The constitution of a progesterone response element.
Progesterone receptors (PRs) recognize and bind to DNA in a sequence-specific manner. To define the sequence-specific determinants of PR binding to DNA, we employed a detailed series of target elements and analyzed both PR binding in vitro and PR-mediated activation of a reporter gene in vivo. These elements represent point substitution or insertion mutants of a progesterone response element derived from the mouse mammary tumor virus (MMTV). Substitution of a G for the first T in the 15-base pair (bp) MMTV progesterone response element core sequence, GTTACAAACTGTTCT, increases PR-DNA binding in vitro. All other tested mutations within this sequence either had no effect or decreased PR binding. From these analyses, we infer an optimal PR recognition sequence, -7RGNACANRNTGTNCY+7, composed of hexameric half-sites separated by precisely 3 bp and exhibiting dyad symmetry. Limited substitution of a suboptimal base for an optimal base, as in the wild type MMTV element, can be tolerated, but further suboptimal substitutions significantly decrease binding by PR. The identity of 1 bp within the hexameric half-site, position +/- 5, is unconstrained. Transition mutations, but not transversions, are permitted at position +/- 7. Here the hydrogen bond acceptor of the N-7 position of the purine ring may be involved in receptor recognition, since this feature is shared by the permitted substitutions. Insertion of a single base pair between the half-sites abolishes detectable binding, suggesting that the spatial orientation of the DNA binding domains of the monomeric receptor subunits are fixed by dimerization of the receptor. This pattern of sequence-specific recognition parallels that previously inferred for the glucocorticoid receptor, indicating that the two receptors may be unable to distinguish individual target sites. Each of the mutant response elements was also assessed for its ability to confer progestin responsiveness to a truncated MMTV promoter when introduced into a PR-containing cell line. Elements exhibiting strong receptor binding in vitro were fully inducible, whereas poorly inducible or uninducible elements displayed little or no recognition by receptor in vitro. However, some elements, though poorly bound in vitro, still activated transcription in vivo in response to hormone. In certain cases activation was as good as that seen with the wild type element. Further examination of in vitro receptor binding by this class of mutant elements using higher levels of baculovirus-expressed receptor revealed that many were indeed recognized by receptor, albeit with a lower affinity than the wild type sequence.(ABSTRACT TRUNCATED AT 400 WORDS)
Lieberman
B A
BA
Department of Pathology, University of Colorado Health Sciences Center, Denver 80262.
Bona
B J
BJ
Edwards
D P
DP
Nordeen
S K
SK
D016367:Baculoviridae / Q000235:genetics; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D001943:Breast Neoplasms; D004247:DNA / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D015735:Mifepristone / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011372:Progestins / Q000494:pharmacology*; D011397:Promegestone / Q000378:metabolism; D011980:Receptors, Progesterone / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D004279:DNA, Viral; D011372:Progestins; D011980:Receptors, Progesterone; D015735:Mifepristone; D004247:DNA; D011397:Promegestone