An alternatively spliced Pit-1 isoform altered in its ability to trans-activate.

Although alternative splicing has been shown to give rise to isoforms of a number of transcription factors, such isoforms have not previously been detected for the POU homeodomain protein Pit-1. Screening of a rat pituitary GH3 cell cDNA expression library yielded a clone, termed pCMVPit-1a, encoding a 35.8 kD protein (Pit-1a) containing a 26 amino acid insert in the Pit-1 trans-activation domain. The position of the insert, plus Southern blot analysis, implied that Pit-1a mRNA arises by alternative splicing of the Pit-1 gene transcript. Pit-1a mRNA was detected in GH3 rat pituitary tumor cells at levels about 1/7 that of Pit-1 mRNA. Pit-1a mRNA-specific sequences were also detected in rat and mouse pituitary, and in mouse thyrotropic tumor TtT cells. DNA mobility shift assays showed that Pit-1a binds specifically to Pit-1 binding sites in the proximal prolactin promoter, but produces DNA-protein complexes of markedly different mobilities than Pit-1. In stably transfected CHO cells which accumulated approximately equal levels of either of the two proteins, Pit-1 trans-activated a prolactin promoter-driven CAT construct, while Pit-1a yielded no detectable transactivation, implying a trans-activation ratio for Pit-1a/Pit-1 of less than 0.05. Thus, the insertion of 26 amino acids of similar composition into the activation domain of Pit-1 has at once affected both the mode of binding of this protein and its ability to function as a trans-activator.

Use of a selectable reporter for the isolation of mammalian regulatory genes.

A general method is described for isolating the genes encoding differentiation-specific activators of transcription using genetic selection. Employing regulation of the prolactin encoding gene (PRL) as a model, we have shown that the hamster dihydrofolate reductase-encoding gene (dhfr) is an effective dominant selectable reporter in this methodology. The dhfr coding region was ligated to the rat PRL promoter, and the resultant construct was stably transfected into DHFR- Chinese hamster ovary (CHO) cells, where it had little or no activity. Transfection of these cells with plasmid DNA, containing the coding region of a pituitary-specific transcription factor (Pit-1/GHF-1) in a eukaryotic expression vector, resulted in transfectants in which activation of the chimeric construct, pPRLdhfr, had occurred, enabling these cells to be selected on the basis of their DHFR+ phenotype. Our results suggest that this strategy could be used to isolate unknown genes that regulate a variety of differentiated functions.