstream repeat in a DR-1 but the downstream repeat in a DR-5.
3 A number of proteins have been identified that interact with receptors in the presence of ligand. To date full-length cDNA clones have been identified for TIF-1, RIP140, SRC-1/RIP160, SUG-1 and TIF-2. TIF-1 is a ring-finger protein that might have a role in chromatin remodelling. RIP-140 is a novel serine threonine rich nuclear protein of unknown function. Both SRC-1 and TIF-2 stimulate the transcriptional activity of many nuclear receptors and appear to function as coactivators. SUG-1 is a protein that has previously been found as a component of the proteosome and RNA polymerase II holenzyme.
4 The following proteins have been expressed in heterologous systems, purified and shown to be functional: all steroid hormone receptors, TBP, TFIIB and a number of TAFs, nuclear factor 1 (NF1) and octamer transcription factor 1 (OTF1). NF-1 acts as a transcription factor but does not synergise with GR or PR whereas OTF-1 is a weak TF but does synergise with these receptors. Furthermore, a protein that interacts with a GR called GRIP170 also promotes the transcriptional activity of the GR.
5 The mitogenic response of MCF-7 cells to estrogen is accompanied by an increase in cyclin D1 gene transcription. DNA sequences have been mapped within the promoter that are both necessary and sufficient for this effect. The site does not bind ER itself but corresponds to a cell-specific, oestrogen induced factor.
6 Sites in the growth hormone promoter have been mapped that mediate ligand independent activation by TR and RAR. A further enhancement in transcriptional activity in the presence of T3 depends on the transcription factor GHF-1. Vitamin D antagonises the retinoid and T3 induced transcription either by competition for DNA binding sites or sequestration of downstream target proteins or both.
The nuclear receptor HNF-4 binds to and regulates transcription of the apoA/C-III/A-IV gene cluster. HNF-1 can antagonise HNF-4 probably by interacting with the LBD of the receptor and blocking AF-2 activity.
7 The low molecular weight urokinase type plasminogen activator has been developed for screening assays employing vitamin D reporters and for analysing expression in individual cells using a casein-agarose overlay. This in vitro technique allows the recovery of such cells for further characterisation.
MAJOR SCIENTIFIC BREAKTHROUGHS:
1 Complete description of the response element repertoire of nuclear receptors and their dimerisation properties on these sites.
2 Identification of five proteins that interact with receptors but have distinct functional roles. SRC-1/RIP160, TIF-2 and probably RIP140 function as co activators while TIF-1 may be involved in chromatin remodelling.
3 A cell-free transcription system has been developed using recombinant and/or purified proteins and the ability of GR and PR to synergise with OTF-1 and not NF-1 demonstrated.
4 Development of high throughput, cell-based screening assays for analysing the activity of ER and GR receptors and individual cell-based assays.
5 Identification of the DNA sequences in the cyclin D1 gene required to mediate a mitogenic response to E2 and characterisation of a cell-cycle-specific, oestrogen-regulated transcription factor.
6 Characterisation of the mechanisms by which vitamin D receptors and HNF-1 can antagonise the activity of other nuclear receptors.
Funding SchemeCSC - Cost-sharing contracts