We have used gel mobility shift assays to examine changes in DNA bending induced by binding of human estrogen receptor (hER) to a series of estrogen response element (ERE) containing DNA fragments. Competition experiments with ERE-containing DNA fragments and antibody supershift experiments demonstrated that ER in crude extracts from MCF-7 human breast cancer cells exhibited specific interaction with the ERE. Using DNA bending standards, we found that binding of ER to a single ERE induced a reproducible DNA bend of 56 degrees. This was 1.65-fold greater than the 34 degrees bending angle we recently reported for binding of bacterially expressed ER DNA binding domain. The DNA bending angle induced was the same whether the salt-extracted receptor was unoccupied, occupied by 17 beta-estradiol, or occupied by trans-hydroxytamoxifen. To determine if proteins associated with ER in MCF-7 cells affect the degree of bending, we examined the ability of partially purified hER expressed in yeast to bend DNA. The degree of bending induced by the partially purified yeast ER was the same as the bending induced by crude MCF-7 cell ER. More highly purified ER from yeast extracts did not bind to an ERE-containing DNA fragment, suggesting that additional proteins may play an important role in the interaction of the ER with the ERE. When two EREs were present in the DNA fragment, a small but reproducible increase in bending was observed. Our demonstration that binding of hER to the ERE induces DNA bending suggests a possible role for DNA bending in ER-induced transcription activation.
ASJC Scopus subject areas
- Molecular Biology