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J. Biol. Chem., Vol. 280, Issue 31, 28749-28760, August 5, 2005

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Alternative Pre-mRNA Splicing Governs Expression of a Conserved Acidic Transactivation Domain in Myocyte Enhancer Factor 2 Factors of Striated Muscle and Brain^*

Bangmin Zhu¶, Bindu Ramachandran, and Tod Gulick||

From the Diabetes Research Laboratory, Department of Medicine, Massachusetts General Hospital, Charlestown, Massachusetts 02129 and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, March 7, 2005 , and in revised form, April 12, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Myocyte enhancer factor 2 (MEF2) transcription factors play pivotal roles in striated muscle, neuron, and lymphocyte gene expression and are targets of stress- and calcium-mediated signaling. All MEF2 gene products have a common DNA binding and dimerization domain, but MEF2 transcripts are alternatively spliced among coding exons to produce splicing isoforms. In vertebrate MEF2A, -C, and -D, a splice versus no-splice option gives forms that include or exclude a short domain that we designate . We show that mRNAs containing are expressed predominantly in striated muscle and brain and that splicing to include is induced during myocyte differentiation. MEF2 + isoforms are more robust than - forms in activating MEF2-responsive reporters despite similar expression levels. One-hybrid transcription assays using Gal4-MEF2 fusions show similar distinctions in the transactivation produced by + versus - isoforms in all cell types tested, including myocytes. function is position-independent and exists in all MEF2 splicing variant contexts. The activity is not due to cis effects on MEF2 DNA binding or dimerization nor are established transcription factor or coactivator interactions involved. Each MEF2 domain contains multiple acidic residues, mutation of which abolishes function. Despite a location between the p38 MAPK docking domain and Thr phosphoacceptors of MEF2A and MEF2C, inclusion of does not influence responses of these factors to this signaling pathway. Thus, a conserved pattern of alternative splicing in vertebrate MEF2 genes generates an acidic activation domain in MEF2 proteins selectively in tissues where MEF2 target genes are highly expressed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Myocyte enhancer factor 2 (MEF2)¹ proteins are members of the MADS (MCM1, agamous, deficiens, serum response factor)-box family of transcriptional regulators (1-3). MEF2 was originally recognized as a sequence-specific DNA-binding activity at conserved elements in the promoters of various genes encoding muscle structural proteins and as products of cDNAs encoding proteins related to serum response factor (4, 5). Four distinct vertebrate genes encoding MEF2 forms were subsequently recognized, MEF2A, MEF2B, MEF2C, and MEF2D (6-9). Initial studies of MEF2 largely considered a role in myogenesis and muscle structural protein expression, but a wider province is now appreciated. Thus, MEF2 target genes include those encoding transporters and metabolic enzymes of striated muscle (10-13) and effectors of stress signaling in various cell types (14, 15). In addition, MEF2 proteins interact directly with neuron-specific transcription factors (16) and play a critical role in differentiation and programmed cell death in this cell type (17-24). Finally, critical roles for MEF2 factors in leukocyte functions have been established, including T lymphocyte apoptosis (25, 26) and activation (27), maintenance of Epstein-Barr virus latency in B cells (28), and macrophage activation (29).

The four MEF2 genes are differentially expressed spatially and temporally during development and in mature tissues (30, 31). MEF2 isotype functions partially overlap, but distinct roles for the different genes remain to be fully elucidated. Murine gene disruption studies provide genetic evidence in support of discrete MEF2 isotype-specific functions (32, 33). Thus, mef2-c null mice die at embryonic day 9 due to failure of cardiac development, and the animals also exhibit vascular defects (34). Because the other mef2 isotype genes are expressed at normal or supraphysiological levels in the mef2-c null animals, lack of compensation by these forms indicates a unique role for mef2-c in cardiac and vascular development. mef2-a null mice survive to the neonatal period, or in some cases to adulthood, but severe myocardial mitochondrial defects are present that predispose to sudden death (33). Expression of the other mef2 genes is also up-regulated in these animals, again indicating lack of compensation in vivo for selective MEF2 gene loss. At present it is not clear whether MEF2 isotype-selective functions relate solely to distinctions in temporospatial expression, or to unique features of the MEF2 protein forms encoded by the different genes, although we have provided recent evidence to suggest that the latter is likely (35).

Regulation of MEF2 function is complex and occurs at many levels. Abundance of MEF2 proteins is controlled at transcriptional (36-39),² translational (41), and degradation (23, 42) steps. The transactivation function of MEF2 proteins is regulated by various means, including through protein-protein interactions with other transcription factors (16, 43-49) and transcriptional co-regulators (25, 50-60); via phosphorylation by mitogen activated protein kinases (MAPK) and cyclin-dependent kinases (cdk) (3, 14, 15, 21); and by determinants of MEF2 and MEF2 co-regulator subcellular localization, including calcium-dependent effects on MEF2 interactors and interactions (2, 51, 61). MEF2 mRNAs, proteins, and sequence-specific DNA-binding activities are widely expressed, but target gene activation is highly restricted among tissues and cell types (20, 62-65). Some of this discordance clearly involves one or more of these regulatory mechanisms, but additional conditions may also be pertinent, including regulated expression of splicing isoforms with distinct functions (35).

Each vertebrate MEF2 gene gives rise to multiple isoforms through alternative splicing patterns that are conserved among vertebrates (35).³ These splicing patterns include use of bona fide alternative exons, a splice versus no-splice option, and use of alternative splice acceptors within one exon. In contrast to extensive work on MEF2 gene and MEF2 protein regulation at the various levels noted above, the differential expression and unique roles and responses of MEF2 splicing variants has only recently been explored (35, 67).³ No MEF2 protein-protein interactions or MEF2 protein modifications have been identified that involve domains unique to the splicing variants. One genetic study of splicing variants of the sole Drosophila MEF2 gene, DMef2, has been reported (68). No significant differences were noted in the ability of the various DMEF2 splicing forms to rescue muscle differentiation defects in a DMef2 mutant. However, distinctions in splicing variant function may have been obscured by the particular conditions in this study. Furthermore, DMef2 gene structure and alternative splicing patterns are not analogous to those of the vertebrate MEF2 genes, such that these findings do not specifically inform function of the vertebrate MEF2 splicing forms.

Previously, we have shown that the MEF2C gene is unique in having alternative splice acceptors at the last exon that generate MEF2C variants that either have or lack a phosphoserine-dependent transrepressor domain called (35).³ In the present report, we show a second distinction among MEF2 splicing isoforms that involves an autonomous acidic transactivation domain that is selectively included in MEF2A, MEF2C, and MEF2D proteins expressed in muscle and nerve. Expression of the domain is governed by tissue-selective inclusion of a short exon in MEF2 mRNAs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Plasmid Construction—Plasmid sequences are available from the corresponding author. All construct sequences were verified by dideoxy sequencing (69). [MEF2_CPT-IB]₃-tk-Luc, [-150/+75]-jun-Luc, and G5Luc were described (35). MEF2 coding regions were obtained using reverse transcription-PCR with human or murine heart, brain or skeletal muscle RNA as templates. pET-MEF2A 1 containing the human MEF2A 1 isoform coding region was described (35). Combination PCR on a pET-MEF2A 1 template was used with vector- and gene-specific mutagenic complementary primers for introduction of the domain with an XhoI restriction site signature. Primers used were 5'-CGGAGGAAGAGGAAcTcGAGTTGAACACCCAAAGGATCAGTAG-3' and 5'-GTTCAACTCgAgTTCCTCTTCCTCCGATAGTGGAGGCATCATGCC-3'. The BglII- and HindIII-restricted amplicons were substituted into similarly restricted pET-MEF2A 1 to give pET-MEF2A 1.. Substitution of a NheI to SphI fragment from expressed sequence tag IMAGE clone 3896491 into these plasmids gave pET-MEF2A 2 and 2.. pET-MEF2C 1 and pET-MEF2C 1., containing the human MEF2C 1 and 1. isoform coding regions, were described (35). Additional MEF2C isoform coding regions were obtained by swapping a PstI to Acc65I fragment of an reverse transcription-PCR amplicon, obtained using a human skeletal muscle RNA template, to give 1. and 1.. forms. The MEF2C SalI site served as a signature. pM-MEF2D 1 was described (35). The MEF2D 1. coding region was obtained by swapping a PstI to XmaI fragment of an reverse transcription-PCR amplicon, obtained using a human skeletal muscle RNA template. The MEF2D BglII site served as a signature. MEF2 isoform coding regions from pET28 were introduced into pCDNA3.hygro (Invitrogen) (pCDNA-MEF2C) and pM (Clontech) (pM-MEF2C) for expression of native factor isoforms and Gal4_DBD fusions, respectively.

pM-N86 MEF2C 1 and 1. were described (35). PstI to NotI fragments from the full-length MEF2C constructs were substituted to give pM-N86 MEF2C 1. and 1... Acc65I to EcoRI segment swapping from a previously described MEF2C mutant gave 1._S388A and 1.._S396A inserts in this N86 MEF2C context. The initial MEF2C domain mutant was created in the pM-N86 MEF2C 1. background using PCR with a forward vector primer and reverse primer 5'-gggcgcGTCGACATCCTCAGcCACTGATGGCATCGTATTCTTGgatCCTGGTG-3', installing a BamHI site 5' to by silent mutation. The PstI- and SalI-digested amplicon was substituted into the vector to give pM-N86 MEF2C 1._S271A. BamHI- and NotI-restricted PCR amplicons were substituted into this construct to make additional MEF2C domain mutants. Amplicons were created using a reverse vector primer with forward primers 5'-gggcgcGGatcCAAGAATACGATGCCATCAGTGTCTcAGGATG-3' (1._E272Q), 5'-gggcgcGGatcCAAGAATACGATGCCATCAGTGTCTGAGaATGTtGACCTG-3' (1._D273N), 5'-gggcgcGGatcCAAGAATACGATGCCATCAGTGTCTGAGGATGTtaACC TGCTTTTG-3' (1._D275N), and 5'-gggcgcGGatcCAAGAATACGATGCCATCAGTGTCTcAGaATGTtaACCTGCTTTTG-3' (1._DENQ), with AlwNI, SalI, and HpaI serving as MEF2C mutant signatures. Human MEF2D sequence 3' of the MADS box/MEF2S domain was amplified by PCR using forward primer 5'-cccgcgccATGgtgACCCTGAGGAAGAAGGGCTTC-3' and reverse vector primer on a pM-MEF2D 1 template. The NcoI- and PstI-digested amplicon was reintroduced into similarly restricted pM-MEF2D 1 and 1. to give pM-N86 MEF2D 1 and 1., respectively. A forward vector primer was used with reverse primers in PCR to generate MEF2D domain mutants. Primers were 5'-cccgcgAGATCTAAATGGTCCTCAGcCAAGTGATGCATTAggCCtTTTCCTGC-3' (1._T286A) and 5'-cccgcgAGATCTAAAgcGTCCTCAGTCAAGTGgTGCATTAACC-3' (1._H289A), and the PstI- and BglII-restricted amplicons were swapped into pM-N86 MEF2D 1. Combination PCR with vector primers and complementary gene-specific mutagenic primers was used to generate an amplicon for PstI to XmaI fragment swapping to give pM-N86 MEF2D 1._DENQ. Primers were 5'-TCACTTGACTcAGaACCATTTAaATCTGAAC-3' and 5'-TTGTTCAGATtTAAATGGTtCTgAGTCAAG-3'. BglII, NsiI, StuI, and DraI served as MEF2D mutant signatures.

pM-_MEF2D was constructed using PCR with primer 5'-gccgtggcggccgctttaCAGATCTAAATGGTCCTCAGTcatgaggaattccggcgatacagtc-3' in combination with a forward SV40 promoter primer on a pM template, followed by introduction of an XhoI- and NotI-restricted amplicon into pM. Complimentary oligonucleotides (5'-aattcACTGAGGACCATTTAGATCTGggcggaACTGAGGACCATTTAGATCTGggcggaACTGAGGACCATTTAGATCTGtaagc-3' and 5'-ggccgcttaCAGATCTAAATGGTCCT CAGTtccgccCAGATCTAAATGGTCCTCAGTtccgccCAGATCTAAATGGTCCTCAGTg-3') were annealed and inserted into EcoRI- and NotI-restricted pM to give pM-(_MEF2D)₃. The reverse primer 5'-gccgtggcggccgctttaCAGATCTAAATGGTCCTCAGTtccacctgcaggCTTTAATGTCCAGGTATCAAGC-3' was used with a forward vector primer on a pET-MEF2D 1 template, followed by introduction of the BamHI- to NotI-restricted amplicon into pM-MEF2D 1 to give pM-N86 MEF2D 1._COOH. The reverse primer 5'-gtggaggcggccgctttaCAAAAGCAGGTCGACATCCTCAGAtcctgcaggTGTTGCCCATCCTTCAG-3' was used with a forward vector primer on pET-MEF2C 1 template, followed by introduction of the EcoRI- to NotI-restricted amplicon into pM-MEF2C 1 to give pM-N86 MEF2C 1/_COOH.

Cultured Cell Transfection—C2C12 cells were maintained and differentiated as described (35). HeLa, COS7, and 293 cells were maintained in DME with 10% fetal bovine serum. Cells were split into 12-well plates 1 day prior to transfection with Superfect (Invitrogen). Triplicate wells received 1.0 µg of reporter plasmid, 0.1 or 0.3 µg of control reporter, and 1.0 µg of expression vector(s) except where otherwise indicated, and cells were harvested for reporter activity determinations 24-48 h after transfection. Luciferase readings were corrected for transfection efficiency using -galactosidase activity from pSV40Gal or Renilla luciferase activity from pRL-tk (Promega).

View larger version (35K): [in this window] [in a new window]   FIG. 1. Vertebrate MEF2 transcripts are alternatively spliced. A, schematic of the highly similar structures of three vertebrate MEF2 genes among coding exons. Splicing patterns of MEF2A and MEF2D differ from MEF2C (top) only at the extreme 3' exons. The MADS box (light gray) and MEF2S domain (dark gray) are DNA binding and dimerization domains. B, schematic of the MEF2C alternative splicing isoforms. Forms are named according to which domain is present (1 or 2), and whether or not the or domains are present (e.g. 1. or 2..) (35). There are eight splicing isoforms of MEF2C. MEF2A and MEF2D each have only four splicing isoforms, because the domain is constitutively present in these isotypes. C, sequences flanking and within (bold) the human MEF2 domains are shown. The most similar region of Drosophila DMEF2 is shown under a consensus vertebrate domain sequence.

RNA Analyses—Murine tissue and C2C12 cell RNA was isolated by conventional procedures (69). RNA was harvested from C2C12 myoblasts daily during growth to confluence in DME with 20% fetal calf serum, and daily during differentiation after cell confluence in DME with 2% horse serum. Ribonuclease protection assays (RPA) and radio-labeled cRNA probe syntheses were carried out as described (35, 74). The template for the mef2-a cRNA probe was a 169-bp SpeI to BglII fragment from a mouse mef2-a + cDNA subcloned into XbaI- and BamHI-restricted pBS-KS (Stratagene). The template for mef2-d cRNA probe was a 198-bp NsiI to PstI - cDNA subcloned into PstI-restricted pBS-SK. The mef2-c template was a 387-bp BglII to HcII -/- cDNA subcloned into BamHI- and HcII-restricted pBS-SK.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Alternative Splicing Patterns Are Conserved among Vertebrate MEF2 Genes—The structures of the four vertebrate MEF2 genes, MEF2A, MEF2B, MEF2C, and MEF2D, have either been described (35, 36, 75) or can be deduced from genomic and cDNA sequences in GenBank^TM. All but MEF2B have highly similar gene structures and alternative splicing patterns among coding exons (Fig. 1, A and B). In each case, coding exon 1 encodes part of the MADS box, whereas exon 2 encodes the carboxyl terminus of this domain and the adjacent MEF2 signature (MEF2S) (1). These domains function together to provide sequence-specific DNA binding and dimerization (76), as well as a platform for interactions with various transcription factors and co-regulators (1-3, 60). Alternative third exons 31 and 32 are present in each gene. Splicing to include either the 1 or 2 exon is regulated among tissues (6),⁴ but functions of the encoded alternative domains remain to be determined.

These vertebrate MEF2 genes each also have an exon between exons 6 and 7, referred to by us as , that is variably included in mRNAs. The exons are short (21 or 24 nucleotides) and are highly conserved at the nucleotide level across species, as well as at the amino acid level across MEF2 gene isotypes (Fig. 1C). We refer to the four distinct coding regions of MEF2A and MEF2D, as well as their cognate encoded protein isoforms, as 1, 1., 2, or 2., based on their composition with respect to alternative domains. We have previously described an MEF2 domain that can be excluded uniquely in the case of MEF2C by use of a splicing to a cryptic acceptor in exon 9 (35). This provides for MEF2C isoforms that either carry or lack a phosphoserine-dependent transrepressor domain. Thus, there are eight potential MEF2C variants, half of which exclude (-) and half of which include (+) a domain (Fig. 1B). Alternative splicing of the DMef2, the sole Drosophila MEF2 gene, is not analogous to that of any of the vertebrate MEF2 genes (68). However, a domain that shares some primary sequence similarity with vertebrate domains is present in all DMEF2 splicing isoforms (Fig. 1C).

Expression of the MEF2 Exons Is Striated Muscle- and Neuron-specific—Although MEF2 gene isoform mRNAs and proteins are widely expressed among tissues, activation of MEF2 targets appears to be largely restricted to skeletal and cardiac muscle, brain, and lymphocytes (1, 20, 62, 63, 65). Among various mechanisms that may pertain to this phenomenon, we speculated that MEF2 alternative splicing could play a role through restricted expression of variants with more robust transactivation function. We examined the distribution of MEF2 splicing isoforms in murine tissues to begin to address this hypothesis. Because the limited size of MEF2 exons prohibits analyses by Northern blotting, the relative abundance of mef2-a, mef2-c, and mef2-d + and - splicing isoforms was determined using ribonuclease protection assays (RPAs). Complementary RNA probes used in these studies spanned the exon 6-7 junction of a + murine mef2-a cDNA and a - mef2-d cDNA (Fig. 2A). The mef2-c cRNA probe encompassed a region extending from 5' of to 3' of of a -/- cDNA, allowing for simultaneous determination of expression of the four possible splicing variants involving these two alternative regions.

In most tissues examined and particularly those with low total mef2-a mRNA levels, mef2-a - vastly exceeded that of mef2-a + mRNA (Fig. 2B). In these cases, however, some distinctions in the ratio of + to - mRNA were apparent, including complete absence of + mRNA in ovary, liver, spleen, and kidney, but easily detected + message in the testis and lung samples. In marked contrast, in adult skeletal muscle, heart, and brain where relatively high mef2-a mRNA expression levels were present, + mRNAs were much more abundant than - messages. In the embryo (day 11 p.c.), total mef2-a mRNA is roughly half - and half +. Virtually identical findings were seen for mef2-d splicing isoform mRNA expression among tissues (Fig. 2C). Thus, mef2-d + mRNA was also largely restricted to skeletal muscle, heart, and brain, the sites of the greatest abundance of mef2-d total mRNA, and embryo RNA also showed both - and + expression. Lung, ovary, and spleen samples each had low levels of mef2-d + message. Therefore, with respect to exon splicing, the sole distinction between mef2-a and mef2-d among adult tissues tested was the distribution of + mRNAs at sites of low expression.

View larger version (33K): [in this window] [in a new window]   FIG. 2. MEF2 exons are selectively included in brain, heart and skeletal muscle. A, schematics of the MEF2 cDNA regions used as templates for cRNA probe syntheses. Sizes of probe fragments protected by the various splicing isoforms are noted. B, results of mef2-a RPA with indicated mouse tissue total RNA samples. Protected probe fragment sizes that are specific for + (169 bases) and - (94 bases) mRNAs are indicated to the right of the autoradiogram. C, results of mef2-d RPA for samples as in B. Protected probe fragment sizes that are specific for + (187 bases) and - (198 bases) mRNAs are indicated to the right. D, results of mef2-c RPA for RNA samples as in B. Protected probe fragment sizes that are specific for +/+ (266 bases), -/+ (317 bases), +/- (336 bases), and -/- (387 bases) mRNAs are indicated to the right. Reactions in B, C, and D each included 5 µg of total RNA.

Inclusion of the MEF2 Exons Is Induced during Myocyte Differentiation—To determine if the ratio of mef2 + to - mRNA is dynamic during myogenesis, RPAs were performed using RNA samples isolated from differentiating C2C12 cells. RNA was obtained at serial time points before and after serum withdrawal at cell confluence, the point at which markers of the skeletal muscle phenotype begin to appear (35). Here, distinctions in both mef2 gene isotype total mRNA and in splicing isoform expression patterns were evident. With respect to total message, mef2-a expression increased gradually from a barely detectable level until serum withdrawal, at which point a robust upsurge was apparent (Fig. 3A). This contrasted with mef2-d mRNA, the level of which remained nearly constant throughout the differentiation process (Fig. 3B). mef2-c message increased from a very low basal level, with a discontinuous burst at cell confluence, but with a continued increase until completion of myotube formation (Fig. 3C).

Patterns of splicing involving exon were similar for mef2-a and mef2-d, each of which showed a rapid switch from exclusive expression of - message to a preponderance of + mRNA at the time of cells reached confluence (Fig. 3, A and B). The ratio of + to - mef2-c message also increased during C2C12 differentiation but remained relatively low in fully differentiated myotubes (Fig. 3C), consistent with the observed ratio in the adult skeletal muscle sample. However, alternative splicing to include the exon was induced during myocyte differentiation for each mef2 gene isotype.

MEF2 Splicing Isoforms Containing the Domain Are More Potent Transactivators—We previously described different magnitudes of transactivation by MEF2C splicing isoforms when addressing the function of the domain (35). To further investigate distinctions in MEF2 splicing isoforms with a specific focus on the domains, we tested the activities of MEF2A splicing isoforms on MEF2-responsive reporters in co-transfected cells. COS7 cells were used in our initial studies because endogenous MEF2 expression is low in this cell type. Fig. 4A shows results of an experiment using a reporter containing three copies of the CPT-IB gene MEF2 element upstream of a minimal herpes simplex tk promoter ([MEF2_CPT-IB]₃-tk-Luc) (35). MEF2A 1. was substantially more transcriptionally active than MEF2A 1, the corresponding - form, with an activity ratio ranging from 2.5 to 8 in a series of COS7 cell transfections. Actual differences in activities of these isoforms may be underrepresented here, because endogenous MEF2 proteins of a fixed isoform composition can dimerize with the overexpressed isoforms to contribute to reporter activity in this assay.

View larger version (62K): [in this window] [in a new window]   FIG. 3. Inclusion of MEF2 exons is induced during myogenesis. C2C12 myoblasts were grown from low density to confluence (day 0) in medium supplemented with 20% fetal bovine serum, followed by growth in medium with 2% horse serum to induce myocyte differentiation. Myotubes were fully formed at day 5. Total RNA was harvested at the indicated times and used in RPA with cRNA probes shown in Fig. 2A. Assay results for the mef2-a (A), mef2-d (B), and mef2-c (C) probes are shown. Reactions each included 5 µg of total RNA.

View larger version (18K): [in this window] [in a new window]   FIG. 4. MEF2 + splicing isoforms have more potent transactivation capacities. A, COS7 cells were cotransfected with [MEF2CPT-IB]3-tk-Luc, control pRL-tk reporter, and pCDNA3 expression vector for the indicated MEF2A splicing isoforms. Each well received 1 µg of each reporter and 0.3 µg of expression vector. After 36 h, firefly luciferase activities in cell extracts were determined and were normalized for transfection efficiency using extract Renilla luciferase and to activity in pCDNA3-transfected cells (= 1.0). B, COS7 cells were cotransfected, processed, and analyzed as in A except with pCDNA3 expression vectors for the indicated MEF2D and MEF2C splicing isoforms.

MEF2 Domain Function Is Cell-type Independent—MEF2 isoforms were expressed as Gal4p DNA binding domain (Gal4_DBD) fusions (Fig. 5A) in tests of transactivation of a Gal4-responsive reporter (pG5Luc). In this one-hybrid system, differences in transactivation produced by the various Gal4_DBD-MEF2A and -MEF2D fusions recapitulated those of the native MEF2 isoforms. In specific, Gal4_DBD-MEF2 + isoforms gave 5- to 15-fold higher activity than the corresponding - form in transfected COS7 cells (Fig. 5B). As for the native MEF2 forms, an influence of the domain was not attributable to differences in levels of expression (Fig. 5C). These observations with isoform fusions to a heterologous DNA binding domain indicate that the presence or absence of does not control MEF2 transcriptional activity via cis effects on formation of a ternary DNA-bound complex.

Having validated the one-hybrid system as an approach to evaluate MEF2 splicing isoform function, we examined whether distinctions in transactivation generalized to cell types in which MEF2 proteins are endogenously expressed. In HeLa (63) and 293 HEK (15) cells, the pattern of Gal4_DBD-MEF2 isoform fusion activities was identical to that seen in COS7 (not shown), and this was also the case in differentiated C2C12 cells (Fig. 5D). Thus, all MEF2 isoforms containing a domain were substantially more transcriptionally active than the corresponding - forms. This indicates that, although + isoforms are selectively expressed among cells and tissues, effectors of function are more widely distributed.

MEF2 Domain Function Does Not Require the MADS or MEF2S Domains—Most previously described MEF2 protein interactors involve the MADS box and MEF2S domains, including the MEF2 coactivators CBP/p300 (26, 53) and peroxisome proliferator-activated receptor gamma coactivator 1 (78), as well as various transcription factors with a constitutive or ligand-induced positive transactivation function (43, 44, 46, 47, 49). Thus, the domain could act in cis to facilitate or stabilize such an interaction with these MEF2 domains, giving enhanced transactivation. To test this possibility, one-hybrid transcription assays were conducted using Gal4_DBD fusions of MEF2 isoforms in which the MADS box and MEF2S domains were omitted (N86 MEF2) (Fig. 6A). In transfected COS7 cells, the ratio of transactivation by Gal4_DBD-N86 MEF2C 1. to Gal4_DBD-N86 MEF2C 1 was similar to that of the corresponding full-length MEF2C isoform fusions (Fig. 6B). Analogous findings were seen with N86 MEF2D constructs, and expression levels of the N86 MEF2 fusion proteins were indistinguishable (not shown). For each MEF2 gene isotype, deletion of the MADS box and MEF2S domain did strengthen activation of the reporter, presumably by virtue of release from histone deacetylase-mediated sequestration (52, 60). Preservation of the distinction in transactivation function of the N86 + versus - splicing isoform fusions indicates that function does not involve established MEF2 co-activator or transcription factor interactions. In addition, mechanisms involving cis effects on DNA binding or dimerization are definitively excluded, because these functions are harbored within the amino-terminal 86 residues of all MEF2 factors (76).

View larger version (17K): [in this window] [in a new window]   FIG. 5. MEF2 domain function is cell type independent. A, schematic of MEF2A and MEF2D splicing isoforms and the Gal4DBD-MEF2 fusions used. B, COS7 cells were cotransfected with pG5Luc, control pRL-tk reporter, and pM vectors expressing indicated Gal4DBD-MEF2 splicing isoform fusions. Each well received 1 µg of each reporter and 1 µg of expression vector. Experiments were analyzed as in Fig. 4, except that normalization was to activity in pM-transfected cells (= 1.0). C, extracts from cells transfected in B were resolved on SDS-PAGE, and Gal4DBD-MEF2 expression was visualized by immunoblotting with monoclonal antibody that recognizes the Gal4DBD (upper panel). Sample loading was normalized using -actin immunoreactivity (lower panel). D, C2C12 myoblasts were transfected and analyzed as in B.

View larger version (17K): [in this window] [in a new window]   FIG. 6. MEF2 domain function does not require the MADS box or MEF2S domain. A, schematic of MEF2C splicing isoforms and the Gal4DBD-MEF2 and deletion mutant fusions used. B, COS7 cells were cotransfected, processed, and analyzed as in Fig. 5 with the indicated pM-MEF2 constructs expressing MEF2 splicing isoforms and deletion mutants.

Like MEF2C, the MEF2A domain contains only Ser, Leu, and acidic residues. By contrast, MEF2D is unique in having a positively charged (His) residue (Fig. 7A). Thus, MEF2D was selected to address whether the functional role of domain acidic residues generalized among MEF2 gene isotypes. As for MEF2C, MEF2D domain mutants were created in the N86 MEF2D 1. background. Here, the T286A and H289A mutants each activated the reporter nearly as well as the wild-type fusion protein, whereas activation by the triple substitution acid to amide mutant (_DENQ) was similar to that of the - fusion protein (Gal4_DBD-MEF2D 1) (Fig. 7D). The previously noted -helical secondary structure in the region of the domains of + MEF2 splicing isoforms may or may not be required for retention of activity, because the various MEF2 domain mutations studied are each compatible with maintenance of a helix. Nonetheless, these data suggest a common mode of domain activity among MEF2 gene isotypes involving acidic amino acid side chains, and not simply bulky polar residues.

MEF2 Functions Independently of p38 MAPK Signaling—MEF2C (29) and MEF2A (15) have previously been shown to be substrates of p38 MAPK. In each case, the corresponding paired Thr phosphoacceptor residues are located directly carboxyl-terminal to the domain (Fig. 8A). Modification of these residues requires a kinase docking (D) domain, present in these MEF2 isotypes but absent in MEF2B, which specifically recognizes the and isoforms of p38 (79). This D domain is also adjacent to but is located to its amino-terminal side (Fig. 8A). Thus, the presence or absence of might govern either accessibility of the D domain for kinase interaction, or the availability of the phosphoacceptor Thr residues for modification by docked p38. Protein secondary structure predictions support the feasibility of this speculation, in that the domain and immediate surrounding region of MEF2A, MEF2C, and MEF2D are expected to form an helix, whereas each - isoform is predicted to lack helical structure in this region (Fig. 8A) (71-73).

View larger version (21K): [in this window] [in a new window]   FIG. 7. MEF2 domain function is conferred by acidic residues. A, alignment of the MEF2A, MEF2C, and MEF2D domains, along with various domain mutants studied. Numbers reference the respective 1. isoforms. B, C2C12 myoblasts were cotransfected, processed, and analyzed as in Fig. 5 using constructs expressing the indicated Gal4DBD-N86 MEF2C 1. wild-type and mutant fusions. C, extracts from cells transfected in B were immunoblotted as in Fig. 5. D, C2C12 myoblasts were co-transfected and analyzed as in B using constructs expressing the indicated Gal4DBD-N86 MEF2D 1. wild-type and mutant fusions.

MEF2 Domain Function Is Position-independent and Exists in Each MEF2 Alternative Splicing Isoform—The position dependence of the domain transactivation function was tested using a fusion of MEF2C to the carboxyl terminus of the Gal4_DBD-N86 MEF2C 1 (-) fusion (Gal4_DBD-N86 MEF2C 1/_COOH) (Fig. 9A). Transactivation by this fusion was virtually identical to that of the wild-type 1. fusion and 5-fold higher than the corresponding wild-type - protein despite similar levels of expression (Fig. 9B). Function of the acidic domain therefore does not require placement in the endogenous site within the MEF2 holoprotein. A Gal4_DBD fusion to the isolated MEF2D domain also stimulated the G5Luc reporter (not shown), as might be expected of an acidic domain.

We have previously described a phosphoserine-dependent transrepressor function within the alternative MEF2C domain (35). This activity also exists in all alternative splicing forms of MEF2A and MEF2D.³ -Mediated repression is seen in MEF2 proteins containing either the 1orthe 2 domain, as well as in forms that have or lack the domain (35). To determine if transactivation function given by the domain is also independent of the domains, transactivation by Gal4_DBD fusions of the four MEF2A splicing isoforms were tested in activation of the G5Luc reporter (Fig. 10A). Here, inclusion of in either MEF2A 1 or MEF2A 2 gave similar results, confirming its function in either domain background (Fig. 10B). Although we have shown that repression exerted by occurs in both + and - contexts (35), lack of functional interdependence between the and domains would also require activity in both + and - contexts. Constructs used to test such functional autonomy of these two domains are shown schematically in Fig. 10C. Gal4_DBD fusions to + variants of N86 MEF2C 1 (-) and 1. each showed a similar -fold activation compared with the corresponding - fusion (Fig. 10D). This was also the case for a MEF2C fusion protein containing a mutation in the domain Ser whose phosphorylation regulates the repressor function (1._S388A versus 1.._S396A). Taken together, this confirms that the domain functions independently in all MEF2 splicing isoforms.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Several splicing variants of the four MEF2 gene transcripts were identified in the original cDNA cloning work (4-9, 83). More recently, alternative splicing of vertebrate MEF2 gene products in both 5' non-coding and coding exons has been more completely described (35-39),² and conservation of patterns of alternative splicing among MEF2A, MEF2C, and MEF2D genes has become clear (35). Despite this, little information has been reported as to distinct functions or expression patterns of the various vertebrate MEF2 splicing isoforms (35, 67, 84). We recently described a potent phosphoserine-dependent transrepression function of a MEF2 domain that is variably included in MEF2C proteins depending on alternative splice acceptor usage (35). This domain is constitutive in other MEF2 forms,³ such that MEF2C and specifically the - variants of MEF2C have unique function within this family. In the present report, we have demonstrated a second distinction among MEF2 splicing isoforms that involves a transactivation domain that is included only in MEF2A, MEF2C, and MEF2D proteins expressed in muscle, heart, and brain. Tissue-selective inclusion of an exon encoding a short conserved acidic domain that we term governs this process (Fig. 11).

View larger version (31K): [in this window] [in a new window]   FIG. 8. p38 MAPK signaling influences the transactivation function of both + and - MEF2 isoforms. A, alignment of segments of human MEF2A, MEF2C, and MEF2D, and Drosophila DMEF2 transactivation domains. Numbers reference residues in the 1. splicing isoform of each human MEF2 isotype, and the DMEF2 splicing isoform c (68). Gray boxes encompass the p38 MAPK docking sites (D domains) (79), the paired Thr phosphoacceptors modified by this kinase (15), and the intervening domains. Hashed gray boxes underlying MEF2D sequences indicate that neither p38 kinase docking nor Thr modification of this isotype has been confirmed, despite sequence similarities with MEF2A and MEF2C. The extent of an -helix in each form predicted by various structural analysis algorithms (71-73) is indicated below each sequence. B, C2C12 myoblasts were cotransfected with pRL-tk, pG5Luc, and the indicated pM-MEF2C isoform fusions, followed by incubation in medium supplemented with 20% fetal bovine serum with or without 10 µM SB203580 prior to harvesting for analysis as in Fig. 5.

View larger version (22K): [in this window] [in a new window]   FIG. 9. MEF2C domain transactivation function is position-independent and autonomous. A, schematic of MEF2C splicing isoforms and the Gal4DBD fusions used. B, C2C12 cells were cotransfected, processed, and analyzed as in Fig. 5 using constructs expressing the indicated Gal4DBD-MEF2C fusions. C, extracts from cells transfected in B were immunoblotted as in Fig. 5.

View larger version (17K): [in this window] [in a new window]   FIG. 10. MEF2 domain-mediated transactivation occurs in all MEF2 splicing form contexts. A, schematic of MEF2A and MEF2C splicing isoforms and the Gal4DBD-MEF2 fusions used. B, C2C12 myoblasts were cotransfected, processed, and analyzed as in Fig. 5 using constructs expressing the indicated Gal4DBD-MEF2A isoform fusions. C, schematic of MEF2C splicing isoforms and the Gal4DBD-MEf2C fusions used. D, C2C12 myoblasts were cotransfected, processed, and analyzed as in Fig. 5 using constructs expressing the indicated Gal4DBD-N86 MEF2C isoform and mutant fusions. MEF2C 1.S388A and MEF2C 1..S396A have Ala substitutions at the identical Ser phosphoacceptor residue of the domain (21, 35).3

View larger version (40K): [in this window] [in a new window]   FIG. 11. Phosphoacceptor sites and cognate kinases, functional domains, and protein interactors of MEF2 proteins. Phosphoacceptor sites for indicated kinases are indicated above a schematic of the MEF2C splicing isoforms. Analogous residues of MEF2A (CKII (94), p38 / (15), and cdk5 (21) and MEF2D (CKII and cdk5) are subject to modification. Different sites within MEF2A and MEF2D are targets of Erk5 (14, 95). Various activities within MEF2 proteins have been established (40, 76). Those known for the alternative domains are indicated, including phosphoserine-dependent transrepression within the domain (35),3 and acidic transactivation function within the domain (see "Results"). Most established interactions with MEF2 proteins involve the MADS box and MEF2S domains (see "Results"). Exceptions are limited to the p38 MAPK interaction with the D domain (79), an interaction with Vgl-2 (66), and a Notch interaction with the 1 domain of MEF2C (84).

Selective or preferential recruitment or stabilization of a transcriptional co-activator complex by + MEF2 factors is one other explanation for the higher activity of these isoforms. However, previously described MEF2 co-activators are not relevant, because p300 (26, 53) and peroxisome proliferator-activated receptor gamma coactivator 1 (78) interact with the MADS/MEF2S domains, which are not required for function, and the steroid receptor coactivator-MEF2 interaction is present in MEF2 deletion mutants that lack (51). Alternatively, domain-mediated interference with a MEF2 co-repressor interaction could contribute to the observed distinctions in + versus - isoform activities. Here, the - isoform or fragment could favor or stabilize the interaction as compared with the corresponding + isoform region. MEF2 contacts the class II histone deacetylase and Cabin1 co-repressors through the MADS and MEF2S domains (56, 57, 60), such that these particular families of transcriptional co-regulators do not participate in function.

As is evident from the foregoing discussion, the transcriptional activity of domains must be transduced by factors that have not previously been identified as MEF2 interactors. The comparable function of the domain in various cell types, differentiation states, and growth conditions suggests the involvement of widely expressed interactor(s) and effector mechanism(s). This view is also supported by the position independence and autonomy of domain-mediated transactivation, as well as by the similar magnitude of the domain effect in different DNA-bound proteins. We therefore favor a direct interaction of with a central component of the transcriptional apparatus, i.e. one whose expression level and capacity for function are omnipresent.

The presence of three (MEF2C and MEF2D) or five (MEF2A) conserved Glu or Asp residues within domains and the predicted helical structure suggest that they may function as "acid blobs" (85, 86). Transduction of "acid blob" activity has been speculated to involve direct interactions with TATA box-binding protein or TFIID (85-87). It has been suggested that solute phase exposure of the polar acidic residues imposes a critical structure to these domains, although there is conflicting evidence to suggest that structural features of acidic domains are less important that the negative charge of the acidic residues, per se (87). Our findings with MEF2 domain mutants are more consistent with the latter view, because the Asn for Asp and Gln for Glu mutations retain polarity and are compatible with maintenance of an helix in the region. The partial loss of function with mutation of single acidic residues, and its complete loss with mutation of all, is similar to observations regarding the ₁ transactivation domain of the glucocorticoid receptor, wherein there exists an additive positive activation function of four acidic residues (88).

The Ser residue of MEF2A can be phosphorylated according to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (89). This phosphoserine was detected in a MEF2A fusion protein isolated from cells in which p38 MAPK signaling was activated. Because this site is clearly not a MAPK phosphoacceptor, the authors of this study speculated that it is instead a target of casein kinase II, the activity of which toward the MEF2A Ser substrate may be stimulated by p38. Although we cannot dispute that this MEF2A domain Ser residue can be phosphorylated, we have shown that mutation of the analogous residue in both MEF2C (MEF2C 1._S271A) and MEF2D (MEF2D 1._T286A) has no impact on transcriptional activity of these isotype forms.

Our studies did not detect distinctions between + and - forms in terms of DNA binding or dimerization, in protein accumulation or degradation when overexpressed, or in subcellular localization of MEF2C isoform-EGFP fusions.⁵ No obvious physiologically relevant protease cleavage sites or predicted protein interaction sites are omitted or introduced in + versus - forms. However, additional roles for may exist, or activities may be created, revealed, lost, or buried in neighboring MEF2 domains with inclusion or exclusion of domains. Protein fragments encoded by very short (<50 nt) alternative spliced exons have different secondary structures compared with the flanking regions much more commonly than not or by chance (90). This is the case for MEF2 domains, wherein along with a number of adjacent residues are predicted to assume an -helical structure. The - isoforms are predicted to lack such a structure in this region. Although transactivation functions of domain mutants may or may not require maintenance of a helical structure, it is likely that some functions of do involve this feature.

Each of the three MEF2 domains is identical or highly similar to polypeptide fragments within a variety of different proteins according to BLAST comparisons against various sequence databases. However, no common classes of proteins are apparent among the proteins detected. Furthermore, despite the conserved primary structures of MEF2A, MEF2C, and MEF2D polypeptides, the three MEF2 isotype domains identify entirely non-overlapping sets of proteins containing similar primary sequence. Finally, there do not appear to be other proteins in which -like domains are the products of alternatively spliced exons. Thus, no hints are provided from sequence comparisons as to either the mechanism of transcriptional activity or any additional roles that may be played by these domains.

MEF2A and MEF2C are established p38 MAPK targets, and modification of a pair of Thr residues within the transactivation domain of these proteins strongly potentiates their transactivation function (15, 29). This is proposed to account for the positive effect of p38 activity on myoblast differentiation (80-82), neuroprotection (22), and the transcriptional response of inflammatory cytokine genes to infection in lymphocytes (29). A docking (D) domain within MEF2A and MEF2C recognizes the and isoforms of p38 and is required for modification of the target phosphoacceptors (79). Docking of p38 to MEF2 was expected to be independent of based on the isolation of both MEF2C - and + isoform partial cDNAs as preys in the yeast two-hybrid screen that originally identified MEF2 as a p38 target (29). However, the predicted helical structure suggested that the interposition of between D and the Thr phosphoacceptors might control Thr phosphorylation by the docked kinase. Although our work cannot exclude differences in the kinetics of this process in + versus - isoforms, splicing isoforms of MEF2A and MEF2C were similarly regulated by p38 activity in our transcription assay. Thus, p38-directed phosphothreonine-mediated and domain-mediated functions are independent and additive in the control of the transcriptional activity of MEF2A and MEF2C factors.

Our work shows that function is also independent of signaling through other pathways known to regulate MEF2 functions. Thus, activity of in a context of mutation of the domain Ser phosphoacceptor (MEF2C 1_S388A and 1._S396A) confirms this for cdk5 (21, 35).³ MEF2 can interact with nuclear factor of activated T cell proteins, the nuclear localization of which is controlled by dephosphorylation given by the phosphoprotein phosphatase calcineurin. MEF2 is sequestered from its DNA targets when interacting with class II histone deacetylase co-repressors. The contact is sensitive to phosphorylation within a histone deacetylase domain that is a target of calcium-calmodulin kinase IV. Both of these calcium-sensitive MEF2 protein interactions involve the MADS and MEF2S domains (2). Maintenance of activity in the N86 fusions establishes that signaling through either calcineurin or calcium-calmodulin kinase IV is neither required for nor affected by . Thus, these established modes of MEF2 regulation through signaling exist in both + and - isoforms, and they are unrelated to transduction of function.

Muscle- and/or neuron-specific alternative splicing has been characterized in a number of mammalian genes. The alternative splice versus no-splice option of MEF2 exons is similar to that of cTnT, which encodes cardiac troponin T, with the evident exception that exon exclusion, rather than inclusion, is acquired during development and muscle differentiation (91). Control of alternative splicing of this type can be exerted either through the activities of positive factors or signals acting at exon or intron splicing enhancers, and/or by loss of negatively acting influences at exon or intron splicing silencing elements (92). Thus, the MEF2 exons could be included through the action of muscle- and neuron-specific factors acting positively. One potentially relevant and well characterized exon splicing enhancer is recognized by ASF/SF2, a positively acting SR splicing factor that recognizes purine-rich (GAR)_n motifs (93). The MEF2A exon contains five conserved GAR repeats (hence the Glu-rich amino acid sequence), but the other MEF2 isotypes have only a single conserved GAR. Thus, ASF/SF2 may or may not play a role in exon inclusion in all or any MEF2 genes. The alternative scenario in which the ground state involves exon exclusion by a ubiquitous factor that is functionally competed in muscle and neuron is most notably comparable to the role of PTB in the highly tissue-specific splicing of various genes (92). It is premature to speculate as to specific alternative splicing mechanisms that may be operational in the MEF2 genes. However, the high degree of homology among vertebrate MEF2 genes of each isotype, the sequence similarity across isotypes, and the similar patterns of exon splicing among these genes should facilitate identification of critical RNA elements and factors that control these splicing choices.

Splicing of MEF2 transcripts involves bona fide alternative splicing of exons (1 versus 2), the splice versus no-splice option, and alternative splice acceptor usage (MEF2C + versus -). Our findings support independence of these alternative splicing events. Thus, RPA with the mef2-c / probe showed that all four possible variants involving the last two options can be expressed, with - forms predominating as we had previously described (35). The various MEF2 isotype 2 splicing isoforms are more prevalent in muscle, and they appear during muscle differentiation,⁴ as do the + isoforms. However, cDNA sequences within the expressed sequence tag databases indicate that - and + forms of both 1- and 2-containing mRNAs exist. The functions of the various domains encoded by alternative splicing are also independent. This indicates that the domain is likely to be functional in all sites where it is expressed, i.e. both in muscle where MEF2 domain isoforms are co-expressed, and in neurons and other tissues where MEF2 proteins appear to have only the 1 domain.

We have demonstrated that alternative splicing is critical to the regulation of MEF2A, MEF2C, and MEF2D function by virtue of tissue-selective inclusion of an exon that encodes an acidic -helical transcriptional activation domain. This work introduces one independent additional level of complexity in the regulation of this important class of transcription factors. Our findings indicate a strategy by which products of the MEF2 genes are made uniquely potent in cardiac and skeletal muscle, the primary sites of MEF2 target gene activation. This work further emphasizes that consideration must be given to splicing isoforms when examining functional distinctions among products of the different vertebrate MEF2 isotypes.

	FOOTNOTES

 
^* This work was supported in part by American Heart Association Grant 0150622N, Juvenile Diabetes Foundation Grant 1998-224, Clinical Nutrition Research Center at Harvard Grant P30 DK40561, and National Institutes of Health Grants DK55875 and HL72713. 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 with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Present address: Ben May Institute for Cancer Research, University of Chicago, 5841 S. Maryland Ave., Box MC6027, Chicago, IL 60637.

^|| Supported by NIDDK Independent Scientist Award DK02461 and by a grant from the Vivien Krantz Memorial Fund. To whom correspondence should be addressed: Diabetes Research Laboratory, Massachusetts General Hospital, CNY 149 8219, Charlestown, MA 02129. Tel.: 617-724-2356; Fax: 617-726-9452; E-mail: gulick{at}helix.mgh.harvard.edu.

¹ The abbreviations used are: MEF2, myocyte enhancer factor 2; MEF2S, MEF2 signature domain; Cabin1, calcineurin binding protein 1; cdk, cyclin-dependent kinase; CPT-IB, carnitine palmitoyltransferase I; DME, Dulbecco's modified Eagle's medium; DMEF2, Drosophila myocyte enhancer factor 2; Erk, extracellular signal responsive protein kinase; Gal4_DBD, DNA binding domain of yeast Gal4p; MADS, MCM1, agamous, deficiens, serum response factor; MAPK, mitogen-activated protein kinase; RPA, ribonuclease protection assay.

² T. Gulick and G.-S. Yu, submitted for publication.

³ B. Ramachandran and T. Gulick, submitted for publication.

⁴ B. Zhu and T. Gulick, unpublished observations.

⁵ B. Ramachandran and T. Gulick, unpublished observations.

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