How protein function is regulated is a fundamental question relevant to many biological processes. Different mechanisms are involved, and one such mechanism operates through modification at the posttranslational level. Lysine acetylation has recently emerged as an important posttranslational modification and has been shown to regulate functions of histones, about 40 transcription factors, and over 30 other proteins (36
). Acetylation of specific lysine residues located at the N-terminal tails of core histones is necessary for controlling chromatin activities in various nuclear processes. Histone deacetylases (HDACs) are the enzymes responsible for reversing the acetylation of histones. Some HDACs also display deacetylase activities towards other proteins. According to sequence similarity and phylogenetic analysis, known HDACs have been grouped into distinct classes (10
). Mammalian class II members have been further divided into two subclasses: IIa (HDAC4, -5, -7, and -9) and IIb (HDAC6 and -10) (66
Class IIa members share a bipartite domain organization and display significant sequence homology in their long N-terminal extensions and C-terminal catalytic domains. A characteristic feature of these deacetylases is dynamic regulation by signal-dependent nucleocytoplasmic trafficking. Ca2+
/calmodulin-dependent kinase (CaMK) and protein kinase D phosphorylate-specific serine residues within the N-terminal extensions of class IIa HDACs to promote 14-3-3 association and CRM1-dependent nuclear export (43
). Other regulatory mechanisms, such as sumoylation (34
), caspase cleavage (40
), ubiquitin-dependent proteosomal degradation (26
), and mitochondrial targeting (3
), have also been reported for some class IIa members. While human HDAC4 is highly sumoylated at Lys559 (34
; unpublished observations), substitution of this residue with arginine modestly affects the deacetylase and transcriptional activities of HDAC4, raising the question whether this modification regulates other functions. Related to this, little is known about potential roles of the regions adjacent to the sumoylation site.
HDAC4 and other class IIa members function as signal-responsive transcriptional corepressors for the myocyte enhancer factor-2 (MEF2) family of transcription factors (43
). In mammals, there are four MEF2 isoforms: MEF2A, -B, -C, and -D. Originally identified as myocyte enhancer factors, MEF2s have been extensively studied as major transcriptional activators for muscle differentiation (43
). Consistent with this, a mutation on the human MEF2A gene plays a potentially causal role in a familial coronary artery disease (71
). Recent studies indicate that MEF2s also play important roles in regulating other cellular programs like growth factor responses, neuronal survival, and T-cell apoptosis (8
). In addition, human MEF2D is expressed in different tissues (43
), and its gene is rearranged in pre-B acute lymphoblastic leukemia patients (78
). Class IIa HDACs interact with the DNA-binding domains of MEF2 proteins and convert them from activators to repressors. Upon activation by Ca2+
/calmodulin, CaMKs phosphorylate class IIa HDACs and promote nuclear export to relieve transcriptional repression, so CaMKs modify these HDACs to stimulate MEF2-dependent transcription. By contrast, MAP kinases directly modify MEF2s. While p38 phosphorylates MEF2A and MEF2C (9
), extracellular signal-regulated kinase 5 (ERK5) phosphorylates MEF2A, -C, and -D (30
). These phosphorylation events activate transcription, whereas cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation of MEF2A and -2D inhibits transcription (17
). Therefore, MEF2 transcription factors are subject to multisite phosphorylation for the integration of diverse signals in the nucleus.
Different covalent modifications are well known to interplay and regulate functions of histones and transcription factors like the p53 tumor suppressor (1
), so we explored whether covalent modifications other than phosphorylation regulate the MEF2 transcriptional activity and, if so, how different modifications may interplay and how class IIa HDACs may be involved. Here, we show that MEF2D, as well as MEF2C, is sumoylated on a single lysine residue located at a consensus sumoylation motif conserved among MEF2 proteins. This modification inhibits transcriptional and myogenic activities. Independent of its deacetylase domain, HDAC4 and other class IIa members potentiate sumoylation, whereas the SUMO protease SENP3 and the ERK5 signal pathway reverse the modification. These results identify sumoylation as a novel regulatory mechanism for MEF2 and suggest the potential interplay of sumoylation with phosphorylation in the control of MEF2-dependent transcription.