Effects of wild type and mutant estrogen receptors on DNA flexibility, DNA bending, and transcription activation.

Abstract

We examined the ability of wild type (WT) estrogen receptor (ER) and mutant ERs to induce distortion and directed bends in DNA fragments containing estrogen response elements and then monitored the ability of these receptors to activate transcription. The ER mutants had either 108 (109-595 ER) or 175 (delta AB ER) amino acids deleted from the amino terminus; 42 (delta F ER) or 65 (1-530 ER) amino acids deleted from the carboxy terminus; or a single point mutation at amino acid 380 (glu-->gln) in the ER hormone binding domain (E380Q ER). Circular permutation analysis was used to determine the degree of distortion induced in estrogen response element-containing DNA fragments (65 degrees for WT ER and E380Q ER, 56 degrees for 109-595 ER, 54 degrees for delta AB ER, 63 degrees for delta F ER, and 60 degrees for 1-530 ER). Phasing analysis delineated the magnitude of directed DNA bends (8.3 degrees for WT ER, 6.9 degrees for 109-595 ER, 6.5 degrees for delta AB ER, 10.6 degrees for delta F ER, 12.4 degrees for 1-530 ER, and 10.2 degrees for E380Q ER) and demonstrated that the direction of the bend was always toward the major groove of the DNA helix. The ability of each receptor to induce transcription of an estrogen-responsive reporter plasmid (E380Q ER > WT ER = delta F ER > 109-595 ER > delta AB ER > 1-530 ER) was related to the ability of the receptor to induce appropriate distortion (63 degrees-65 degrees) and directed DNA bending (8 degrees-10 degrees) angles and the presence of transcription activation functions AF-1 and AF-2. These studies suggest that ER-induced DNA bending is one part of a multistep process involved in regulating estrogen-responsive genes.