microRNAs (miRNAs) are short non-coding RNAs that can mediate changes in gene expression and are required for the formation of skeletal muscle (myogenesis). With the goal of identifying novel miRNA biomarkers of muscle disease, we profiled miRNA expression using miRNA-seq in the gastrocnemius muscles of dystrophic mdx4cv mice. After identifying a down-regulation of the miR-30 family (miR-30a-5p, -30b, -30c, -30d and -30e) when compared to C57Bl/6 (WT) mice, we found that overexpression of miR-30 family miRNAs promotes differentiation, while inhibition restricts differentiation of myoblasts in vitro. Additionally, miR-30 family miRNAs are coordinately down-regulated during in vivo models of muscle injury (barium chloride injection) and muscle disuse atrophy (hindlimb suspension). Using bioinformatics tools and in vitro studies, we identified and validated Smarcd2, Snai2 and Tnrc6a as miR-30 family targets. Interestingly, we show that by targeting Tnrc6a, miR-30 family miRNAs negatively regulate the miRNA pathway and modulate both the activity of muscle-specific miR-206 and the levels of protein synthesis. These findings indicate that the miR-30 family may be an interesting biomarker of perturbed muscle homeostasis and muscle disease.

Post-translational modification by small ubiquitin-like modifier 1 (SUMO-1) is a highly conserved process from yeast to humans and plays important regulatory roles in many cellular processes. Sumoylation occurs at certain internal lysine residues of target proteins via an isopeptide bond linkage. Unlike ubiquitin whose carboxyl-terminal sequence is RGG, the tripeptide at the carboxyl terminus of SUMO is TGG. The presence of the arginine residue at the carboxyl terminus of ubiquitin allows tryptic digestion of ubiquitin conjugates to yield a signature peptide containing a diglycine remnant attached to the target lysine residue and rapid identification of the ubiquitination site by mass spectrometry. The absence of lysine or arginine residues in the carboxyl terminus of mammalian SUMO makes it difficult to apply this approach to mapping sumoylation sites. We performed Arg scanning mutagenesis by systematically substituting amino acid residues surrounding the diglycine motif and found that a SUMO variant terminated with RGG can be conjugated efficiently to its target protein under normal sumoylation conditions. We developed a Programmed Data Acquisition (PDA) mass spectrometric approach to map target sumoylation sites using this SUMO variant. A web-based computational program designed for efficient identification of the modified peptides is described. Molecular & Cellular Proteomics 4:1626 -1636, 2005.Protein modification by SUMO 1 is emerging as an important regulatory event in many cellular processes (1-3). Although SUMO-1 is only 18% identical to ubiquitin, they display high structural homology, and sumoylation occurs by a mechanism closely related to that of ubiquitination. As such, it involves an E1-activating enzyme, which in the case of SUMO is a heterodimer, Aos1/Uba2. Like the ubiquitination pathway, there is one E1 common to all SUMO substrates. The E1 is charged with SUMO in an ATP-dependent fashion via a thioester linkage between the active site Cys of Uba2 and the carboxyl-terminal Gly of SUMO. Subsequently SUMO is passed to an E2-conjugating enzyme where it is again covalently linked through a thioester bond, paralleling once more the ubiquitination mechanism (1-3). However, an interesting difference arises here; in the case of ubiquitination, there are dozens of E2s with known conjugating activity, whereas in the case of SUMO, there is only one known E2, Ubc9 (1-3). An additional intriguing divergence between the two pathways is that although ubiquitination requires an E3 ligase enzyme to complete the transfer of ubiquitin to the substrate protein, sumoylation of many substrates apparently does not (1-3). This has been ascribed to Ubc9 binding directly to many SUMO substrates and is displayed by the fact that sumoylation can occur in the absence of any E3 in a totally reconstituted in vitro system. Although a number of E3s specific to the sumoylation pathway have now been identified, they do not appear to be essential for the transfer of SUMO to the target molecule. However, when an E3 specific to th...

Myocyte enhancer factor 2 (MEF2) transcription factors are crucial regulators controlling muscle-specific and growth factor-inducible genes. Numerous studies have reported that the activity of these transcription factors is tightly modulated by posttranslational modifications such as activation by specific phosphorylation as well as repression by class II histone deacetylases (HDACs). We hypothesized that MEF2 could also be regulated by covalent modification by SUMO-1, a reversible posttranslational modification which has been shown to regulate key proteins involved in cell proliferation, differentiation and tumor suppression. In this study, we demonstrate that MEF2A undergoes sumoylation primarily at a single lysine residue (K395) both in vitro and in vivo. We also show that the nuclear E3 ligase, PIAS1, promotes sumoylation of MEF2A. Mutation of lysine 395 to arginine abolishes MEF2A sumoylation and the sumoylation incompetent mutant protein has enhanced transcriptional activity compared to the wild type protein. Our results suggest that protein sumoylation could play a pivotal role in controlling MEF2 transcriptional activity. . A growing list of E3 enzymes, including RanBP2, Pc2 and the PIAS family proteins [7][8][9], has been identified. Although they do not appear to be essential for SUMO conjugation, they usually enhance the rate and degree of sumoylation of the substrate. SUMO modification is a transient regulatory mechanism provided by a dynamic equilibrium between conjugation and deconjugation. The last process is driven by enzymes known as isopeptidases that can specifically remove SUMO [3,10]. KeywordsMost of the reported SUMO targets are predominantly nuclear residents including transcription factors, transcription co-activators, transcription corepressors, nuclear pore complex proteins, nuclear body proteins and genome integrity proteins [11,12]. In these cases, sumoylation may affect subnuclear localization, transcription activation capacity, and protein stability by interfering with ubiquitination [3][4][5].In this study we investigated whether protein sumoylation plays a role in regulating MEF2A transcriptional activity. We found that the MEF2 family harbors a sumoylation consensus sequence and we demonstrate that MEF2A can be sumoylated in vitro and in vivo at a single lysine residue, K395. Our data also indicate that covalent SUMO modification of MEF2A could inhibit its transcriptional activity. These data point toward a role for sumoylation as a regulatory mechanism to control expression of MEF2-dependent target genes. Materials and methods Plasmids and mutagenesispCMV-Sport6_hMEF2A and pCMV-Sport6-mUbc9 were purchased from Open Biosystem. The mutant MEF2A K395R was generated by a QuikChangeXL mutagenesis kit (Stratagene). pET23a-hUbc9, pET11d-Uba2, and pET28a-Aos1 were kind gifts from Dr. Frauke Melchior. SUMO-1 (1-97) cDNA, kindly provided by Drs. Seeler and Dejean, was subcloned into the NdeI and NotI sites of pET23a. Dr. Stephan Müller kindly provided expression plasmids for GST-...

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The host factor, nuclear factor of activated T-cells (NFAT), regulates the transcription and replication of HIV-1. Here, we have determined the crystal structure of the DNA binding domain of NFAT bound to the HIV-1 long terminal repeat (LTR) tandem kappaB enhancer element at 3.05 A resolution. NFAT binds as a dimer to the upstream kappaB site (Core II), but as a monomer to the 3' end of the downstream kappaB site (Core I). The DNA shows a significant bend near the 5' end of Core I, where a lysine residue from NFAT bound to the 3' end of Core II inserts into the minor groove and seems to cause DNA bases to flip out. Consistent with this structural feature, the 5' end of Core I become hypersensitive to dimethylsulfate in the in vivo footprinting upon transcriptional activation of the HIV-1 LTR. Our studies provide a basis for further investigating the functional mechanisms of NFAT in HIV-1 transcription and replication.