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Introduction: The Molecular Biology of Steroid Hormone Action

The control of gene transcription and mRNA levels is critical to investigations of cell growth and development, malignant transformation and hormone action. The effects of steroid hormones, such as estrogens and testosterone, thyroid hormone, retinoids, ecdysone, some fatty acids and prostaglandins, bile acids and oxygenated cholesterols are mediated through specific receptor proteins termed steroid/nuclear receptors. This large super-family of gene regulatory proteins represents the fundamental system for ligand-regulated transcription in multicellular eukaryotes. Steroid/nuclear receptors are found in organisms ranging from nematodes (with only 1,000 cells) to Drosophila to humans, but have not been found in yeast.

Until recently the steroid/nuclear receptors were thought to be distinct from cell surface receptors acting through signal transduction pathways, with the two signaling systems interacting at many levels to control and mediate each others activity. However, recent studies describe membrane-based actions of steroid hormone receptors that involve activation of plasma membrane associated signal transduction pathways. These activities are often referred to as non-genomic actions of estrogen and other steroid hormones. Non-genomic actions of estrogens include the induction of DNA replication or apoptosis (programmed cell death) in different cell contexts, and the induction of nitric oxide in cells of the arterial wall.

Steroid/nuclear receptors act to regulate mRNA levels by controlling gene transcription and by regulating the stability of mRNAs. To regulate gene transcription in response to a hormone ligand, steroid/nuclear receptors must carry out three basic functions. (1) The receptor must be activated, which usually involves binding by a specific small molecule hormone ligand. Phosphorylation or dephosphorylation of the receptor in response to cell signaling pathways also modulates receptor activity, as does binding to DNA. (2) The receptor usually binds to a specific DNA sequence, often termed a hormone response element. In some genes, the receptor does not bind directly to the DNA, but interacts with another gene regulatory protein which binds to a specific DNA sequence, and "tethers" the receptor to the DNA. (3) The receptor must undergo a conformational change as a result of ligand binding, DNA binding, and phosphorylation by cell signaling pathways. This conformational change enables it to activate transcription. Transcription activation (or repression) is brought about by the interaction of the receptor with other proteins termed coregulators, which can activate or repress transcription. Coregulators form a multi-protein complex with the receptor, that interacts with components of the transcription apparatus, carries out histone acetylation (or deaceylation) and interacts with other multi-protein complexes that facilitate chromatin remodeling. To induce the transcription of a gene, the receptor-coactivator complex must facilitate a loosening of the histone-DNA interactions that repress transcription in chromatin, and stimulate or stabilize formation of a transcription complex at the promoter.

Our transcription research centers primarily on the estrogen receptor (ER), which mediates the actions of estrogens in target tissues. Estrogens, acting via the ER, regulate the growth and differentiation of cells of the female and male reproductive systems and the growth, proliferation and metastatic potential of breast cancer cells. Estrogens also have important effects on brain development, autoimmune diseases, and bone metabolism. We are studying the actions of estrogen receptor in breast cancer cells, liver cells and in other systems at its three of its major sites of action; the cell membrane, the cytoplasm and the nucleus.

Cellular Sites of Estrogen Receptor Action are Illustrated

Although the hormone ligand clearly plays a key role in determining the potential of a steroid receptor to activate transcription, we and others have proposed that steroid receptor proteins function as integrator proteins receiving and interpreting signals derived from diverse sources, including the hormone response element on the DNA, the promoter context, the cell type, extracellular signals, and the hormone ligand. In our hypothesis, estrogen receptor gains the ability to activate transcription through an 揳ctivation pathway? in which ligand binding, phosphorylation, interaction with coactivators and corepressors, and binding to the estrogen response element all contribute to the receptor抯 activation potential. Because we, and others, suggested that the DNA sequence recognized by the estrogen receptor (termed the estrogen response element, or ERE) is a kind of allosteric effector, regulating receptor conformation, we focus most of our attention on the role of the estrogen response element estrogen receptor interaction in estrogen-mediated transcription.

In initial studies we focused on development of new expression systems for production of recombinant estrogen receptor, including stably transfected cultured human cells (Zhang, C-C., Krieg, S. and Shapiro D.J. (1999) HMG-1 Stimulates Estrogen Response Element Binding by Estrogen Receptor from Stably Transfected HeLa Cells. Molec. Endocrinol., 13: 642-643) and E. coli (Zhang, C.C., Glenn, K.A., Kuntz, M.A. and Shapiro, D.J. (2000) High Level Expression of Full-length Estrogen Receptor in Escerichia coli is Facilitated by the Uncoupler of Oxidative Phosphorylation. CCCP. J. Steroid Biochem. and Mol. Biol. 74: 169-178). The activation pathway hypothesis predicts that enhancing binding of estrogen receptor to the ERE would move the ER further down the activation pathway, decreasing the requirement for a strongly estrogenic ligand. We showed that the DNA binding protein HMG-1 I (High Mobility Group Protein 1) enhanced binding of ER to the ERE in vitro, enhanced ER-mediated transcription in intact cells and dramatically increased the ability of the weak estrogen, 4-hydroxytamoxifen (the activity metabolite of the anti-cancer drug, tamoxifen) to activate transcription (Zhang, C-C., Krieg, S. and Shapiro D.J. (1999) HMG-1 Stimulates Estrogen Response Element Binding by Estrogen Receptor from Stably Transfected HeLa Cells. Molec. Endocrinol., 13: 642-643.)

To further analyze the role of the ERE in estrogen receptor-mediates transcription we developed a genetic selection system that enabled us to produce a large pool of mutants and to select from that mutant pool steroid receptors DNA binding domains with altered ability to recognize hormone response elements (Chusalcultanachai, S., Glenn, K.A., Rodriguez, A.O., Read, E.K., Gardner, J.F., Katzenellenbogen, B.S. and Shapiro, D.J. (1999) Analysis of Estrogen Response Element Binding by Genetically Selected Steroid receptor DNA Binding Domain Mutants Exhibiting Altered Specificity and Enhanced Affinity. J. Biol. Chem., 274: 23591-23598.) Development of these mutants enables us to engineer a specific chimeric repressor able to carry out ligand-dependent repression of a specific gene (de Haan, G., Chusacultanachai, S., Mao, C., Katzenellenbogen, B.S. and Shapiro, D.J. (2000) Estrogen Receptor-KRAB Chimeras are Potent Ligand-dependent Repressors of Estrogen-regulated Gene Expression. J. Biol. Chem. 275,: 13493-13501.)

We currently pursue three lines of research in this area. Using information from the mutant estrogen receptors and from in vitro binding studies we show that information from the ERE sequence can be communicated to the activation functions of the estrogen receptor and convert a basically inactive ER ligand, 4-hydroxy-tamoxifen into a potent transcription activator. These observations enabled us to produce a novel type of tamoxifen and 4-hydroxytamoxifen regulated promoter for regulated production of proteins in eukaryotic cells. (Mao, C and Shapiro. D.J., A Tamoxifen and 4-Hydroxytamoxifen-Activated System for Regulated Production of Proteins in Eukaryotic Cells, U.S. and International Patents Pending; and Mao, C., Wang, C., Rodriguez, A., Katzenellenbogen. B.S. and Shapiro, D.J. (2003) Communication Between the DNA Binding and Activation Domains of Estrogen Receptor: A Novel Estrogen Response Element Converts 4-Hydroxytamoxifen Into a Potent Transcription Activator, Manuscript in preparation). Additional evidence that the ERE plays a direct role in transcription activation stems from our observation that binding to the ERE reverses the ability of cell extracts to interfere with estrogen receptor binding to coactivator proteins (Wang, S. and Shapiro. D.J. (2003) Binding to the Estrogen Response Element Reverses Cell Exract Mediated Repression of Coactivator Binding to Estrogen Receptor, Manuscript in Preparation).

Ligands that bind to the estrogen receptor and compete with estrogen for binding are called Selective Estrogen receptor Modulators, or SERMs, form an important class of drugs. These drugs seek to elicit the beneficial effects of estrogens in bone and other tissues while blocking the harmful effects of estrogens in breast and uterine cells. The breast cancer therapeutic, Tamoxifen and the anti-osteoporosis agent Raloxifene, are two examples of therapeutically important SERMs. Previously it was not possible to test large numbers of estrogen receptor ligands for the ability to modify or interfere with binding of ER to estrogen response elements. Very recently, we developed an assay for ER:ERE interactions suitable for large-scale screening. We are beginning to use this system for analysis of ligand-dependent ER:ERE interactions (S. Wang, B. Ahn, R. Harris, and D. Shapiro unpublished research).

Because most previous studies of ER:ERE interactions were in vitro studies, they did not take into account the fact that the ERE-containing promoters are packaged into chromatin. To analyze the effect of ERE sequence on binding of liganded estrogen receptors to promoters in intact cells, we used quite different EREs in the pS2 gene and in the PI-9 gene that direct potent estrogen induction of gene expression (Krieg et al. 2001). We used Chromatin Immunoprecipitation (ChIP) to analyze the interaction with these genes of estrogen receptor bound to different ligands.

In ChiP assays, DNA-protein complexes in the cells are effectively fixed in place by cross liking with formaldehyde, the DNA with its bound proteins is fragmented into small pieces, and an antibody against the regulatory protein of interest (here the estrogen receptor), is used to precipitate the DNA:protein complex, the cross linking is reversed and primers specific for the promoter region of interest are used to amplify the precipitated DNA. Because PCR amplification is so specific it is possible to analyze what is happening at one specific gene in the entire genome.

Schematic of Chromatin Immunoprecipitation (ChIP)

Our studies demonstrated that binding of ER to the ERE is rapid and does not exhibit the periodic oscillation proposed by some researchers. We find that binding of ER associated with different ligands to the two genes is unrelated to the ligands ability to activate transcription and is highly dependent on the nature of the ERE binding site. These data demonstrate that ERE sequence and ligands work together to regulate intracellular binding of estrogen receptor to estrogen responsive promoters (Krieg et al 03).

Estradiol-ER Complex Rapidly Associates with the pS2 ERE in MCF-7, Human Breast Cancer Cells

MCF-7 cells (a widely studied line of human breast cancer cells that contains functional estrogen receptor and depends on estrogen for growth) were incubated in estrogen and then fixed with formaldehyde at the indicated times. ChIP assays were carried out using antibodies specific for ER-α, for the transcription coactivator CBP, for acetylated histones (Ac-H3 and Ac-H4), and a control non-specific antibody (M2) and the immunoprepcipitated chromatin was amplified with primers specific for the pS2 ERE as shown at the bottom. The data shows that unliganded estrogen receptor is not pre-bound at the pS2 ERE. Following addition of estrogen to the cell culture medium, the estrogen-ER complex rapidly binds to the pS2 gene. Estrogen-ER bound to the ERE rapidly recruits the transcription coactivator CBP. Binding of estrogen-ER and of CBP is largely complete by 20 minutes after addition of estrogen and does not exhibit time-dependent oscillation. There is also a rapid increase in histone acetylation on the promoter (Ac-H4). Immunoprecpitation with the control non-specific M2 antibody produces a low and constant signal at all times. ( Krieg et al., 2003)

Some Recent Papers:

Krieg, S.A., Krieg, A.J., and Shapiro D.J. (2001) A unique Downstream Estrogen Responsive Unit Mediates Estrogen induction of proteinase Inihibitor-9, A Cellular inhibitor of IL-1beta Converting Enzyme (Capsase 1). Molec. Endocrinol., 15: 1971-1982.

Krieg, A.J., Krieg, S.A. Ahn, B.S. and Shapiro, D.J. (2003) Interplay Between Estrogen Response Element Sequence and Ligands Controls In Vivo Binding of Estrogen receptor to Regulated Genes. J. Biol. Chem., Provisionally accepted, Subject to minor revision.