MEF2 is a family of four transcription factors, MEF2A-D, that were initially implicated in gene transcription leading to muscle cell differentiation (57
). Although they were found to be ubiquitously expressed in all tissues and cell types including T cells, their role in calcium signaling and particularly in T cells remained unknown. It was after MEF2 was identified as an interacting protein of Cabin1 (and HDACs), and through the ensuing characterization of the interaction between Cabin1 and MEF2 that a role of MEF2 in calcium signaling in general, and in mediating intracellular calcium signaling in T cells in particular, was unraveled using TCR-mediated T-cell hybridoma apoptosis as a model system (58
MEF2 is composed of a highly conserved N-terminal MADS/MEF2S domain responsible for homo-or heterodimerization and DNA binding and a divergent C-terminal domain involved in transactivation that is unique for each isoform. It is constitutively bound to DNA in the nucleus regardless of the activation status of T cells (60
). In the absence of calcium signaling, it is associated with Cabin1 along with its associated class I HDACs and a histone methyltransferase or class II HDACs (59
). Together, the HDAC and methyltransferase-containing ternary complex on MEF2 silence the promoter containing MEF2 binding sites. Upon an increase in intracellular calcium concentration during TCR signaling, the nuclear subset of calmodulin binds to Cabin1 and class II HDACs, releasing them from MEF2 (58
). This leads to the association of the transcriptional coactivator p300 (59
). The calcium-dependent dynamic switch in partners from HDAC/methyltransferase to HAT is made possible by another built-in molecular switch in the form of a merged overlapping MEF2-binding domain and calmodulin-binding domain in Cabin1 and class II HDACs, which converts MEF2 from a transcriptional repressor to a transcriptional activator (). These findings also revealed an important role of MEF2 in sensing and transducing calcium signal in various cellular processes. They pointed to the existence of another calcium signaling module consisting of MEF2, Cabin1/class II HDACs and p300 that is independent of the well-established calcineurin-NFAT signaling module (). In addition to extensive biochemical evidence for the MEF2-based calcium signaling module, the crystal structure of the Cabin1-MEF2-DNA complex provided further support for this model (63
The calcineurin-NFAT vs. MEF2-Cabin1/class II HDAC signaling modules in the regulation of IL-2 transcription
The role of the MEF2-Cabin1-p300 in calcium sensing and signaling was initially identified and characterized in the context of TCR-mediated thymocyte apoptosis and the induction of the orphan nuclear receptor Nur77 (58
). In an effort to assess the importance of Cabin1 in thymocyte apoptosis, a knockout mouse strain was produced that expressed a truncation mutant of Cabin1 lacking the C-terminal calcineurin-binding domain and the MEF2-binding domain (48
). Given the critical role of both calcineurin and MEF2 in mediating Nur77 expression and apoptosis of T-cell hybridoma, it was expected that deletion of the two C-terminal domains would cause a defect in thymocyte negative selection. Surprisingly, the Cabin1ΔC mutant displayed no deficiency in thymocyte development with normal populations of double positive thymocytes and single positive T cells (48
). These observations ruled out a role of Cabin1 and MEF2 as well as Nur77 in TCR-mediated thymocyte apoptosis and negative selection. Unexpectedly, when the response of peripheral CD4+
T cells were stimulated with either PMA/ionomycin or anti-CD3 antibodies, a dramatic increase in the production of IL2, IFN-γ and other cytokines was observed relative to the same T-cell population isolated form wildtype animals, suggesting a previously unknown role of Cabin1 and MEF2 in signal transduction in helper T cells (48
). As both the calcineurin-binding domain and the MEF2-binding domain were missing in the Cabin1ΔC mutant animal, the abnormal upregulation of cytokine production could have been attributed to either an upregulation of calcineurin or MEF2, the latter due to the absence of the associated Cabin1-HDAC repressive complex. An examination of NFAT, however, revealed no difference in its dephosphorylation and nuclear translocation between the Cabin1ΔC mutant and wild type animals, leaving the dysregulation of MEF2 as the remaining cause of the observed phenotype.
Although MEF2 has been found to be expressed in T cells, it was not known to be involved in TCR signaling leading to cytokine gene expression. In particular, MEF2 sites had not been found in the well-characterized IL-2 promoter or promoters of other cytokines that were upregulated in the Cabin1ΔC T cells. Thus, the hint from the Cabin1ΔC mutant animal raised another question as to how MEF2 participates in the regulation of cytokine gene expression. A reexamination of the IL-2 proximal promoter revealed a site that bears some, but not all, characteristics of a consensus MEF2 binding site (64
). This site happens to be in a region of long-standing interest and confusion as it contains a TATA element upstream of the established TATA box. It had been thought that this region might represent a unique second TATA box in the IL-2 gene. The sequence, ‘CATAATATTT’ in the human gene and ‘CATATTATTT’ in the mouse gene, is reminiscent of the consensus MEF2 binding sequence ‘G(A/T)8
C’ in that they do contain a purine base pair followed by at least eight contiguous pyrimidine base pairs. But, they lack a purine cap at the 3′ end. Using gel mobility shift assay with oligonucleotide probes corresponding to the putative human MEF2 binding sequence, it was shown that it indeed bound to MEF2 specifically (64
). Using a promoter sequence with a mutation in the putative MEF2-binding site, it was shown by CHIP assay that this site is required for MEF2 binding. Most importantly, when MEF2D, the most abundant isoform of MEF2 in peripheral T cells, was knocked down using lentivirus-mediated shRNA, it was found that levels of IL-2 transcripts as well as secreted IL-2 in response to stimulation by anti-CD3 and anti-CD28 antibodies were dramatically reduced in primary human T lymphocytes. These results established a key role of MEF2 and its partner proteins Cabin1/mSin3/HDAC1/2 in the regulation of calcium-dependent transcription of IL-2 and other cytokines. The association of the repressive MEF2-Cabin1-mSin3-HDAC/methyltransferase complex with the IL-2 promoter in the resting naïve T cells also offers an explanation on how this and the promoters of other cytokines are kept in a silenced state in the absence of TCR activation and the ensuing calcium signaling. The demonstration of MEF2 as a critical factor independent of NFAT for TCR-mediated cytokine production also suggests that MEF2 may serve as an alternative target for discovering and developing novel immunosuppressive agents.
In addition to Cabin1, class II HDACs including HDAC4, 5, 7, and 9, have been shown to directly bind to MEF2 via the same N-terminal MADS/MEF2S domain (65
). More recent structural studies revealed that class II HDACs binds to the same pocket in MEF2 as Cabin1 (63
). Interestingly, HDAC4, and by prediction based on sequence similarity, all class II HDACs, binds to calmodulin via similar bifunctional domains that also mediate their interaction with MEF2 (62
). Like Cabin1, the binding of class II HDACs is mutually exclusive to their binding to MEF2. Thus, upon calcium signaling, class II HDACs are dissociated from MEF2 in a similar manner as the Cabin1-mSin3-HDAC1/2 complex. In addition to regulation directly by calmodulin, class II HDACs as well as Cabin1, are subject to regulation by CaMKIV and other kinases, which constitute another mechanism of regulation of their interactions with MEF2. The regulation of the interaction between class II HDACs in the context of muscle cell differentiation by CaMKIV has been extensively investigated (70
). However, the role of class II HDACs in calcium signaling in T cells remains to be investigated, especially in light of the existence of multiple members within the class II HDAC family and their potential redundancy with the Cabin1-mSin3-HDAC1/2 complex. The generation of conditional knockout animals for different members of class II HDAC family and different isoforms of MEF2 offers new opportunities to address those questions.