Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2011) 25 P244

SFEBES2011 Poster Presentations Pituitary (41 abstracts)

BAC recombineering to understand the role of the alternative promoter in the regulation of prolactin expression

Raheela Awais 1 , Anne McNamara 1, , Claire Harper 1 , Anthony Adamson 2 , Dave Spiller 1 , Sabrina Semprini 3 , John Mullins 3 , Julian Davis 4 & Michael R H White 1,

1Institute of Integrative Biology, University of Liverpool, Liverpool, UK; 2Faculty of Life Sciences, University of Manchester, Michael Smith Building, Manchester, UK; 3Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, UK; 4Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK.

Alternative promoters control many genes including prolactin (PRL), which in man is expressed at extra-pituitary sites controlled by an alternative promoter located 5.8 Kbp upstream of the pituitary transcription start site. Previous studies using short promoter fragments may be misleading, as the human hPRL genomic locus has many conserved far-upstream regions. To study the function of the alternative promoter, we engineered a bacterial artificial chromosome (BAC) expressing Luciferase (Luc) under the control of a 163 Kb hPRL genomic fragment (1), deleting the entire 5 kbp pituitary promoter by BAC-recombineering, leaving intact the upstream exon 1a and alternative promoter. The alternative splice acceptor site was reinserted after mutating three adjacent Pit-1 binding sites. This alternative PRL promoter BAC- Luc reporter gene (AP-BAC-Luc) was used to generate stably-transfected rat pituitary GH3 and Jurkat lymphoblastoid cell lines, representing cellular models of pituitary and extra-pituitary sites of expression.

Surprisingly, AP-BAC-Luc was active in both GH3 and Jurkat cell lines, with greater signal measured in pituitary cells. PRL-regulating stimuli, including FGF2, forskolin and PMA, induced an increase in AP-BAC-Luc expression in the GH3 recombinant stable clones (similar fold-activation to that seen from the pituitary PRL promoter). Oestrogen and TNFα failed to induce AP-BAC-Luc expression, in contrast to their strong induction of PRL pituitary promoter activity in GH3 cells. Real-time luminescence imaging of living cells showed dynamic patterns of expression, as seen with pituitary promoter constructs, indicating that cyclical transcriptional function is shared by the alternative promoter.

These results provide new insights into how alternative promoters differentially or coordinately regulate PRL expression. Our data indicate that both promoters share activity within the same cell, and both display cyclical function. In this context, the AP-BAC-Luc generated in this study offers a powerful model approach to understand mechanisms responsible for differential activation of alternative promoters in alternative sites of gene expression.

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