The overall objective is to identify nuclear regions within which estrogen receptors interact with chromatin in order to define the molecular mechanisms through which estrogenic hormone action is mediated. Estrogen receptor activity in mouse uterine chromatin will be fractionated using a DNAase I-mediated methodology which has already defined a Mg-soluble fraction that demonstrates receptor activity after exposure to estrogen but not after exposure to anti-estrogen. The physiological significance of estrogen receptor activity in the Mg-soluble fraction will be explored by examining how receptor activity in this chromatin fraction is affected by treatment with agents which modulate estrogenicity. Because 3H-4OH-tamoxifen receptor complexes may be unable to penetrate the chromatin in the Mg-soluble fraction due to physical differences mediated by antagonistic ligands, receptors complexed with 3H-4OH-tamoxifen will be characterized. These characterizations will involve analyses by SEHPLC, hydrophobic HPLC and evaluation of the potential for receptor activation by examinations of binding to DNA-cellulose, ATP-sepherose, slowed ligand dissociation and induced positive cooperative binding. Efforts will be directed at defining additional chromatin subfractions within the bulk chromatin which remains after nuclease-mediated fractionation in order to see if the majority of nuclear 3H-4OH-tamoxifen receptors becomes differentially localized within the nucleus. As a consequence of this abnormal intranuclear localization these receptors may be unable to penetrate chromatin in the Mg-soluble fraction. Efforts will also be directed at defining the transcriptional activity of the Mg-soluble fraction by examining the mRNA in this fraction which is available for in vitro translation. This effort will focus on the question of proximal relationships between estrogen receptors and the initiation of chromatin events that culminate in new protein synthesis. The unique feature of this proposal is that, in addition to its attempt to identify chromatin regions involved in the mechanism of hormone action in a model system, it develops an approach which will have direct relevance to hormone responsive malignancy. Some tumors are characterized by high receptor levels but are unresponsive to hormone therapy because they may have undergone changes which deny receptor access to chromatin regions from which therapeutically productive responses can be initiated. The approaches outlined herein are relevant to this problem and should result in the development of methods which identify tumors with receptors that fail to initiate therapeutic responses.
