Transcriptional Regulation of Promoters Containing Different Estrogen Response Elements

104 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2000.The actions of estrogen are mediated through estrogen receptor alpha (ERalpha), and ER beta (ERbeta), which bind to estrogen response elements (EREs) present in target genes and activate transcription, In order to gain a better understanding of how individual EREs influence the ability of (ERalpha), to activate transcription, transient transfections were carried using the consensus A2 ERE, the imperfect B1 or oxytocin (OT) EREs which differ from the A2 ERE in the 5' half site, or the imperfect pS2 ERE which differs from the A2 ERE in the 3' half site. (ERalpha), activated transcription from the A2 ERE to the greatest extent. Furthermore, the receptor was a more potent activator of transcription from the OT ERE than from the pS2 or B1 ERE. To delineate how these different ERE sequences bring about varied levels of transcription, the interaction of ERalpha with each of the four EREs was compared. Gel mobility shift assays, DNase I footprinting experiments and methylation interference experiments demonstrated that the ERalpha DBD bound as a dimer to the A2 ERE but bound first as a monomer and then as a dimer to the pS2 ERE. Partial proteolytic cleavage experiments of the A2, pS2, B1, or OT ERE-bound ERalpha provide evidence that differences in ERE sequence leads to changes in full-length receptor conformation and suggests that individual EREs act as allosteric modulators of receptor conformation. Interestingly, nuclear proteins from ERalpha-containing MCF-7 breast cancer cells formed different complexes with DNA fragments containing the A2, pS2, B1, or OT ERE in gel mobility shift assays suggesting that ERE-induced receptor conformation may dictate which nuclear proteins are recruited to the promoter region of target genes. When the affinity of ERalpha for each of the four EREs was assessed by gel mobility shift competition assays, ERalpha had a higher affinity for the A2 ERE than the pS2, B1, or OT ERE but had a similar affinity for the three imperfect EREs. These combined experiments indicate that both DNA-induced changes in ERalpha conformation and the affinity of the receptor for an ERE influence the regulation of target gene expression.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD