Structure of the MADS-box/MEF2 Domain of MEF2A Bound to DNA and Its Implication for Myocardin Recruitment

Abstract

Myocyte enhancer factor 2 (MEF2) regulates specific gene expression in diverse developmental programs and adaptive responses. MEF2 recognizes DNA and interacts with transcription cofactors through a highly conserved N-terminal domain referred to as the MADS-box/MEF2 domain. Here we present the crystal structure of the MADS-box/MEF2 domain of MEF2A bound to DNA. In contrast to previous structural studies showing that the MEF2 domain of MEF2A is partially unstructured, the present study reveals that the MEF2 domain participates with the MADS-box in both dimerization and DNA binding as a single domain. The sequence divergence at and immediately following the C-terminal end of the MEF2 domain may allow different MEF2 dimers to recognize different DNA sequences in the flanking regions. The current structure also suggests that the ligand-binding pocket previously observed in the Cabin1–MEF2B–DNA complex and the HDAC9 (histone deacetylase 9)–MEF2B–DNA complex is not induced by cofactor binding but rather preformed by intrinsic folding. However, the structure of the ligand-binding pocket does undergo subtle but significant conformational changes upon cofactor binding. On the basis of these observations, we generated a homology model of MEF2 bound to a myocardin family protein, MASTR, that acts as a potent coactivator of MEF2-dependent gene expression. The model shows excellent shape and chemical complementarity at the binding interface and is consistent with existing mutagenesis data. The apo structure presented here can also serve as a target for virtual screening and soaking studies of small molecules that can modulate the function of MEF2 as research tools and therapeutic leads.

Introduction

The myocyte enhancer factor 2 (MEF2) family of proteins was originally identified as a regulator of muscle gene expression in vertebrates;¹ its function has now been expanded to diverse cellular processes in eukaryotic cells.² In yeast Saccharomyces cerevisiae, the MEF2 homolog Rlm1 regulated specific gene expression in response to mitogen-activated protein kinase activation.³ In Drosophila, genetic analyses have demonstrated the central role of MEF2 in myogenesis.4, 5, 6, 7, 8 In vertebrates, where four different genes of MEF2 are encoded in the genome (mef2a, b, c, and d), MEF2 has been shown to play critical roles in controlling the differentiation, proliferation, and survival/apoptosis of a wide range of cell types including muscle, T cells, and neurons.2, 9, 10, 11, 12, 13 In adult tissues, MEF2 also serves as a key regulator of stress responses and adaptive programs in response to environmental signals, including fiber-type switch of skeletal muscle, cardiac hypertrophy, and activity-dependent remodeling of neuronal synapses.14, 15, 16, 17, 18, 19

MEF2 is emerging as a potential therapeutic target for a number of human diseases. MEF2 and its associated transcription cofactors such as class IIa histone deacetylases (HDACs) and p300 have been shown to modulate transcription programs involved in muscle metabolism and cardiac growth,16, 20, 21, 22 suggesting that MEF2 modulators could be used to treat muscle diseases and cardiac hypertrophy. MEF2 regulates cytokine expression and apoptosis in T cells.9, 12 Recent studies show that HDAC9 knockout leads to enhanced function of regulatory T cells.²³ Since HDAC9 and MEF2 often function as a complex in vivo, these observations raise the possibility that the function of MEF2 might be modulated for treating autoimmune diseases and transplant rejection. A series of studies have established a key role of MEF2 in neuronal survival and synapses.13, 17, 18, 24, 25 These findings suggest that MEF2 could be targeted for treating neurodegenerative diseases such as Alzheimer disease, Huntington disease, and other psychiatric disorders such as autism, schizophrenia, and drug addiction. Finally, there is increasing evidence linking MEF2 to cancer development, pointing to a potential role of MEF2 as an oncogene.26, 27 MEF2 is highly expressed in certain leukemias and is apparently required for the self-renewal of the leukemia stem cell.²⁸ Moreover, MEF2 activity is elevated through chromosome translocation and gene fusion in some leukemia cell lines.29, 30 These results suggest that inhibition of the function of MEF2 in the contexts of tumor cells could be a potential approach to cancer therapy. To develop small molecules that bind MEF2 and modulate its transcription function specifically, it is important to analyze systematically the structure and function of MEF2 and its various complexes in detail.

The MEF2 family of proteins (MEF2A–D) of vertebrates share a highly conserved N-terminal region (residues 1–93) followed by a more divergent C-terminal region. The N-terminal region contains the DNA-binding domain, the MADS-box (residues 1–58), which is named after MCM1, Agamous, Deficiens, and SRF.³¹ The sequence immediately following the MADS-box (residues 59–93) is unique to the MEF2 family and is thus called the MEF2-specific domain (MEF2 domain). MEF2 recognizes a core consensus DNA sequence of YTA(A/T)4TAR (Y, pyrimidine; R, purine).32, 33, 34 MEF2 expressed in different tissues appear to have distinct preferences for sequences flanking the core region, but how such selectivity is achieved is not clear.³² The activity of MEF2 is modulated by posttranslational modifications such as phosphorylation, sumoylation, and acetylation, as well as interactions with a variety of other proteins. These proteins include signaling molecules such as Erk5,³⁵ transcription factors such as MyoD,³⁶ NFAT,¹⁴ and Smad3,³⁷ transcriptional corepressors such as Cabin1,9, 10 HDAC338, 39 and class IIa HDACs,40, 41, 42 and transcriptional coactivators such as p300 and myocardin.22, 43, 44, 45, 46, 47 Most of these MEF2-interacting proteins have been shown to bind to the MADS-box/MEF2 region.

Given the important roles of the MADS-box/MEF2 domain in DNA-binding and protein–protein interactions, substantial effort has been put into characterizing the structure and function of this domain. The structure of the MADS-box/MEF2 domain of MEF2A bound to DNA has been characterized by X-ray crystallography and NMR.48, 49 These studies revealed that the MADS-box of MEF2A is similar to that of SRF and MCM1 but also with some key differences. In the crystal structure,⁴⁸ the C-terminal half of the MEF2 domain (residues 79–86) was truncated off. This region was included in the NMR study but was shown to be disordered.⁴⁹ The crystal structures of two ternary MEF2 transcription factor complexes, the Cabin1–MEF2B–DNA complex and the HDAC9–MEF2–DNA complex, have also been characterized.50, 51 These studies reveal a general corepressor recruiting mechanism by MEF2 wherein an amphipathic helix from Cabin1 and class IIa HDACs bind to a concave hydrophobic groove on the MADS-box/MEF2 domain.

Based on the systematic structural and biochemical studies of MEF2 complexes,50, 51 it is possible to analyze if an MEF2-interacting protein uses a similar binding mechanism described above. This can be achieved by first identifying if the protein of interest has a short amphipathic helix that matches with consensus MEF2-binding motif and then docking the helix into the hydrophobic groove of MEF2. Such analysis will help to interpret existing data and guide functional studies of a variety of MEF2 complexes. However, since the C-terminal half of the MEF2 domain is not observed in the previous crystal structure and is disordered in the NMR study,48, 49 it is not clear if the structure of the hydrophobic groove observed in the Cabin1–MEF2B–DNA complex and the HDAC9–MEF2B–DNA complex is induced by the corepressor binding. In the present study, we have solved the crystal structure of the MADS-box/MEF2 domain of human MEF2A bound to DNA in the absence of any cofactor. Our study shows that the entire MADS-box/MEF2 domain of MEF2A is stably folded. The structure of the intact MEF2 domain (residues 59–93) is stabilized by extensive interactions with the MADS-box and DNA. The hydrophobic groove is preformed in the “apo” structure of the MEF2A–DNA complex and is therefore poised to bind transcription factor partners. Using this structure, we analyzed a number of proteins that have been shown to interact with MEF2 physically using the docking approach described. We show that a short amphipathic helix in Myocardin and MASTR, which have been shown to activate MEF2-depdent gene expression and binds to the MADS-box/MEF2 domain,46, 47 fits perfectly into the hydrophobic groove on MEF2. The structure of the MEF2–DNA complex presented here will facilitate similar computer-based analyses of other MEF2 interacting proteins. This structure can also be used for virtual screen and soaking studies of small molecules that can bind MEF2 and modulate its transcription function inside cells.