Estrogen Receptor Binding to DNA Is Not Required for Its Activity through the Nonclassical AP1 Pathway

Abstract

In the classical signaling pathway, the estrogen receptor (ER) binds directly to estrogen response elements (EREs) to regulate gene transcription. To test the hypothesis that the nonclassical pathway involves ER interactions with other proteins rather than direct binding to DNA, mutations were introduced into the DNA binding domain (DBD) of the mouse ER␣. The effects of these DBD mutations were examined in DNA binding assays using reporter constructs containing either EREs (classical) or AP1 (nonclassical) response elements. Using the AP1 reporter, there was a reversal of ER action relative to that seen with the ERE reporter. Estradiol induced suppression, and the antiestrogen ICI 182,780 stimulated transcription of the AP1 reporter. DBD mutations in the proximal (P-box) of the first zinc finger of the ER (E207A/G208A and E207G/G208S) eliminated ERE binding. These mutants were inactive using the ERE reporter but retained partial or full activity with the AP1 reporter. The DBD mutant ERs interacted with Jun when tested in mammalian cell two-hybrid assays. Two mutations (K366D and I362R) in the ER ligand binding domain known to alter coactivator interactions impaired transcriptional responses using either the ERE or AP1 reporters. We concluded that ER action through the AP1 response element involves interactions with other promoter-bound proteins instead of, or in addition to, direct binding to DNA. Interactions with coactivators were required for both pathways. These data supported a model in which ER-mediated transcriptional activation or repression is dependent on the ligand and the nature of the response element in the target gene.Estrogen has a wide range of physiologic activities, including the control of development, reproduction, and metabolism as well as effects on cell growth and differentiation. Most, if not all, actions of estrogen occur through its receptors, ER␣ 1 and ER␤. The functional domains of the ER are relatively well In the traditional model of ER action, the receptor binds as homodimers (3) or heterodimers (4 -7) to estrogen response elements (EREs) in the promoters of many, though not all, estrogen-responsive genes. Similar to other nuclear receptors, the ER recruits an array of transcriptional cofactors (coactivators and corepressors) that bind to the receptor and also interact with other transcription factors, including components of the general transcription factor apparatus. Some of the cofactors also possess chromatin-remodeling activities or recruit additional proteins to the complex to mediate transcription (reviewed in Ref. 8).It is now recognized that the type of ligand bound to the ER influences its interaction with cofactors. The crystal structures of the ER LBD when bound to an agonist (estradiol) or an antagonist (raloxifene) have been solved. Comparison of these structures suggests a molecular basis for the differential liganddependent cofactor binding (9). The binding of 17␤-estradiol induces a major shift in the position of helix 12, one of several helices that form the coac...

Mutations in the Estrogen Receptor DNA-binding Domain Discriminate between the Classical Mechanism of Action and Cross-talk with Stat5b and Activating Protein 1 (AP-1)

“…Whether AP-1-regulated promoters are actively transrepressed or induced in response to E 2 may depend on the composition of the AP-1 complex (25). In accordance with our results, cellspecific ER␤-mediated transrepression of the collagenase promoter in response to E 2 has been demonstrated (9) and has been recently described for ER␣ (11). Fig.…”

New insights into the classical and non-classical actions of estrogen: Evidence from estrogen receptor knock-out and knock-in mice

“…To better define non-classical signaling mechanisms of ERα action, Jakacka and colleagues generated selective DNA binding mutations in the mouse ERα and observed their effects in vitro (Jakacka et al, 2001). One such mutation within the first zinc finger of the DNA binding domain, E207A/G208A ("AA"), completely eliminated ERE binding and activation of EREcontaining reporter genes, but retained full transcriptional activity of reporter genes containing AP1 response elements and interacted with Jun when tested in mammalian cell two-hybrid assays (Fig.…”

“…The rescue of negative feedback in ERα −/AA females may also reflect rapid, non-genotropic actions of E 2 originating at the plasma membrane, as the knock-in mutation is specific to the DNA-binding domain and leaves the membrane-localization domain intact (Jakacka et al, 2001). Evidence from ovariectomized ewes (Nett et al, 1984), monkeys (Pau et al, 1990), and guinea pigs (Condon et al, 1988) demonstrates that E 2 can rapidly decrease LH secretion, indicating non-genotropic mechanisms of E 2 action.…”

Distinct Transcriptional Regulation of Human Large Conductance Voltage- and Calcium-activated K+ Channel Gene (hSlo1) by Activated Estrogen Receptor α and c-Src Tyrosine Kinase

“…Two point mutations were introduced into hER␣ (NM_000125) utilizing QuikChange (Stratagene) to change the glutamic acid residue 203 and glycine residue 204 to alanine (E203A, G204A). These mutations have previously been shown to disrupt ER␣ DNA binding (7,24). The mutated hER␣ is called here, DNA binding domain mutant of hER␣ (DBDM-hER␣).…”

“…A direct binding of hER␣ to the hSlo1 promoter was tested by utilizing a hER␣ mutant that does not bind to DNA (E203A, G204A DNA binding domain mutant called here DBDMhER␣) (7,24). HeLa cells were transfected with 2.5-and 5-kb hSlo1 constructs together with empty pcDNA3 vector (Control), hER␣, or DBDM-hER␣, and induced with 1 nM estrogen (light gray bars) or vehicle alone (dark gray bars) (Fig.…”