Transcription factor interactions at the GH promoter

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DESCRIPTION (provided by applicant): Growth hormone is a central regulator of linear growth and metabolic homeostasis. GH synthesis is restricted transcriptionally to a subpopulation of ceils in the anterior pituitary gland. Our prior studies showed transcription of the evolutionary-related GH and prolactin (PRL) genes to be cooperatively activated by the pituitary-specific transcription factor Pit-1 and other transcription factors including CCAAT/enhancer binding protein alpha (C/EBPalpha). We found molecular interactions of Pit-1, C/EBPalpha, the coactivator CBP and the basal factors TBP and TFIIB to participate in cooperative activation. We also uncovered a pan-genomic layer to cooperative activity: C/EBPalpha is held inactive at pericentromeric heterochromatin through C/EBPalpha binding to alpha-satellite repetitive DNA. Pit-1 prevents C/EBPalpah binding to a-satellite DNA and thereby relocates C/EBPalpha to the transcriptionally active subcompartment of the cell nucleus. Such functional compartmentalization is emerging as an overlooked, epigenetic regulator of transcription Thus, Pit-1 both grants C/EBPalpha access to active genes and directly cooperates with C/EBPalpha at the GH and PRL promoters. The biochemical and functional inter-relationships between Pit- 1 release of C/EBPalpha from heterochromatin and direct synergy at the promoters remain to be defined. Our hypothesis is that pituitary-specific gene transcription results from distinct molecular interactions at the promoter that are regulated by the subnuclear compartmentalization of the interacting transcription factors and co-factors. Our goal is to define the molecular basis of the promoter-targeted and epigenetic layers and their relative contributions to synergy. We will: Aim 1. Compare the effects of mutations in specific Pit- l and C/EBPalpha activities on transcriptional synergy, release of C/EBPalpha binding to alpha-satellite DNA, and Pit-1 re-location of C/EBPalpha to euchromatin, Aim 2. Define the effects of the Pit-1 and C/EBPalpha mutants on the biochemical interactions of C/EBPalpha, Pit-l, and their relevant co-factors, Aim 3. Determine the effects of Pit- l and C/EBPalpha expression on biochemical recruitment of Pit- 1, C/EBPalpha and relevant co-factors specifically at the GH and PRL promoters and at alpha-satellite DNA.