S. Thenot et al., Effect of ligand and DNA binding on the interaction between human transcription intermediary factor 1 alpha and estrogen receptors, MOL ENDOCR, 13(12), 1999, pp. 2137-2150
Hormonal regulation of gene activity is mediated by nuclear receptors actin
g as ligand-activated transcription factors. To achieve efficient regulatio
n of gene expression, these receptors must interact with different type of
molecules: 1) the steroid hormone, 2) the DNA response element, and 3) vari
ous proteins acting as transcriptional cofactors, In the present study, we
have investigated how ligand and DNA binding influence the in vitro interac
tion between estrogen receptors (ERs) and the transcription intermediary fa
ctor hTIF1 alpha (human transcriptional intermediary factor 1 alpha). We fi
rst optimized conditions for the coactivator-dependent receptor ligand assa
y to lower ED50, and we then analyzed the ability of various natural and sy
nthetic estrogens to allow the binding of the two types of proteins. Result
s were compared with the respective affinities of these ligands for the rec
eptor. We then developed a protein-protein-DNA assay allowing the quantific
ation of cofactor-ER-estrogen response element (ERE) complex formation in t
he presence of ligand and used measurements of fluorescence anisotropy to d
efine the equilibrium binding parameters of the interaction. We demonstrate
d that the leucine-charged domain of hTIF1 alpha is sufficient to interact
with ERE-bound ER alpha in a ligand-dependent manner and showed that bindin
g of ER alpha onto DNA does not significantly effect its hormone-dependent
association with TIF1 alpha. Finally, we show that, mainly in the absence o
f hormone, hTIF1 alpha interacts better with ERP than with ER alpha indepen
dently of the presence of ERE.