Functional Characterization of Nuclear Receptor and Co-activator Binding Loci in the Human Genome

Steroid hormones, such as oestrogen, mediate their effects via activation of oestrogen regulated genes using nuclear receptors. Selective oestrogen receptor modulators (SERMs), such as Tamoxifen, are used to treat oestrogen responsive breast cancers, hctioning to act as oestrogen antagonists, preventing the oestrogen receptor biding DNA and blocking gene expression. However, Tamoxifen has been identified as an oestrogen agonist in other tissues which can often lead to secondary tumors in the years following the treatment. Identification of the genomic regions where SERMs can act as oestrogen agonists can possibly lead to the development of gene targeted therapies or other alternatives to prevent this from occurring. Investigations of a previously constructed ERE reporter library were directed towards the isolation of SERM responsive plasmids. Identified SERM responsive plasmids were compared to oestrogen responsive reporter piasmids on the basis of their ability to mediate trauscription in conjunction with other nuclear -tors and coactivators to establish potential novel interactions. ERa mutation analysis was performed to attempt to identify the mechanism by which these sequences have the ability to be activated with the addition of SERMs. It was found that SRC-3, a member of the SRC family of coactivators, had the ability to corepress SERMs via oestrogen receptors, a response not documented previously. Further to results obtained from the investigations of the SERM responsive reporter plasmids, DamID technology was employed to attempt to construct two libraries of sequences that associated, indirectly, with SRC-1A or SRC-3, via ligand bound oestrogen or progesterone. Gateway technology was used to transfer cloned sequences in to reporter plasmids to enable the luciferase assay to be employed in order to determine the functionality of the isolated sequences. - ~.... - Transactivation assays suggested that the attempt to direct the SRC-1A:Dam fusion protein to bind to DNA via either ligand bound oestrogen or progesterone failed, however following reporter plasmid sequencing bioinformatical studies were performed q d a number of possible transcription factor binding,motifs were identified in the sequences. Further transactivation assays indicated SRC-1A:Dam had, in a subset of the reporter plasmids, bound via the orphan nuclear receptors RORa and COUP TFI. Results suggest that SRC-1A may mediate this association and coactivate gene transcription; the mechanism behind this novel finding remains to be elucidated