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      ERα and ERβ Homodimers in the Same Cellular Context Regulate Distinct Transcriptomes and Functions

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          Abstract

          The two estrogen receptors ERα and ERβ are nuclear receptors that bind estrogen (E2) and function as ligand-inducible transcription factors. They are homologues and can form dimers with each other and bind to the same estrogen-response element motifs in the DNA. ERα drives breast cancer growth whereas ERβ has been reported to be anti-proliferative. However, they are rarely expressed in the same cells, and it is not fully investigated to which extent their functions are different because of inherent differences or because of different cellular context. To dissect their similarities and differences, we here generated a novel estrogen-dependent cell model where ERα homodimers can be directly compared to ERβ homodimers within the identical cellular context. By using CRISPR-cas9 to delete ERα in breast cancer MCF7 cells with Tet-Off-inducible ERβ expression, we generated MCF7 cells that express ERβ but not ERα. MCF7 (ERβ only) cells exhibited regulation of estrogen-responsive targets in a ligand-dependent manner. We demonstrated that either ER was required for MCF7 proliferation, but while E2 increased proliferation via ERα, it reduced proliferation through a G2/M arrest via ERβ. The two ERs also impacted migration differently. In absence of ligand, ERβ increased migration, but upon E2 treatment, ERβ reduced migration. E2 via ERα, on the other hand, had no significant impact on migration. RNA sequencing revealed that E2 regulated a transcriptome of around 800 genes via each receptor, but over half were specific for either ERα or ERβ (417 and 503 genes, respectively). Functional gene ontology enrichment analysis reinforced that E2 regulated cell proliferation in opposite directions depending on the ER, and that ERβ specifically impacted extracellular matrix organization. We corroborated that ERβ bound to cis-regulatory chromatin of its unique proposed migration-related direct targets ANXA9 and TFAP2C. In conclusion, we demonstrate that within the same cellular context, the two ERs regulate cell proliferation in the opposite manner, impact migration differently, and each receptor also regulates a distinct set of target genes in response to E2. The developed cell model provides a novel and valuable resource to further complement the mechanistic understanding of the two different ER isoforms.

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          Most cited references59

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          Genome engineering using the CRISPR-Cas9 system.

          Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.
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            Estrogen synthesis and signaling pathways during aging: from periphery to brain.

            Estrogens are the primary female sex hormones and play important roles in both reproductive and non-reproductive systems. Estrogens can be synthesized in non-reproductive tissues such as liver, heart, muscle, bone and brain, and tissue-specific estrogen synthesis is consistent with a diversity of estrogen actions. In this article we review tissue and cell-specific estrogen synthesis and estrogen receptor signaling in three parts: (i) synthesis and metabolism, (ii) the distribution of estrogen receptors and signaling, and (iii) estrogen functions and related disorders, including cardiovascular diseases, osteoporosis, Alzheimer's disease (AD), and Parkinson disease (PD). This comprehensive review provides new insights into estrogens by giving a better understanding of the tissue-specific estrogen effects and their roles in various diseases. Copyright © 2012 Elsevier Ltd. All rights reserved.
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              Hormone-replacement therapy: current thinking

              Roger Lobo (2016)
              Clinical practice regarding the use of hormone-replacement therapy (HRT) has undergone many changes since its introduction in the 1940s. Here, Roger Lobo frames the current thinking on the use of HRT in postmenopausal women, beginning with a historical perspective and then discussing how the interpretation of HRT data has changed over time.
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                Author and article information

                Contributors
                Journal
                Front Endocrinol (Lausanne)
                Front Endocrinol (Lausanne)
                Front. Endocrinol.
                Frontiers in Endocrinology
                Frontiers Media S.A.
                1664-2392
                06 July 2022
                2022
                : 13
                : 930227
                Affiliations
                [1] 1 Clinical Medical Research Center for Women and Children Diseases, Maternal and Child Health Care Hospital of Shandong Province , Jinan, China
                [2] 2 Department of Biosciences and Nutrition, Karolinska Institutet , Huddinge, Sweden
                [3] 3 School of Public Health, Jilin University , Changchun, China
                [4] 4 Science for Life Laboratory, Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), KTH Royal Institute of Technology , Solna, Sweden
                [5] 5 Department of Women’s and Children’s Health, Karolinska Institutet , Stockholm, Sweden
                Author notes

                Edited by: Victoria Fabris, CONICET Instituto de Biología y Medicina Experimental (IBYME), Argentina

                Reviewed by: Marine ADLANMERINI, Institut National de la Santé et de la Recherche Médicale (INSERM), France; Guan Chen, Medical College of Wisconsin, United States

                *Correspondence: Cecilia Williams, cecilia.williams@ 123456scilifelab.se

                †These authors have contributed equally to this work

                This article was submitted to Cancer Endocrinology, a section of the journal Frontiers in Endocrinology

                Article
                10.3389/fendo.2022.930227
                9299245
                35872983
                d551d663-45bc-4cf6-ab89-e27026fd8fa2
                Copyright © 2022 Song, He, Indukuri, Huang, Stepanauskaite, Sinha, Haldosén, Zhao and Williams

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 27 April 2022
                : 03 June 2022
                Page count
                Figures: 5, Tables: 1, Equations: 0, References: 60, Pages: 15, Words: 8188
                Funding
                Funded by: Karolinska Institutet , doi 10.13039/501100004047;
                Award ID: 2-3707/2013 , 2-3591/2014 , 2021-00501
                Funded by: Cancerfonden , doi 10.13039/501100002794;
                Funded by: Vetenskapsrådet , doi 10.13039/501100004359;
                Funded by: Stockholms Läns Landsting , doi 10.13039/501100004348;
                Categories
                Endocrinology
                Original Research

                Endocrinology & Diabetes
                estradiol (17ß-estradiol),estrogen receptor beta (erß),estrogen receptor alpha (erα),rna-seq - rna sequencing,cistrome,proliferation

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