Differential utilization of transcription activation subdomains by distinct coactivators regulates Pit-1 basal and Ras responsiveness

Abstract

The POU-homeodomain transcription factor Pit-1 governs ontogeny and cell-specific gene expression of pituitary lactotropes, somatotropes, and thyrotropes. The splice isoform, Pit-1β, inserts a 26-amino acid (AA) repressor at AA48 in the Pit-1 transcription activation domain (TAD). The Pit-1 TAD contains a basal regulatory subregion, R1 (AA1-45), and a basal and Ras-responsive region, R2 (AA46-80). To precisely map these activities, we generated GAL4-Pit-1/Pit-1βTAD fusions and, in full-length HA-Pit-1, a series of substitution mutants of R2. Analysis in GH4 cells identified an activation domain at AA50-70, followed by an overlapping, dual-function, Ras-responsive-inhibitory domain, located from AA60-80. In contrast, GAL4-Pit-1βTAD repressed both basal and Ras-mediated TAD activity. To determine the functional interplay between TAD subregions and the β-domain, we inserted the β-domain every 10 AA across the 80-AA Pit-1 TAD. Like wild-type Pit-1β, each construct retained transcriptional activity in HeLa cells and repressed the Ras response in GH4 cells. However, β-domain insertion at AA61 and 71 resulted in greater repression of Ras responsiveness, defining a critical R2 TAD spanning AA61-71 of Pit-1. Furthermore, Ras activation is augmented by steroid receptor coactivator 1, whereas cAMP response element binding protein-binding protein is not a Ras mediator in this system. In summary, the Pit-1/Pit-1β TADs are composed of multiple, modular, and transferable subdomains, including a regulatory R1 domain, a basal activation region, a selective inhibitory-Ras-responsive segment, and a β-specific repressor domain. These data provide novel insights into the mechanisms by which the Pit-1 TAD integrates DNA binding, protein partner interactions, and distinct signaling pathways to fine-tune Pit-1 activity. Copyright © 2007 by The Endocrine Society.