MEF2 Family Of Transcription Factors Research Articles

Research Resource: The Androgen Receptor Modulates Expression of Genes with Critical Roles in Muscle Development and Function

Androgen signaling through the androgen receptor (AR), a ligand-dependent transcription factor within the steroid receptor superfamily, plays an important role in the development and maintenance of many tissues. In muscle, androgens act as anabolic agents that increase both muscle mass and strength; however, a key unanswered question is the mechanism through which AR-mediated gene expression leads to these effects. To gain further insight into the mechanism of AR action in muscle, we identified AR-binding sites in primary human muscle cells using ChIP-on-Chip (chromatin immunoprecipitation coupled with tiling microarray detection of genomic fragments). Through this analysis, we identified 32,518 potential AR-binding sites throughout the genome that were enriched upon androgen treatment. Sequence analysis of these regions indicated that approximately 90% possess a consensus androgen response element or half-site. Among the identified AR-binding sites are genes known to be directly regulated by AR, confirming the validity of our methodology. Additionally, we identified a number of novel AR targets, including genes and micro-RNAs implicated in muscle differentiation and function, suggesting a direct role for AR-mediated transcription in muscle development. Intriguingly, binding sequences for the Mef2 family of transcription factors were enriched in the AR-bound regions, and we show that several Mef2c-dependent genes are direct targets of AR, suggesting a functional interaction between Mef2c and AR in skeletal muscle. Our results provide new insights into the mechanisms by which androgens promote muscle growth and validate AR as a potential therapeutic target for sarcopenia, muscle wasting, and other androgen-related muscle disorders.

The Transcriptional Coactivator PGC-1β Drives the Formation of Oxidative Type IIX Fibers in Skeletal Muscle

Skeletal muscle must perform different kinds of work, and distinct fiber types have evolved to accommodate these. Previous work had shown that the transcriptional coactivator PGC-1alpha drives the formation of type I and IIA muscle fibers, which are "slow-twitch" and highly oxidative. We show here that transgenic expression of PGC-1beta, a coactivator functionally similar to but distinct from PGC-1alpha, causes a marked induction of IIX fibers, which are oxidative but have "fast-twitch" biophysical properties. PGC-1beta coactivates the MEF2 family of transcription factors to stimulate the type IIX myosin heavy chain (MHC) promoter. PGC-1beta transgenic muscle fibers are rich in mitochondria and are highly oxidative, at least in part due to coactivation by PGC-1beta of ERRalpha and PPARalpha. Consequently, these transgenic animals can run for longer and at higher work loads than wild-type animals. Together, these data indicate that PGC-1beta drives the formation of highly oxidative fibers containing type IIX MHC.

A Temporal Map of Transcription Factor Activity: Mef2 Directly Regulates Target Genes at All Stages of Muscle Development

Dissecting components of key transcriptional networks is essential for understanding complex developmental processes and phenotypes. Genetic studies have highlighted the role of members of the Mef2 family of transcription factors as essential regulators in myogenesis from flies to man. To understand how these transcription factors control diverse processes in muscle development, we have combined chromatin immunoprecipitation analysis with gene expression profiling to obtain a temporal map of Mef2 activity during Drosophila embryonic development. This global approach revealed three temporal patterns of Mef2 enhancer binding, providing a glimpse of dynamic enhancer use within the context of a developing embryo. Our results provide mechanistic insight into the regulation of Mef2's activity at the level of DNA binding and suggest cooperativity with the bHLH protein Twist. The number and diversity of new direct target genes indicates a much broader role for Mef2, at all stages of myogenesis, than previously anticipated.

MEF2-dependent Recruitment of the HAND1 Transcription Factor Results in Synergistic Activation of Target Promoters

HAND proteins are tissue-restricted members of the basic helix-loop-helix transcription factor family that play critical roles in cell differentiation and organogenesis including placental, cardiovascular, and craniofacial development. Nevertheless, the molecular basis underlying the developmental action of HAND proteins remains undefined. Within the embryo, HAND1 is first detected in the developing heart where it becomes restricted to the atrial and left ventricular compartments, a pattern identical to that of the Nppa gene, which encodes atrial natriuretic factor, the major secretory product of the heart. We hereby report that the cardiac atrial natriuretic factor promoter is directly activated by HAND1, making it the first known HAND1 transcriptional target. The action of HAND1 does not require heterodimerization with class I basic helix-loop-helix factors or DNA binding through E-box elements. Instead, HAND1 is recruited to the promoter via physical interaction with MEF2 proteins. MEF2/HAND1 interaction results in synergistic activation of MEF2-dependent promoters, and MEF2 binding sites are sufficient to mediate this synergy. MEF2 binding to DNA is not enhanced in the presence of HAND1. Instead, cooperativity likely results from corecruitment of co-activators such as CREB-binding protein. The related HAND2 protein can also synergize with MEF2. Thus, HAND proteins act as cell-specific developmental co-activators of the MEF2 family of transcription factors. These findings identify a novel mechanism for HAND action in the heart and provide a general paradigm to understand the mechanism of HAND action in organogenesis.

The Mef2c gene is a direct transcriptional target of myogenic bHLH and MEF2 proteins during skeletal muscle development.

Members of the MEF2 family of transcription factors are upregulated during skeletal muscle differentiation and cooperate with the MyoD family of myogenic basic helix-loop-helix (bHLH) transcription factors to control the expression of muscle-specific genes. To determine the mechanisms that regulate MEF2 gene expression during skeletal muscle development, we analyzed the mouse Mef2c gene for cis-regulatory elements that direct expression in the skeletal muscle lineage in vivo. We describe a skeletal muscle-specific control region for Mef2c that is sufficient to direct lacZ reporter gene expression in a pattern that recapitulates that of the endogenous Mef2c gene in skeletal muscle during pre- and postnatal development. This control region is a direct target for the binding of myogenic bHLH and MEF2 proteins. Mutagenesis of the Mef2c control region shows that a binding site for myogenic bHLH proteins is essential for expression at all stages of skeletal muscle development, whereas an adjacent MEF2 binding site is required for maintenance but not for initiation of Mef2c transcription. Our findings reveal the existence of a regulatory circuit between these two classes of transcription factors that induces, amplifies and maintains their expression during skeletal muscle development.

ERK5 is a novel type of mitogen-activated protein kinase containing a transcriptional activation domain.

Previous studies have shown that upregulation of the orphan steroid receptor Nur77 is required for the apoptosis of immature T cells in response to antigen receptor signals. Transcriptional upregulation of Nur77 in response to antigen receptor signaling involves two binding sites for the MEF2 family of transcription factors located in the Nur77 promoter. Calcium signals greatly increase the activity of MEF2D in T cells via a posttranslational mechanism. The mitogen-activated protein (MAP) kinase ERK5 was isolated in a yeast two-hybrid screen using the MADS-MEF2 domain of MEF2D as bait. ERK5 resembles the other MAP kinase family members in its N-terminal half, but it also contains a 400-amino-acid C-terminal domain of previously uncharacterized function. We report here that the C-terminal region of ERK5 contains a MEF2-interacting domain and, surprisingly, also a potent transcriptional activation domain. These domains are both required for coactivation of MEF2D by ERK5. The MEF2-ERK5 interaction was found to be activation dependent in vivo and inhibitable in vitro by the calcium-sensitive MEF2 repressor Cabin 1. The transcriptional activation domain of ERK5 is required for maximal MEF2 activity in response to calcium flux in T cells, and it can activate the endogenous Nur77 gene when constitutively recruited to the Nur77 promoter via MEF2 sites. These studies provide insights into a mechanism whereby MEF2 activity can respond to calcium signaling and suggest a novel, unexpected mechanism of MAP kinase function.

14-3-3 Proteins Corral HDAC Proteins

Acetylated histones yield a favorable environment for the transcription of genes; acetylation allows for greater accessibility to gene promoters by transcription factors. On the other hand, histone deacetylases (HDACs) inhibit transcription by limiting accessibility to chromatin. HDACs bind to several transcription factors including the MEF2 family of transcription factors, which are involved in muscle cell differentiation. Grozinger and Scheiber investigate the regulation of HDAC4 and HDAC5, and observe that 14-3-3 proteins, which bind to phosphoserine residues, mediate the localization of HDACs within the cell. Immunocomplexes of overexpressed HDAC4 and HDAC5 contained 14-3-3 proteins, whereas overexpression of HDAC1, representing a different class of HDAC, did not. Fluorescence assays revealed that overexpressed HDAC4 localized to the nucleus, but in the presence of overexpressed 14-3-3β, HDAC4 remained in the cytoplasm. The association of 14-3-3 and HDAC4 was phosphoserine-dependent, and prevented the ability of importin α, a nuclear translocator protein, to bind HDAC4. Finally, increased nuclear localization of HDAC4 correlated with decreased MEF2-dependent transcription. Thus, these results suggest that 14-3-3 proteins can function to regulate transcription by associating with HDAC proteins. Grozinger, C.M., and Schreiber, S.L. (2000) Regulation of histone deacetylase 4 and 5 and transcriptional activity by 14-3-3-dependent cellular localization. Proc . Natl . Acad . Sci . U.S.A. 97 : 7835-7840. [Abstract] [Full Text]

Ca2+-dependent Gene Expression Mediated by MEF2 Transcription Factors

Ca(2+) induction of a subset of cellular and viral immediate-early activation genes in lymphocytes has been previously mapped to response elements recognized by the MEF2 family of transcription factors. Here, we demonstrate that Ca(2+) activation of MEF2 response elements in T lymphocytes is mediated in synergy by two Ca(2+)/calmodulin-dependent enzymes, the phosphatase calcineurin, and the kinase type IV/Gr (CaMKIV/Gr), which promote transcription by the MEF2 family members MEF2A and MEF2D. Calcineurin up-regulates the activity of both factors by an NFAT-dependent mechanism, while CaMKIV/Gr selectively and independently activates MEF2D. These results identify MEF2 proteins as effectors of a pathway of gene induction in T lymphocytes which integrates diverse Ca(2+) activation signals and may be broadly operative in several tissues.

The Transcription Factor MEF2C-Null Mouse Exhibits Complex Vascular Malformations and Reduced Cardiac Expression of Angiopoietin 1 and VEGF

The MEF2 family of transcription factors has been implicated in transcriptional regulation in a number of different cell types. Targeted deletion of the MEF2C gene, in particular, revealed its importance for early cardiogenesis (Q. Lin et al., 1997, Science 276, 1404–1407). We report here that this deletion also resulted in vascular anomalies characterized by extreme variability in lumen size and defects in remodeling. While primary vascular networks formed in the yolk sac of the mutants, they failed to remodel into more complex vascular structures. Likewise, although the primordia of the dorsal aortae formed normally, anomalies were observed in these vessels later in development. Dorsal and anterior to the heart, the aortae exhibited abnormally small lumens, as did the anterior cardinal veins and intersegmental arteries. In contrast, the dorsal aortae and intersegmental arteries caudal to the heart were grossly enlarged. Cranial vessels were also enlarged and less branched than normal. Endocardiogenesis in the mutant was abnormal with the endothelial cells exhibiting a number of aberrant phenotypes. These endocardial defects were accompanied by a notable reduction in angiopoietin 1 and VEGF mRNA production by the myocardium, indicating that MEF2C is required for myocardial expression of these important endothelial-directed cytokines and thus for correct endocardial morphogenesis.

Regulation of the MEF2 family of transcription factors by p38.

Members of the MEF2 family of transcription factors bind as homo- and heterodimers to the MEF2 site found in the promoter regions of numerous muscle-specific, growth- or stress-induced genes. We showed previously that the transactivation activity of MEF2C is stimulated by p38 mitogen-activated protein (MAP) kinase. In this study, we examined the potential role of the p38 MAP kinase pathway in regulating the other MEF2 family members. We found that MEF2A, but not MEF2B or MEF2D, is a substrate for p38. Among the four p38 group members, p38 is the most potent kinase for MEF2A. Threonines 312 and 319 within the transcription activation domain of MEF2A are the regulatory sites phosphorylated by p38. Phosphorylation of MEF2A in a MEF2A-MEF2D heterodimer enhances MEF2-dependent gene expression. These results demonstrate that the MAP kinase signaling pathway can discriminate between different MEF2 isoforms and can regulate MEF2-dependent genes through posttranslational activation of preexisting MEF2 protein.

High Level Expression in Soluble Form, One Step Purification, and Characterization of the DNA Binding Domain of MEF-2C

Members of the MEF-2 family of transcription factors act as coregulators of basic helix–loop–helix (BHLH) proteins in the control of lineage specific gene expression in many cell types through direct interaction between the respective DNA binding domains. To make possible a thorough biochemical, biophysical, and structural characterization of the properties of myocyte enhancer factor (MEF) proteins and of their interactions with BHLH-proteins, a simple system for high level expression and rapid purification of myocyte enhancer factor-2C (MEF-2C) was developed. A T7 expression system was used to produce in high yield inEscherichia colian N-terminal fragment of MEF-2C comprising both the MADS box and the MEF domain. Purification by a single round of cation-exchange chromatography on a Resource-S HPLC column at elevated pH afforded an essentially pure protein. Recombinant MEF-2C(1–117) bound with high affinity to the MEF consensus DNA binding site (CTATAAATAG). Mutations in this sequence that replaced adenines with thymine or vice versa did not significantly alter the affinity for MEF-2C(1–117). The introduction of G–C pairs into the core of the MEF-site, however, dramatically increased the concentration of MEF-2C(1–117) needed for half maximal DNA binding. We propose an explanation of the DNA binding specificity of MEF-2C based on the intrinsic bending properties of the unbound DNA.

Structures and chromosome locations of the human MEF2A gene and a pseudogene MEF2AP

The MEF2 family of transcription factors control the expression of muscle-specific and mitogen-induced genes. Here we describe the isolation and structure of the human MEF2A gene. The protein coding region of MEF2A is divided by 10 introns. The 3' untranslated region (UTR) is 3.7 kb in length, and it contains a region that is highly homologous with a portion of the 3' UTR of Xenopus MEF2A. A partially processed pseudogene (MEF2AP) corresponding to MEF2A was also isolated and characterized. Human MEF2A was mapped by fluorescence in situ hybridization to chromosome 15q26, and MEF2AP was mapped to chromosome 1q24 --> q25.

Mef2 gene expression marks the cardiac and skeletal muscle lineages during mouse embryogenesis

Members of the MEF2 family of transcription factors bind a conserved A/T-rich sequence in the control regions of many skeletal and cardiac muscle genes. To begin to assess the roles of the different Mef2 genes in the control of muscle gene expression in vivo, we analyzed by in situ hybridization the expression patterns of the Mef2a, Mef2c and Mef2d genes during mouse embryogenesis. We first detected MEF2C expression at day 7.5 postcoitum (p.c.) in cells of the cardiac mesoderm that give rise to the primitive heart tube, making MEF2C one of the earliest markers for the cardiac muscle lineage yet described. By day 8.5, MEF2A, MEF2C and MEF2D mRNAs are all detected in the myocardium. By day 9.0, MEF2C is expressed in rostral myotomes, where its expression lags by about a day behind that of myf5 and several hours behind that of myogenin. MEF2A and MEF2D are expressed at a lower level than MEF2C in the myotome at day 9.5 and are detected in more embryonic tissues than MEF2C. Expression of each of the MEF2 transcripts is observed in muscle-forming regions within the limbs at day 11.5 p.c. and within muscle fibers throughout the embryo at later developmental stages. The expression of MEF2C in the somites and fetal muscle is distinct from that of MEF2A, MEF2D and the myogenic bHLH regulatory genes, suggesting that it may represent a distinct myogenic cell type. Neural crest cells also express high levels of MEF2 mRNAs between days 8.5 and 10.5 of gestation. After day 12.5 p.c., MEF2 transcripts are detected at high levels in specific regions of the brain and ultimately in a wide range of tissues. The distinct patterns of expression of the different Mef2 genes during mouse embryogenesis suggest that these genes respond to unique developmental cues and support the notion that their products play roles in the regulation of muscle-specific transcription during establishment of the cardiac and skeletal muscle lineages.