The role of CBP/p300 interactions and Pit-1 dimerization in the pathophysiological mechanism of combined pituitary hormone deficiency.
BACKGROUND
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.


METHODS
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.
Cohen
Ronald N
RN
Section of Pediatric and Adult Endocrinology, Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA. roncohen@medicine.bsd.uchicago.edu
Brue
Thierry
T
Naik
Karuna
K
Houlihan
Christine A
CA
Wondisford
Fredric E
FE
Radovick
Sally
S
D001483:Base Sequence; D002478:Cells, Cultured; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D007018:Hypopituitarism / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D008958:Models, Molecular; D010907:Pituitary Hormones / Q000172:deficiency*; D011485:Protein Binding; D050817:Transcription Factor Pit-1 / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection; D050880:p300-CBP Transcription Factors / Q000502:physiology*
D010907:Pituitary Hormones; D050817:Transcription Factor Pit-1; D008156:Luciferases; D050880:p300-CBP Transcription Factors