Activation of Estrogen Receptor-a by E2 or EGF Induces Temporally Distinct Patterns of Large-Scale Chromatin Modification and mRNA Transcription Valeria Berno1, Larbi Amazit1, Cruz Hinojos1, Jeannie Zhong 1 , Maureen G. Mancini1*, Zelton Dave Sharp2, Michael A. Mancini1 1 Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, United States of America, 2 Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center San Antonio, San Antonio, Texas, United States of America Abstract Estrogen receptor-a (ER) transcription function is regulated in a ligand-dependent (e.g., estradiol, E2) or ligand-independent (e.g., growth factors) manner. Our laboratory seeks to understand these two modes of action. Using a cell line that contains a visible prolactin enhancer/promoter array (PRL-HeLa) regulated by ER, we analyzed ER response to E2 and EGF by quantifying image-based results. Data show differential recruitment of GFP-ER to the array, with the AF1 domain playing a vital role in EGF-mediated responsiveness. Temporal analyses of large-scale chromatin dynamics, and accumulation of array- localized reporter mRNA over 24 hours showed that the EGF response consists of a single pulse of reporter mRNA accumulation concomitant with transient increase in array decondensation. Estradiol induced a novel cyclical pattern of mRNA accumulation with a sustained increase in array decondensation. Collectively, our work shows that there is a stimuli- specific pattern of large-scale chromatin modification and transcript levels by ER. Citation: Berno V, Amazit L, Hinojos C, Zhong J, Mancini MG, et al. (2008) Activation of Estrogen Receptor-a by E2 or EGF Induces Temporally Distinct Patterns of Large-Scale Chromatin Modification and mRNA Transcription. PLoS ONE 3(5): e2286. doi:10.1371/journal.pone.0002286 Editor: Dong-Yan Jin, University of Hong Kong, China Received December 23, 2007; Accepted April 14, 2008; Published May 28, 2008 Copyright: 2008 Berno et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported from NIH grants DK55622 (M.A.M.), and a Department of Defense Synergistic Idea Award GRANT00210313 to MAM and ZDS; Integrated Microscopy Core support was provided by HD-07495 (Center for Reproductive Biology, B.W. O’Malley) and the Baylor College of Medicine Dan L. Duncan Cancer Center. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mancini@bcm.tmc.edu Introduction The estrogen receptor-a (ER) integrates signals from different stimuli in its role as a transcription regulator. The transcriptional activity of ER is regulated directly through binding of estrogenic or anti-estrogenic molecules [1]. Indirectly it is regulated by growth factor signaling pathways [2,3]. Although there is overlap with direct activation, growth factors activate kinase cascades resulting in phosphorylation and activation of the ER, which is distinguish- able from ligand activation [4]. Indeed, cross talk between the regulatory pathways indicates that direct and indirect regulation of ER are not mutually exclusive [4–6]. Epidermal growth factor (EGF) and its receptor, EGFR, initiate an important signaling cascade leading to an activated ER [7]. It has been hypothesized that the combined over-stimulation of ER and the EGF receptor (EGFR) may provide a strong stimulus for breast tumor growth and may contribute to the resistance of tumor cells to antagonist therapy. The molecular mechanism of this activation remains poorly understood and is an area of continued study [8–10]. EGF signaling is initiated by binding and activation of the EGFR at the plasma membrane [11]. Tyrosine autophosphoryla- tion by EGFR initiates multiple kinase cascades, targets of which include the ER. EGF induces ER-dependent stimulation of estrogen responsive element (ERE) reporter expression [12]. In this model, the extracellular EGF signal is transduced to genes regulated by the ER, the physiological relevance of which is underlined by estrogen-like effects of EGF on the mouse uterus do not occur in ER-deficient transgenic mice [13]. ERK1 and ERK2, kinases belonging to the MAPK pathway, have been shown to phosphorylate the ER at serine 118 in the activation function-1 (AF-1) domain of ER [3,14]. This post- translational modification has a strong impact on ER-mediated transcriptional activation induced by both direct (estradiol) and indirect signaling [15,16]. Interestingly, ER phosphorylation at serine 118 is also a marker of an activated ER signaling pathway in breast cancer, and provides a precise biomarker of responsiveness to endocrine therapy [17,18]. Therefore, elucidation of the mechanism of EGF-dependant activation of ER could be important in the development of new therapeutic targets for overcoming the resistance of breast tumor cells to hormone- therapy. We have developed a model system, PRL-HeLa, for the single- cell study of multiple mechanistic aspects of ER regulation of transcription [19]. This cell line contains a multi-copy integrated prolactin (PRL) enhancer/promoter reporter construct, which is responsive to E2. When ER is expressed as a GFP-fusion protein (GFP-ER), the integration site can be easily visualized allowing spatial and temporal analyses of promoter/enhancer targeting by ER, large-scale chromatin modification and accumulation of reporter mRNA. In our initial studies, we used PRL-HeLa to examine ligand-dependent ER regulation [19]. Treatment of these cells with E2 induces an ER-dependent large-scale chromatin PLoS ONE | www.plosone.org 1 May 2008 | Volume 3 | Issue 5 | e2286