title: 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-1beta, 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-1betaTAD 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-1betaTAD repressed both basal and Ras-mediated TAD activity. To determine the functional interplay between TAD subregions and the beta-domain, we inserted the beta-domain every 10 AA across the 80-AA Pit-1 TAD. Like wild-type Pit-1beta, each construct retained transcriptional activity in HeLa cells and repressed the Ras response in GH4 cells. However, beta-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-1beta 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 beta-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.
authors.affiliation: Department of Medicine, University of Colorado Health Sciences Center, Aurora, Colorado 80045, USA. dawn.duval@uchsc.edu
authors.forename: Dawn L
authors.initials: DL
authors.lastname: Duval
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Jonsen
authors.forename: Scott E
authors.initials: SE
authors.lastname: Diamond
authors.forename: Patience
authors.initials: P
authors.lastname: Murapa
authors.forename: Annie
authors.initials: A
authors.lastname: Jean
authors.forename: Arthur
authors.initials: A
authors.lastname: Gutierrez-Hartmann
mesh_terms: D000818:Animals; D001665:Binding Sites; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D016296:Mutagenesis; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D050817:Transcription Factor Pit-1 / Q000502:physiology*; D015533:Transcriptional Activation; D018631:ras Proteins / Q000378:metabolism / Q000502:physiology*
chemical_list: D016271:Proto-Oncogene Proteins c-myc; D050817:Transcription Factor Pit-1; D018631:ras Proteins