Differential abilities of the chicken Pit‐1 isoforms to regulate the chicken growth hormone promoter

Abstract

Pit1, a pituitary-specific transcription factor, regulates differentiation of cells of the PIT1 lineage in the anterior pituitary. PIT1 also regulates the synthesis of peptide hormones from these cell types, including growth hormone (GH). A founding member of the POU-homeodomain family of transcription factors, PIT1 is characterized by a serinethreonine rich N-terminal transactivation domain and a C-terminal POU-domain.Alternative forms of PIT1, differing from each other in the N-terminal domain have been reported in several species, but the functional implication of having multiple isoforms is not known. Several Pit1 isoform mRNAs exist in chickens which have not been characterized. The main aim of this study was to determine which, if any, of the chicken PIT1 isoforms regulated the chicken Gh (cGh) promoter. PIT1β2, a novel isoform of chicken PIT1 was discovered, and known and novel isoforms (PIT1α, PIT1β1, PIT1β2 and PIT1γ) were characterized. A luciferase reporter construct containing 1775bp of the cGh promoter driving expression of firefly luciferase was used to determine the ability of the isoforms to regulate the target gene promoter activity in chicken LMH cells. We showed that three of the isoforms, PIT1α, PIT1β1 and PIT1β2, expressed from recombinant plasmids, regulated the cGh promoter, while PIT1γ did not. All the isoforms localized to the nucleus in both non-pituitary and pituitary cells. Results from gel-shift assays show that PIT1γ did not bind the proximal PIT1-binding site of the cGh promoter as well as the other isoforms, suggesting a possible mechanism behind the inactivity. Our result did not suggest a negative regulatory role for this isoform. In contrast, we found a functional advantage for having multiple isoforms. PIT1β1, the isoform that activated the promoter most strongly, when co-transfected with other activating isoforms, such as PIT1α and PIT1β2, induced significantly higher level of activation than one isoform alone. Whether this increased activation required, or was facilitated by, heterodimerization of two isoforms is not known. Nevertheless, identification of isoforms © Copyright by Malini Mukherjee 2011 work that much harder to never fail. Dear daidamoni, my brother, what would I do without you?! Not only do we share our parents, we share our each and every happiness and loss, sadness and elation, together. Sarmila, thank you for joining our family and making our house a home again. Sonali, why didn't I get to know you better years ago? If I had, I am certain I would have not made many of the mistakes I did in my graduate career. Anyway, now that you are here, you are not going anywhere. Thank you! Susmita, I would never have reached this milestone without your help! How can I thank you enough? Thank you for being you; keep smiling, always! I would also like to thank a few dear friends and cousins, many of whom have already been or are currently pursuing the same degree in me. Thank you Tannistha, Sangeeta, Pragati, Dan, Oli, Veena, Piu. Thank you all for all your support. And l...