The Journal of clinical endocrinology and metabolism · 2005
The role of CBP/p300 interactions and Pit-1 dimerization in the pathophysiological mechanism of combined pituitary hormone deficiency.
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Cohen Ronald N, Brue Thierry, Naik Karuna, Houlihan Christine A, Wondisford Fredric E, Radovick Sally
Аннотация
CONTEXT: Combined pituitary hormone deficiency (CPHD) in humans is caused by mutations of pituitary-specific transcription factors such as Pit-1. Although many patients with CPHD have an autosomal recessive disorder caused by a Pit-1 DNA-binding mutation, there are a number of reports of mutant Pit-1 molecules that either by prediction or through experimentation bind normally to DNA.
OBJECTIVE: The objective of this study was to understand the pathophysiological mechanisms of mutant Pit-1 molecules with intact DNA binding.
DESIGN: DNA-binding and functional studies were used to assess five Pit-1 mutations: F135C, R143Q, A158P, K216E, and R271W.
RESULTS: In gel-shift studies using well-characterized DNA-binding elements from the GH and prolactin genes, the K126E mutant displayed markedly enhanced Pit-1 dimer binding to either element, whereas the R271W mutant bound with high avidity, but only as a monomer. In contrast, the R143Q mutant was unable to bind these elements, and the F135C and A158P mutants displayed near-normal DNA-binding characteristics. We observed that CBP/p300 bound poorly to the A158P and K216E mutant Pit-1 molecules, but bound normally to the F135C, R143Q, and R271W mutants. In functional assays, CBP/p300 cotransfection with mutant Pit-1 expression vectors resulted in less transactivation of either the GH or prolactin reporter genes.
CONCLUSIONS: From these studies, we suggest that CBP/p300 recruitment and Pit-1 dimerization are necessary for Pit-1 target gene activation and are important in the pathogenesis of CPHD.
OBJECTIVE: The objective of this study was to understand the pathophysiological mechanisms of mutant Pit-1 molecules with intact DNA binding.
DESIGN: DNA-binding and functional studies were used to assess five Pit-1 mutations: F135C, R143Q, A158P, K216E, and R271W.
RESULTS: In gel-shift studies using well-characterized DNA-binding elements from the GH and prolactin genes, the K126E mutant displayed markedly enhanced Pit-1 dimer binding to either element, whereas the R271W mutant bound with high avidity, but only as a monomer. In contrast, the R143Q mutant was unable to bind these elements, and the F135C and A158P mutants displayed near-normal DNA-binding characteristics. We observed that CBP/p300 bound poorly to the A158P and K216E mutant Pit-1 molecules, but bound normally to the F135C, R143Q, and R271W mutants. In functional assays, CBP/p300 cotransfection with mutant Pit-1 expression vectors resulted in less transactivation of either the GH or prolactin reporter genes.
CONCLUSIONS: From these studies, we suggest that CBP/p300 recruitment and Pit-1 dimerization are necessary for Pit-1 target gene activation and are important in the pathogenesis of CPHD.