Elsevier

Molecular Immunology

Volume 35, Issue 8, 1 May 1998, Pages 445-458
Molecular Immunology

Characterization of myocyte enhancer factor 2 (MEF2) expression in B and T cells: MEF2C is a B cell-restricted transcription factor in lymphocytes

https://doi.org/10.1016/S0161-5890(98)00058-3Get rights and content

Abstract

Our studies examined the expression and DNA binding activity of myocyte enhancer factor 2 (MEF2A-D) transcription factors in lymphopoietic tissues, cell lines, and primary lymphocytes. Our analyses demonstrate that mef2C expression is restricted to B cells within the lymphocyte lineage. Using in situ hybridization, mef2C is detected in foci in fetal liver and postnatal thymic medulla, and both mef2B and mef2C are expressed in areas of the postnatal spleen and lymph node that also express kappa light chain (Cκ), a B cell-specific marker. Reverse transcriptase-PCR (RT-PCR) analyses demonstrate that all mef2 family members are expressed in B cell lines, and all except mef2C are expressed in T cell lines. Immunoblot analyses of cell lines and primary thymic and splenic lymphocytes show that MEF2C and MEF2D proteins are expressed in B cells and that MEF2D is expressed in T cells; however, MEF2A protein is not detected in lymphocytes. Electrophoretic mobility shift assays (EMSA) demonstrate that B cell lines have MEF2C-containing, MEF2-specific DNA binding complexes whereas T cells do not. Our data is the first to describe mef2C expression in the lymphocyte lineage, and this finding suggests possible roles for MEF2C activity in B cell development and function.

Introduction

In recent years the roles that transcription factors play in lymphocyte development have become more defined (Dorshkind, 1994;Storb and Kruisbeek, 1996). Several of these transcription factors are expressed specifically in the B lymphocyte lineage. Many of these factors have been functionally disrupted by homologous recombination in mice, and several null mutant phenotypes reveal blocks in B lymphocyte differentiation at specific stages of development (Clevers and Grosschedl, 1996). B lymphocytes in both the Pax5 and EBF knockout mice do not differentiate beyond very early stages in B cell development (Urbanek et al., 1994; Lin and Grosschedl, 1995). These and other data demonstrate the importance of identifying and studying new B lymphocyte-specific transcription factors to facilitate better understanding of the development and function of B lymphocytes.

The MEF2 family of transcription factors was originally described as a muscle-specific DNA binding activity that recognizes A⧹T rich regions in the promoter and enhancer elements of several muscle-specific genes (Gossett et al., 1989;Cserjesi and Olson, 1991). To date, there are four known mef2 genes in mice (mef2A-D), each possessing multiple splice isoforms at the RNA level. These factors share a high degree of homology in their MADS domains, which mediate dimerization and DNA binding. They possess a unique 28-amino acid motif, known as the MEF2 domain, which is adjacent to the MADS box and also influences dimerization (Olson et al., 1995). Previous studies demonstrate that mef2A, -2B, and -2D mRNAs are expressed in a widespread fashion at a low level during mouse embryogenesis, but are enriched in muscle and neurons (Edmondson et al., 1994;Molkentin et al., 1996a,Molkentin et al., 1996b). Further studies show that mef2 factors are also expressed in a spatially restricted fashion in the brain (Lyons et al., 1995) Alternatively, mef2C mRNA has been previously shown to be expressed exclusively in muscle and neurons during embryogenesis, and in these tissues and the spleen in adults (Martin et al., 1993; Leifer et al., 1994).

Regulation of MEF2 protein expression and activity is complex. Each gene has alternatively spliced transcripts (Olson et al., 1995). One hypothesis suggests that tissue-specific splice variants of the mef2 mRNAs are required for protein expression. MEF2A protein expression can be regulated at the posttranscriptional level (Suzuki et al., 1995), and the 3′ UTR of mef2A has been shown to mediate this posttransciptional control of MEF2A protein expression within muscle cells during differentiation (Black et al., 1997). Recent studies have shown that MEF2C DNA binding and transactivation activities can be regulated posttranslationally by phosphorylation (Molkentin et al., 1996a, Molkentin et al., 1996b;Han et al., 1997). Thus this multigene family can be regulated at the transcriptional, posttranscriptional, and posttranslational levels.

Previous studies suggest that MEF2 can regulate transcription of target genes in cooperation with other transcription factors. At the molecular level, the interaction of MEF2 factors with other transcription factors is best understood in myocyte development. These factors have been shown to interact with the myogenic basic region, helix-loop-helix (bHLH) factors to activate muscle-specific gene expression in a cooperative fashion (Kaushal et al., 1994;Molkentin et al., 1995). Conversion of non-muscle cells to muscle cells is also cooperatively enhanced by cotransfection of mef2C and myoD compared to myogenic conversion with either factor independently (Molkentin et al., 1995). The ETS-domain transcription factor polyoma enhancer activator 3 (PEA3) has also been shown to most efficiently activate muscle gene expression in differentiating myoblasts in cooperation with MEF2 (Taylor et al., 1997). These and other studies demonstrate that MEF2 factors can interact with a diverse spectrum of transcription factors to mediate their biological activity, thus adding another level of regulation to the functions of this gene family.

Following up on the results ofMartin et al., 1993our initial studies of the expression of mef2C in spleen showed that the mRNA was expressed specifically in the peripheral white pulp, an area predominantly comprised of B lymphocytes. This paper shows that mef2C is expressed at the mRNA and protein level in a restricted fashion within B cells. Several previous studies published during the course of this work have implicated MEF2 factors in important physiological processes in T cells, B cells, and monocytes⧹macrophages (Woronicz et al., 1995;Han et al., 1997;Liu et al., 1997). Our research described in this paper demonstrates that myocyte enhancer factor 2C (MEF2C) is expressed specifically in B cells in the lymphocyte lineage, and that MEF2D is expressed in both B and T lymphocytes. Since it has been shown that MAP kinase p38 activates MEF2C in macrophages (Han et al., 1997), MEF2C may also be a target for p38 activation in B cells to carry out p38 mediated physiological processes such as proliferation and apoptosis.

Section snippets

In situ hybridization

In situ hybridization was performed as outlined in Lyons et al., 1995. Briefly, mouse embryos and tissues were fixed in paraformaldehyde, embedded in paraffin, and 5–7 μm sections were mounted onto gelatinized slides. The sections were deparaffinized in xylene, rehydrated and postfixed in paraformaldehyde. After digestion with proteinase K, the sections were treated with triethanolamine⧹acetic anhydride followed with a final dehydration in ethanol before hybridizing with the gene specific

Mef2B and mef2C mRNAs are enriched within lymphoid tissues

We investigated the expression of mef2 mRNAs in lymphoid tissues of developing embryos and postnatal and adult mice. Mef2C transcripts were the only mef2 transcripts to be detected above background using the in situ hybridization technique in lymphoid tissues during embryonic development. Mef2C mRNAs were detected in a punctate fashion in 12.5 day post coitum (d.p.c.) fetal liver (Fig. 1A, B). Expression in the fetal liver persisted through 15.5 d.p.c. (Fig. 1C, D), but the number of mef2C

Discussion

Previous studies of MEF2 factors have focused on their expression in muscle and brain or on their transactivation activity for expression of muscle-specific genes. Our work shows that these factors are also likely to be essential regulators of lymphocyte differentiation and function. We describe the expression of mef2 mRNAs within lymphoid tissues and cell lines and provide the first evidence that MEF2C is expressed specifically within B cells in the lymphocyte lineage. Mef2C mRNAs are

Unknown BIBs

Ornatsky et al., 1997

Acknowledgements

We thank Dr. John Schwarz, Dr. Youngsook Lee, and Vanessa Ott for helpful comments on the manuscript, and Dr. Matyas Sandor, Dr. Steve Clark, Dr. Jeff Molkentin, and Dr. Robert Auerbach for helpful discussions and Bruce Micales for technical support. We thank Dr. Ursula Storb for providing us with the Cκ probe, Dr. Eric Olson for providing us with the mef2 cDNAs and the MEF2C and MEF2C expression vectors, and Dr. John Schwarz and Dr. Ron Prywes for providing the MEF2C and MEF2D antibodies

References (47)

  • Bain, G., Maandag, E.C., Izon, D.J., Amsen, D., Kruisbeek, A.M., Weintraub, B.C., Krop, I., Schlissel, M.S., Feeney,...
  • Baker, S.J., Reddy, E.P., 1995. B cell differentiation: Role of E2A and Pax5/BSAP transcription factors. Oncogene 11,...
  • Barbara, E., Mishell, B. and Shiigi, S.M., 1980. Lysis of red blood cells with TRIS-buffered ammonium chloride. In...
  • Black, B.L., Lu, J., Olson, E.N., 1997. The MEF2A 3′ untranslated region functions as a cis-acting translational...
  • Bornemann, K.D., Brewer, J.W., Beck-Engeser, G.B., Corley, R.B., Haas, I.G., Jack, H.M., 1995. Roles of heavy and light...
  • Clevers, H.C., Grosschedl, R., 1996. Transcriptional control of lymphoid development: Lessons from gene targeting....
  • Cserjesi, P., Olson, E.N., 1991. Myogenin induces the myocyte-specific enhancer binding factor MEF-2 independently of...
  • Davidson, W.F., Fredrickson, T.N., Rudikoff, E.K., Coffman, R.L., Hartley, J.W., Morse, H.C.d., 1984. A unique series...
  • Dorshkind, K., 1994. Transcriptional control points during lymphopoiesis. Cell 79,...
  • Edmondson, D.G., Lyons, G.E., Martin, J.F., Olson, E.N., 1994. Mef2 gene expression marks the cardiac and skeletal...
  • Firulli, A.B., Miano, J.M., Bi, W., Johnson, A.D., Casscells, W., Olson, E.N., Schwarz, J.J., 1996. Myocyte enhancer...
  • Gillies, S.D., Morrison, S.L., Oi, V.T., Tonegawa, S., 1983. A tissue-specific transcription enhancer element is...
  • Gossett, L.A., Kelvin, D.J., Sternberg, E.A., Olson, E.N., 1989. A new myocyte-specific enhancer-binding factor that...
  • Graves, J.D., Draves, K.E., Craxton, A., Saklatvala, J., Krebs, E.G., Clark, E.A., 1996. Involvement of...
  • Gutman, G.A., Warner, N.L., Harris, A.W., 1981. Immunoglobulin production by murine B-lymphoma cells. Clin. Immunol....
  • Han, T.H., Prywes, R., 1995. Regulatory role of MEF2D in serum induction of the c-jun promoter. Mol. Cell. Biol. 15,...
  • Horibata, K., Harris, A.W., 1970. Mouse myelomas and lymphomas in culture. Exp. Cell. Res. 60,...
  • Han, J., Jiang, Y., Li, Z., Kravchenko, V.V., Ulevitch, R.J., 1997. Activation of the transcription factor MEF2C by the...
  • Jäck, H.M., Beck-Engeser, G., Lee, G., Wofsy, D., Wabl, M., 1992. Tumorigenesis mediated by an antigen receptor. Proc....
  • Kaushal, S., Schneider, J.W., Nadal-Ginard, B., Mahdavi, V., 1994. Activation of the myogenic lineage by MEF2A, a...
  • Keyna, U., Beck-Engeser, G.B., Jongstra, J., Applequist, S.E., Jack, H.M., 1995. Surrogate light chain-dependent...
  • Kitamura, D., Kudo, A., Schaal, S., Muller, W., Melchers, F., Rajewsky, K., 1992. A critical role of lambda 5 protein...
  • Leifer, D., Golden, J., Kowall, N.W., 1994. Myocyte-specific enhancer binding factor 2C expression in human brain...
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