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The molecular basis for estrogen-induced tight nuclear binding of estrogen receptor [alpha] (ER) is described. The effects of estrogen binding on altering DNA binding in whole cells have occurred through estrogen-induced changes in ER-chaperone protein interactions, which have changed the DNA accessibility of ER but have not directly changed the affinity of the ER for DNA.

Coactivators are believed to mediate estrogen-induced gene responses via interaction with estrogen receptors (ER). Currently, a major challenge is to determine the importance of each coactivator in a specific cell type and promoter context in response to a particular ligand. The potential of ER to interact with a growing list of coactivators has been shown in a variety of in vitro and gene transfer assays, yet very few data have demonstrated the interaction of endogenous coactivators with ER in intact cells. We report here a ligand-specific interaction of endogenous human ER (hER) and the AIB1 coactivator in MCF-7 human breast cancer cells by using immunoprecipitation analyses. Complexes between endogenously expressed hER and AIB1 were detected in estradiol-treated cells and to a much lesser extent in cells treated with the partial agonist, monohydroxytamoxifen. We were unable to detect an hER-SRC-1 complex in our immunoprecipitations from MCF-7 cells. The in vitro-binding affinity for mouse ER interaction with AIB1 was estimated to be 40-120 nM. We conclude that AIB1 is a major coactivator for hER in MCF-7 human breast cancer cells.

A study on estrogen receptors (ER) and the molecular basis for the estrogen-induced tight nuclear binding of ER was conducted to find a way to biochemically isolate the transformed ER that is bound to the nucleus and to identify associated proteins. The experiment presents a new alternate technique to the use of salt buffers to release the occupied ER from the nucleus. Data suggests that the mechanism of ER release is by decavanadate competition with nuclear DNA.

The human progesterone receptor (PgR) gene has a complex promoter that produces alternate mRNAs encoding the PgRA (94 kDa) and PgRB (120 kDa) protein isoforms. Expression of PgR is induced by estradiol (E2) in the breast, reproductive tract and many cell lines despite the lack of a classical estrogen responsive element (ERE) in the promoter regions. We employed chromatin immunoprecipitation (ChIP) to analyze the sites of estrogen receptor alpha (ERα) and Sp1 occupancy of the PgR promoters in vivo. We also assessed the functional relevance of histone acetylation levels on the accessibility of transcription factors to the promoter and subsequent hormone-induced transcription. We utilized MCF-7 human breast cancer cells that express PgR in response to E2 and the MCF-7 derived C4 cell strain that has lost PgR expression as a model system. We found that promoter-wide levels of histone acetylation were not decreased in C4 cells, but that access was partially blocked for Sp1 and completely blocked for ERα. The basal level of histone acetylation at six localized regions of the promoter did show some differences between cell lines, but it did not correlate with transcription factor binding. Furthermore, we found only a modest and highly localized change in histone acetylation levels in response to E2 at only one of three sites of ERα binding in MCF-7 cells. This was at the B1 site at the distal 5′ end of the promoter. This site also showed a significant decrease in basal histone acetylation in C4 compared to MCF-7 cells. We speculate that the histone acetylation level at this site may be a marker for chromatin structure that affects the access of transcription factors to the whole promoter. [Copyright &y& Elsevier]

We have examined the role of histone acetylation in the very earliest steps of differentiation of mouse embryonic stem cells in response to withdrawal of leukemia inhibitory factor (LIF) as a differentiation signal. The cells undergo dramatic changes in morphology and an ordered program of gene expression changes representing differentiation to all three germ layers over the first 3-5 days of LIF withdrawal. We observed a global increase in acetylation on histone H4 and to a lesser extent on histone H3 over this time period. Treatment of the cells with trichostatin A (TSA), a histone deacetylase inhibitor, induced changes in morphology, gene expression, and histone acetylation that mimicked differentiation induced by withdrawal of LIF. We examined localized histone acetylation in the regulatory regions of genes that were transcriptionally either active in undifferentiated cells, induced during differentiation, or inactive under all treatments. There was striking concordance in the histone acetylation patterns of specific genes induced by both TSA and LIF withdrawal. Increased histone acetylation in local regions correlated best with induction of gene expression. Finally, TSA treatment did not support the maintenance or progression of differentiation. Upon removal of TSA, the cells reverted to the undifferentiated phenotype. We concluded that increased histone acetylation at specific genes played a role in their expression, but additional events are required for maintenance of differentiated gene expression and loss of the pluripotent state. [ABSTRACT FROM AUTHOR]