Abstract

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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK054345-06
Application #
7020650
Study Section
Endocrinology Study Section (END)
Program Officer
Malozowski, Saul N
Project Start
1999-07-01
Project End
2009-02-28
Budget Start
2006-03-01
Budget End
2007-02-28
Support Year
6
Fiscal Year
2006
Total Cost
$312,893
Indirect Cost

  Institution

Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143

  Publications

Kofoed, Eric M; Guerbadot, Martin; Schaufele, Fred (2010) Structure, affinity, and availability of estrogen receptor complexes in the cellular environment. J Biol Chem 285:2428-37
Kofoed, Eric M; Guerbadot, Martin; Schaufele, Fred (2008) Dimerization between aequorea fluorescent proteins does not affect interaction between tagged estrogen receptors in living cells. J Biomed Opt 13:031207
Liu, Xiaowei; Wu, Bo; Szary, Jaroslaw et al. (2007) Functional sequestration of transcription factor activity by repetitive DNA. J Biol Chem 282:20868-76
Day, Richard N; Schaufele, Fred (2005) Imaging molecular interactions in living cells. Mol Endocrinol 19:1675-86
Ross, Sarah E; Radomska, Hanna S; Wu, Bo et al. (2004) Phosphorylation of C/EBPalpha inhibits granulopoiesis. Mol Cell Biol 24:675-86
Enwright 3rd, John F; Kawecki-Crook, Margaret A; Voss, Ty C et al. (2003) A PIT-1 homeodomain mutant blocks the intranuclear recruitment of the CCAAT/enhancer binding protein alpha required for prolactin gene transcription. Mol Endocrinol 17:209-22
Schaufele, Fred; Wang, Xia; Liu, Xiaowei et al. (2003) Conformation of CCAAT/enhancer-binding protein alpha dimers varies with intranuclear location in living cells. J Biol Chem 278:10578-87
Day, Richard N; Voss, Ty C; Enwright 3rd, John F et al. (2003) Imaging the localized protein interactions between Pit-1 and the CCAAT/enhancer binding protein alpha in the living pituitary cell nucleus. Mol Endocrinol 17:333-45
Weatherman, R V; Chang, C-Y; Clegg, N J et al. (2002) Ligand-selective interactions of ER detected in living cells by fluorescence resonance energy transfer. Mol Endocrinol 16:487-96
Liu, Weiqun; Enwright 3rd, John F; Hyun, William et al. (2002) CCAAT/enhancer binding protein alpha uses distinct domains to prolong pituitary cells in the growth 1 and DNA synthesis phases of the cell cycle. BMC Cell Biol 3:6

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