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...

No abstract

Kisspeptin is a product of the Kiss1 gene and is expressed in the forebrain. Neurons that express Kiss1 play a crucial role in the regulation of pituitary luteinizing hormone secretion and reproduction. These neurons are the direct targets for the action of estradiol-17␤ (E 2 ), which acts via the estrogen receptor ␣ isoform (ER␣) to regulate Kiss1 expression. In the arcuate nucleus (Arc), where the dynorphin gene (Dyn) is expressed in Kiss1 neurons, E 2 inhibits the expression of Kiss1 mRNA. However, E 2 induces the expression of Kiss1 in the anteroventral periventricular nucleus (AVPV). The mechanism for differential regulation of Kiss1 in the Arc and AVPV by E 2 is unknown. ER␣ signals through multiple pathways, which can be categorized as either classical, involving the estrogen response element (ERE), or nonclassical, involving ERE-independent mechanisms. To elucidate the molecular basis for the action of E 2 on Kiss1 and Dyn expression, we studied the effects of E 2 on Kiss1 and Dyn mRNAs in the brains of mice bearing targeted alterations in the ER␣ signaling pathways. We found that stimulation of Kiss1 expression by E 2 in the AVPV and inhibition of Dyn in the Arc required an ERE-dependent pathway, whereas the inhibition of Kiss1 expression by E 2 in the Arc involved ERE-independent mechanisms. Thus, distinct ER␣ signaling pathways can differentially regulate the expression of identical genes across different brain regions, and E 2 can act within the same neuron through divergent ER␣ signaling pathways to regulate different neurotransmitter genes.

Problem definition is a package of ideas that includes, at least implicitly, an account of the causes and consequences of undesirable circumstances and a theory about how to improve them. As such, it serves as the overture to policymaking, as an integral part of the process of policymaking, and as a policy outcome. In each of these roles it seems to exert influence on government action. Distinguishing among the roles clarifies the nature of that influence. A case study examines the transition from one problem definition to another in the domain of information collection by the federal government. The rise of the Paperwork Reduction definition illustrates the variety of ways in which problem definition has powerful consequences.