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Enhancer Λ Ig Differentiation, Bind an Essential Site in the Mef2 Proteins

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Enhancer Λ Ig Differentiation, Bind an Essential Site in the Mef2 Proteins Mef2 Proteins, Required for Muscle Differentiation, Bind an Essential Site in the Ig λ Enhancer This information is current as Ebenezer Satyaraj and Ursula Storb of September 24, 2021. J Immunol 1998; 161:4795-4802; ; http://www.jimmunol.org/content/161/9/4795 Downloaded from References This article cites 52 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/161/9/4795.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 24, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Mef2 Proteins, Required for Muscle Differentiation, Bind an Essential Site in the Ig l Enhancer1 Ebenezer Satyaraj2* and Ursula Storb3† The Ig l light chain gene enhancer has two unique essential motifs, lA and lB. The transcription factors that bind the lB motif have been identified as Pu.1 and Pu.1-interacting partner (Pip). We report here that the lA site includes a binding site for the myocyte-specific enhancer factor 2 (Mef2) family of transcription factors. Mef2 proteins were first described in muscle cells and, in vertebrates, include four known members designated A to D. Using a lA electrophoretic-mobility shift assay (EMSA), in conjunction with a high affinity Mef2 binding site and anti-Mef2 Abs, we show that members of the Mef2 family are present in nuclear extracts of l-producing B cells and bind the lA site. Functional assays using the chloramphenicol acetyltransferase (CAT) reporter construct containing three copies of the lA motif demonstrate that the lA sequence can function as an enhancer in conjunction with the thymidine kinase (TK) promoter and is regulated by Mef2 proteins. Extrapolating from other systems where Downloaded from transcriptional regulation by Mef2 has been studied, other transcription factors may be involved along with Mef2 in transcrip- tional regulation at the lA site. The Journal of Immunology, 1998, 161: 4795–4802. mmunoglobulins are heteromeric molecules composed of while the other is bound by Pip (Pu.1-interacting partner) recruited light (L)4 and heavy (H) chains. There are two light chains: through specific interaction with Pu.1 (16). Analysis of the human l k l l l I lambda ( ) and kappa ( ). The Ig H and L chains are encoded Ig enhancers has also identified similar A and B domains (17). http://www.jimmunol.org/ by multiple segments that must be somatically recombined to form Analysis of the lA site using a transcription factor database a functional gene (1–2). In B cells, expression of Ig genes is (Transcription Factor Search, 1995, Yutaka Akiyama, Kyoto Uni- strictly regulated for cell-type specificity and development stage versity; http://pdap1.trc.rwcp.or.jp/research/db/TFSEARCH.html) specificity. This is accomplished by multiple cis-regulatory ele- identified an A1T rich consensus binding site for Mef2 (Fig. 1A). ments, promoters, and enhancers (2–4). Additionally, these tran- The Mef2 transcription factor was first identified as a protein scriptional elements have a significant role in V(D)J recombination that binds an AT-rich sequence in the muscle-specific enhancer (1, 5, 6). Enhancers have been identified in the J-C introns of both of the muscle creatine kinase (MCK) gene (18). Mef2 factors k 9 H (7) and genes (8) and also 3 of the C exons of H (9, 10), and bind as homo- and heterodimers to the consensus sequence k k by guest on September 24, 2021 (11) genes. Unlike the H chain and light chain gene locus, the CTA(A/T) TA(G/A), which is found in the control regions of nu- l light chain gene locus is organizationally distinct (12). In the 4 merous muscle-specific genes and growth factor-induced genes murine l locus, two transcriptional enhancers, El2-4 and El3-1, (19). In the muscle cell, cooperative interaction between Mef2 fac- have been identified and lie 39 of the Jl-Cl gene clusters (13). No tors and myogenic basic-helix-loop-helix (bHLH) factors has been intronic enhancers have been found in the l gene locus. El2-4 and shown to regulate muscle-specific transcription (20). Although El3-1, which are .90% homologous, are thought to have evolved Mef2 binding sites are present in many muscle-specific promoters by gene duplication and are believed to function similarly. Two and are important for skeletal and cardiac muscle development (18, distinct domains, lA and lB, that are essential for function have been defined in the l2-4 enhancer (14, 15). Both lA and lB sites 21), several findings suggest that Mef2 proteins may play a role in bind B cell-specific factors in nuclear extracts (14, 15). The lB site nonmuscle gene expression. Four different Mef2 genes have been has two juxtaposed but distinct binding sites that are bound by a identified by molecular cloning experiments and are designated pair of interacting transcription factors (15). One of the composite Mef2A, Mef2B, Mef2C, and Mef2D (22–24). Mef2 proteins belong elements is bound by Pu.1, an Ets family transcription factor (15), to the MADS family of transcription factors (25) (named after the first four proteins in which the MADS domain was first identified: minichromosome maintenance 1 (MCM1), which regulates mat- *Department of Molecular Genetics and Cell Biology and †Committee on Immunol- ogy, University of Chicago, Chicago, IL 60637 ing-specific genes in yeast; AGAMOUS and DEFICIENS, which have homeotic function in flower development; and serum-re- Received for publication April 23, 1998. Accepted for publication June 29, 1998. sponse element (SRE), which regulates serum-inducible and mus- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance cle gene expression) and share a 56-aa N-terminal MADS box with 18 U.S.C. Section 1734 solely to indicate this fact. domain followed by a 27-aa Mef2 domain, which extends C-ter- 1 This work was supported by National Institutes of Health Grant AI 39535. minal of the MADS domain (26). These protein domains together 2 Current address: Department of Medicine, Northwestern University, Chicago, IL mediate DNA-binding, homo- and heterodimerization, and inter- 60611. action with bHLH proteins (27). 3 Address correspondence and reprint requests to Dr. Ursula Storb, Department of The expression of Mef2 gene products marks early myogenic Molecular Genetics and Cell Biology, 920 East 58th Street, Chicago, IL 60615. E- mail address: [email protected] lineages during embryogenesis (28) and follows specific expres- 4 Abbreviations used in this paper: L, light; H, heavy; EMSA, electrophoretic mo- sion patterns in different tissues (29). After birth, Mef2A, Mef2B, bility shift assay; Mef2, myocyte specific enhancer factor 2; MCK, muscle creatine and Mef2D are expressed ubiquitously (22–24, 26, 30, 31), while kinase; TK, thymidine kinase; CAT, chloramphenicol acetyl transferase; bHLH, ba- sic-helix-loop-helix; MADS, minichromosome maintenance 1 (MCM1) 1 agamous Mef2C transcripts are restricted to skeletal muscle, brain, and 1 deficiens 1 serum-response element (SRE). spleen (26, 31). Mef2C null mice show cardiogenic defects and an Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 4796 Mef2 PROTEINS BIND THE lA SITE IN THE Ig l ENHANCER buffer on ice for 30 min before the addition of the remaining components of the binding reaction. Western blotting For Western blotting, 20 to 40 mg of nuclear extract or 5 ml of in vitro translated proteins were boiled for 5 min with an equal volume of SDS sample buffer and chilled on ice and resolved on an 8% SDS-PAGE fol- lowed by blotting onto nitrocellulose membranes (Hybond ECL, Amer- sham, Buckinghamshire, U.K.). Rabbit anti-Mef2A (cross-reacts with Mef2C), anti-Mef2B, and anti-Mef2D (kind gift of Dr. Ron Prywes, Co- lumbia University, New York) were used as the first Ab followed by goat anti-rabbit-horseradish peroxidase (HRP) (Amersham) conjugate. Immu- noblots were developed using a chemiluminescent ECL Western blotting kit (Amersham) and visualized by exposing to x-ray film. In vitro transcription and translation Mef2A and Mef2C cDNAs cloned into the CMV promoter-driven expres- sion vector pcDNAI (Invitrogen, San Diego, CA) (a kind gift of Dr. E. N. FIGURE 1. The lA site of the El3-1 and El2-4 enhancers includes a Olson, Dallas, TX) were used. Mef2A and Mef2C proteins were made by Mef2 binding site. A, Sequences of El3-1 and El2-4 are shown with the T7-directed in vitro transcription-translation (TNT Kit, Promega, Madison, l l l l WI) in the presence of [35S]methionine and expression confirmed by footprinted sites, A and B motif, and the two E boxes, E1 and E2 (14). Downloaded from B, The Mef2 binding site included in the lA site of both El3-1 and El2-4 SDS-PAGE. are shown in boldface along with the consensus Mef2 binding site. Functional assays using a CAT reporter construct The A3-TKCAT construct was made as follows.
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