A genome-scale genetic interaction map was constructed by examining 5.4 million gene-gene pairs for synthetic genetic interactions, generating quantitative genetic interaction profiles for ~75% of all genes in the budding yeast, Saccharomyces cerevisiae. A network based on genetic interaction profiles reveals a functional map of the cell in which genes of similar biological processes cluster together in coherent subsets, and highly correlated profiles delineate specific pathways to define gene function. The global network identifies functional cross-connections between all bioprocesses, mapping a cellular wiring diagram of pleiotropy. Genetic interaction degree correlated with a number of different gene attributes, which may be informative about genetic network hubs in other organisms. We also demonstrate that extensive and unbiased mapping of the genetic landscape provides a key for interpretation of chemical-genetic interactions and drug target identification.

The phosphorylation status of the myocyte enhancer factor 2 (MEF2) transcriptional regulator is a critical determinant of its tissue-specific functions. However, due to the complexity of its phosphorylation pattern in vivo, a systematic inventory of MEF2A phosphorylation sites in mammalian cells has been difficult to obtain. We employed modern affinity purification techniques, combined with mass spectrometry, to identify several novel MEF2 phosphoacceptor sites. These include an evolutionarily conserved KSP motif, which we show is important in regulating the stability and function of MEF2A. Also, an indirect pathway in which a protein kinase casein kinase 2 phosphoacceptor site is phosphorylated by activation of p38 MAPK signaling was documented. Together, these findings identify several novel aspects of MEF2 regulation that may prove important in the control of gene expression in neuronal and muscle cells. Myocyte enhancer factor 2 (MEF2)1 is a transcriptional regulatory complex mediating diverse cellular functions in neurons (1, 2), skeletal (reviewed in Ref.3) and cardiac muscle (4 -6), and T cells (7,8). It is now well established that MEF2 plays a role in the differentiation of these cell types as well as functioning in a protective role against neuronal apoptosis.To respond to diverse developmental and physiological cues, MEF2 is structurally organized to receive and respond to multiple signals from several intracellular signaling pathways (reviewed in Refs. 3 and 9). In this regard, perhaps the best characterized is the p38 MAPK-MEF2 axis, in mammals (10, 11) and in yeast (12), although other kinase-catalyzed cascades mediated by big MAP kinase (13,14), protein kinase C (10), and protein kinase CK2 (15) are known to target MEF2. Moreover, consistent with its role as a signal sensor, putative phosphoacceptor motifs in the carboxyl terminal MEF2 transactivation domain may prove to further modulate MEF2 function in response to extracellular cues.Given that MEF2, and the biological processes it regulates, are intrinsically governed by MEF2 phosphorylation status, we undertook to systematically document MEF2 phosphorylation patterns in mammalian cells; previous phosphopeptide mapping studies used in vitro phosphorylated MEF2 protein. The purpose thus being to detect physiologically relevant, and possibly novel, in vivo MEF2 phosphorylation sites. To accomplish this we used several state-of-the-art mass spectrometric techniques to detect phosphorylation sites from MEF2 expressed in mammalian cells. To this end, we have made use of a mammalian tandem affinity purification (TAP) method (16, 17) for low-abundance nuclear transcription factors that allows purification to homogeneity and provides amounts compatible with mass spectrometric analysis of phosphorylation sites.In these studies, we have identified two important and novel aspects of MEF2 regulation. One is a highly conserved phosphoacceptor motif that regulates MEF2 stability and function. The second is an indirect pathway of MEF2 regulation by p38 MAPK me...

The myocyte enhancer factor 2 (MEF2) transcription factors play important roles in neuronal, cardiac, and skeletal muscle tissues. MEF2 serves as a nuclear sensor, integrating signals from several signaling cascades through protein-protein interactions with kinases, chromatin remodeling factors, and other transcriptional regulators. Here, we report a novel interaction between the catalytic subunit of protein phosphatase 1␣ (PP1␣) and MEF2. Interaction occurs within the nucleus, and binding of PP1␣ to MEF2 potently represses MEF2-dependent transcription. The interaction utilizes uncharacterized domains in both PP1␣ and MEF2, and PP1␣ phosphatase activity is not obligatory for MEF2 repression. Moreover, a MEF2-PP1␣ regulatory complex leads to nuclear retention and recruitment of histone deacetylase 4 to MEF2 transcription complexes. PP1␣-mediated repression of MEF2 overrides the positive influence of calcineurin signaling, suggesting PP1␣ exerts a dominant level of control over MEF2 function. Indeed, PP1␣-mediated repression of MEF2 function interferes with the prosurvival effect of MEF2 in primary hippocampal neurons. The PP1␣-MEF2 interaction constitutes a potent locus of control for MEF2-dependent gene expression, having potentially important implications for neuronal cell survival, cardiac remodeling in disease, and terminal differentiation of vascular, cardiac, and skeletal muscle.The myocyte enhancer factor 2 (MEF2) transcription factors play important roles in T-cell selection, neuronal survival, and terminal differentiation of cardiac and skeletal muscle (3, 47). The MEF2 family proteins are encoded by four genes, MEF2A to -D, which demonstrate tissue-specific and temporally dependent developmental expression patterns. Expression and activity of MEF2 factors in both cardiac and skeletal muscle lineages are vital for activation and maintenance of genes representing the structural components of sarcomeric muscle. Gene-targeted ablation of MEF2A or MEF2C results in aberrant heart formation and premature death (32, 46), whereas loss of the single MEF2 gene in Drosophila melanogaster (Dmef2) results in complete loss of all muscle tissues (31). In neurons, MEF2 transcriptional activity plays a critical role for prevention of apoptosis due to neurocytotoxicity signals (1,5,20,37).The amino-terminal (NT) region of MEF2 transcription factors is composed of a highly conserved MADS (MCM1, agamous, deficiens, and serum response factor) domain, responsible for DNA binding to the consensus DNA binding element (T/C)TA(A/T) 4 TA(G/A), and the MEF2 domain, which is required for homo-and heterodimerization of MEF2 factors. The carboxyl terminus (CT) of all MEF2 factors is subject to alternative splicing, represents the target for several signal transduction pathways, and is required for transcriptional activation properties (3).MEF2 transcriptional activity is stimulated by the mitogenactivated protein kinase (MAPK) p38 signaling module (25, 50), Ca 2ϩ /calmodulin kinases (CaMKs) (52), extracellular signal-regulated kinase ...