Three fundamental issues in postgenomic biology are (i) how the amino acid sequence of a given human protein predicates its structure, function, and regulation; (ii) how a protein is compared to its paralogs, as well as to its orthologs and other homologous proteins in model organisms; and (iii) how related studies contribute to the understanding of human pathology and the development of efficacious diagnostic and therapeutic means. These fascinating issues have inspired us to conduct a comprehensive analysis of information available on class II histone deacetylases (HDACs). In what follows, we will start with a brief description of different classes of HDACs and then compare class II HDACs from yeast and higher organisms in terms of domain organization, function, and regulation. We will also discuss evidence that links class II human HDACs to cardiomyopathy, osteodystrophy, neurodegenerative disorders, and cancer and will propose that, in addition to inhibitors, activators of these HDACs are of potential therapeutic value.

Rationale Direct reprogramming of fibroblasts into cardiomyocytes is a novel strategy for cardiac regeneration. However, the key determinants involved in this process are unknown. Objective To assess the efficiency of direct fibroblast reprogramming via viral overexpression of GATA4, Mef2c, and Tbx5 (GMT). Methods and Results We induced GMT overexpression in murine tail tip fibroblasts (TTFs) and cardiac fibroblasts (CFs) from multiple lines of transgenic mice carrying different cardiomyocyte lineage reporters. We found that the induction of GMT overexpression in TTFs and CFs is inefficient at inducing molecular and electrophysiological phenotypes of mature cardiomyocytes. In addition, transplantation of GMT infected CFs into injured mouse hearts resulted in decreased cell survival with minimal induction of cardiomyocyte genes. Conclusions Significant challenges remain in our ability to convert fibroblasts into cardiomyocyte-like cells and a greater understanding of cardiovascular epigenetics is needed to increase the translational potential of this strategy.

A eukaryotic protein is often subject to regulation by multiple modifications like phosphorylation, acetylation, ubiquitination, and sumoylation. How these modifications are coordinated in vivo is an important issue that is poorly understood but is relevant to many biological processes. We recently showed that human MEF2D (myocyte enhancer factor 2D) is sumoylated on Lys-439. Adjacent to the sumoylation motif is Ser-444, which like Lys-439 is highly conserved among MEF2 proteins from diverse species. Here we presented several lines of evidence to demonstrate that Ser-444 of MEF2D is required for sumoylation of Lys-439. Histone deacetylase 4 (HDAC4) stimulated this modification by acting through Ser-444. In addition, phosphorylation of Ser-444 by Cdk5, a cyclin-dependent kinase known to inhibit MEF2 transcriptional activity, stimulated sumoylation. Opposing the actions of HDAC4 and Cdk5, calcineurin (also known as protein phosphatase 2B) dephosphorylated Ser-444 and inhibited sumoylation of Lys-439. This phosphatase, however, exerted minimal effects on the phosphorylation catalyzed by ERK5, an extracellular signal-regulated kinase known to activate MEF2D. These results identified an essential role for Ser-444 in MEF2D sumoylation and revealed a novel mechanism by which calcineurin selectively "edits" phosphorylation at different sites, thereby reiterating that interplay between different modifications represents a general mechanism for coordinated regulation of eukaryotic protein functions in vivo.In higher eukaryotes, each cell type has a unique gene expression pattern that is ultimately determined by a specific network of transcription factors. The question how cell signaling regulates activities of transcription factors is thus of central importance to many biological processes. The MEF2 3 family of transcription factors comprises four members in mammals, MEF2A, -B, -C, and -D. They were originally identified as major transcriptional activators for muscle differentiation (1, 2). Indeed, some of them were subsequently shown to be important for cardiac myogenesis; null mutation of the murine MEF2A or MEF2C gene led to cardiac death (3-5), and a mutation on the human MEF2A gene was recently suggested to play a role in coronary artery disease (6). MEF2 proteins also have important roles in non-muscle cells by regulating growth factor response, viral gene expression, neuronal survival, T-cell apoptosis, and tumorigenesis (7-12). Consistent with this, the human MEF2D gene is rearranged in pre-B acute lymphoblastic leukemia (13,14), and large scale retrovirus-mediated insertion mutagenesis identified the mouse MEF2D gene as a potential oncogene (15, 16). Therefore, MEF2 transcription factors are key players in diverse cellular programs.At the molecular level, MEF2 is composed of a highly conserved N-terminal domain responsible for DNA recognition and a C-terminal domain with trans-acting function. Regulation of MEF2 function is complex and occurs at multiple levels, including tissue-specific expression (1, 5), altern...

The myocyte enhancer factor 2 (MEF2) family of transcription factors is not only important for controlling gene expression in normal cellular programs, like muscle differentiation, T-cell apoptosis, neuronal survival, and synaptic differentiation, but has also been linked to cardiac hypertrophy and other pathological conditions. Lysine acetylation has been shown to modulate MEF2 function, but it is not so clear which deacetylase(s) is involved. We report here that treatment of HEK293 cells with trichostatin A or nicotinamide upregulated MEF2D acetylation, suggesting that different deacetylases catalyze the deacetylation. Related to the trichostatin A sensitivity, histone deacetylase 4 (HDAC4) and HDAC5, two known partners of MEF2, exhibited little deacetylase activity towards MEF2D. In contrast, HDAC3 efficiently deacetylated MEF2D in vitro and in vivo. This was specific, since HDAC1, HDAC2, and HDAC8 failed to do so. While HDAC4, HDAC5, HDAC7, and HDAC9 are known to recognize primarily the MEF2-specific domain, we found that HDAC3 interacts directly with the MADS box. In addition, HDAC3 associated with the acetyltransferases p300 and p300/CBP-associated factor (PCAF) to reverse autoacetylation. Furthermore, the nuclear receptor corepressor SMRT (silencing mediator of retinoid acid and thyroid hormone receptor) stimulated the deacetylase activity of HDAC3 towards MEF2 and PCAF. Supporting the physical interaction and deacetylase activity, HDAC3 repressed MEF2-dependent transcription and inhibited myogenesis. These results reveal an unexpected role for HDAC3 and suggest a novel pathway through which MEF2 activity is controlled in vivo.Protein lysine acetylation refers to transfer of the acetyl moiety from acetyl coenzyme A (acetyl-CoA) to the ε-amino group of a lysine residue and is an important posttranslational modification that has recently emerged and rivals phosphorylation (41, 61). Proteins known to be subject to lysine acetylation include histones, over 50 transcription factors, and various other proteins (10,40,41,61,77). This dynamic modification is controlled by the opposing actions of acetyltransferases and deacetylases in vivo. Histones were the first substrates identified, so these two families of enzymes are known as histone acetyltransferases (HATs) and histone deacetylases (HDACs), although most of them also act on nonhistone proteins. In the past decade, many proteins have been shown to possess HDAC activity (4,22,39,66,78). On the basis of homology to budding yeast counterparts, human HDACs are grouped into four classes, with HDAC1, -2, -3, and -8, homologs of yeast Rpd3, forming class I. Class II comprises HDAC4, -5, -6, -7, -9, and -10, which possess deacetylase domains highly related to that of yeast Hda1. HDAC4, -5, -7, and -9 have similar domain organization and thus belong to a subgroup known as class IIa.Class III consists of SIRT1 and other Sir2-related proteins. A recent phylogenetic analysis revealed that HDAC11 represents class IV (21). Members of classes I, II, and IV are zinc-depend...

The myocyte enhancer factor-2 (MEF2) family of transcription factors plays an important role in regulating cellular programs like muscle differentiation, neuronal survival, and T-cell apoptosis. Multisite phosphorylation is known to control the transcriptional activity of MEF2 proteins, but it is unclear whether other modifications are involved. Here, we report that human MEF2D, as well as MEF2C, is modified by SUMO2 and SUMO3 at a motif highly conserved among MEF2 proteins from diverse organisms. This motif is located within the C-terminal transcriptional activation domain, and its sumoylation inhibits transcription. As a transcriptional corepressor of MEF2, histone deacetylase 4 (HDAC4) potentiates sumoylation. This potentiation is dependent on the N-terminal region but not the C-terminal deacetylase domain of HDAC4 and is inhibited by the sumoylation of HDAC4 itself. Moreover, HDAC5, HDAC7, and an HDAC9 isoform also stimulate sumoylation of MEF2. Opposing the action of class IIa deacetylases, the SUMO protease SENP3 reverses the sumoylation to augment the transcriptional and myogenic activities of MEF2. Similarly, the calcium M kinase and extracellular signal-regulated kinase 5 signaling pathways negatively regulate the sumoylation. These results thus identify sumoylation as a novel regulatory mechanism for MEF2 and suggest that this modification interplays with phosphorylation to promote intramolecular signaling for coordinated regulation in vivo.

How multisite posttranslational modification coordinates dynamic regulation of protein function is an issue fundamental to many biological processes. Related to this, a composite sequence motif has recently been identified that couples phosphorylation, sumoylation, and perhaps also deacetylation to control transcriptional repression in stress response, mitogen and nuclear hormone signaling, myogenesis, and neuronal differentiation. This motif is present in many proteins, integrates cellular signals from diverse pathways, and serves as a valuable signature for in silico identification of proteins regulated by adjacent phosphorylation and sumoylation.

Rational Cardiogenesis is regulated by a complex interplay between transcription factors. However, little is known about how these interactions regulate the transition from mesodermal precursors to cardiac progenitor cells (CPCs). Objective To identify novel regulators of mesodermal cardiac lineage commitment. Methods and Results We performed a bioinformatic-based transcription factor binding site analysis on upstream promoter regions of genes that are enriched in embryonic stem cell (ESC)-derived CPCs. From 32 candidate transcription factors screened, we found that YY1, a repressor of sarcomeric gene expression, is present in CPCs in vivo. Interestingly, we uncovered the ability of YY1 to transcriptionally activate Nkx2.5, a key marker of early cardiogenic commitment. YY1 regulates Nkx2.5 expression via a 2.1 kb cardiac-specific enhancer as demonstrated by in vitro luciferase-based assays and in vivo chromatin immunoprecipitation (ChIP) and genome-wide sequencing analysis. Furthermore, the ability of YY1 to activate Nkx2.5 expression depends on its cooperative interaction with Gata4 at a nearby chromatin. Cardiac mesoderm-specific loss-of-function of YY1 resulted in early embryonic lethality. This was corroborated in vitro by ESC-based assays where we show that the overexpression of YY1 enhanced the cardiogenic differentiation of ESCs into CPCs. Conclusion These results demonstrate an essential and unexpected role for YY1 to promote cardiogenesis as a transcriptional activator of Nkx2.5 and other CPC-enriched genes.

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