miR-30 family microRNAs regulate myogenic differentiation and provide negative feedback on the microRNA pathway.

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.

Structure and function of steroid receptor RNA activator protein, the proposed partner of SRA noncoding RNA.

In a widely accepted model, the steroid receptor RNA activator protein (SRA protein; SRAP) modulates the transcriptional regulatory activity of SRA RNA by binding a specific stem-loop of SRA. We first confirmed that SRAP is present in the nucleus as well as the cytoplasm of MCF-7 breast cancer cells, where it is expressed at the level of about 10(5) molecules per cell. However, our SRAP-RNA binding experiments, both in vitro with recombinant protein and in cultured cells with plasmid-expressed protein and RNA, did not reveal a specific interaction between SRAP and SRA. We determined the crystal structure of the carboxy-terminal domain of human SRAP and found that it does not have the postulated RRM (RNA recognition motif). The structure is a five-helix bundle that is distinct from known RNA-binding motifs and instead is similar to the carboxy-terminal domain of the yeast spliceosome protein PRP18, which stabilizes specific protein-protein interactions within a multisubunit mRNA splicing complex. SRA binding experiments with this domain gave negative results. Transcriptional regulation by SRA/SRAP was examined with siRNA knockdown. Effects on both specific estrogen-responsive genes and genes identified by RNA-seq as candidates for regulation were examined in MCF-7 cells. Only a small effect (~20% change) on one gene resulting from depletion of SRA/SRAP could be confirmed. We conclude that the current model for SRAP function must be reevaluated; we suggest that SRAP may function in a different context to stabilize specific intermolecular interactions in the nucleus.

Estrogenic compounds are not always cardioprotective and can be lethal in males with genetic heart disease.

Hypertrophic cardiomyopathy (HCM) is more severe in male than female mice eating a soy-based diet. We sought to determine whether the detrimental effects are mediated by the phytoestrogens present in soy, the mechanism by which phytoestrogens act, and to test whether estrogen modulates the sexually dimorphic phenotype. A soy-free diet (casein based) supplemented with the predominant phytoestrogens in soy, genistein and daidzein, recapitulated the fibrotic, proapoptotic and negative hemodynamic effects of soy in male hearts. As with the soy diet, the hearts of female HCM mice were not negatively affected by the phytoestrogen-containing diet. To determine the role of estrogen in the sex differences mediated by diet in HCM, gonadectomies were performed and estrogen was administered to male and female HCM mice on a casein- or phytoestrogen-supplemented diet. Somewhat surprisingly, estrogen was not protective in male or female mice with HCM and, in fact, was lethal in phytoestrogen-fed male mice with HCM. Because genistein is a potent tyrosine kinase inhibitor and tyrosine kinase inhibition has been associated with cardiotoxicity, we tested its effects in isolated adult cardiac myocytes. Genistein inhibited different tyrosine kinases depending on sex and, in combination with estrogen, resulted in apoptosis only in adult male cardiac myocytes. Finally, we show that phytoestrogens led to distinct programs of gene expression in hearts from males vs. females with HCM, suggesting mechanisms by which males are more sensitive to the detrimental effects of phytoestrogens and females are protected. These results implicate the phytoestrogen genistein in mediating cardiac pathology in males with HCM and, importantly, establish that estrogen is not protective in the setting of HCM.

Remodeling the cardiac transcriptional landscape with diet.

The perception that soy food products and dietary supplements will have beneficial effects on cardiovascular health has led to a massive consumer market. However, we have previously noted that diet profoundly affects disease progression in a genetic model of hypertrophic cardiomyopathy (HCM). In this model, a soy-based diet negatively impacts cardiac function in male mice. Given the frequent connection between functional changes and transcriptional changes, we investigated the effect of diet (soy- vs. milk-based) on cardiac gene expression and how it is affected by the additional factors of sex and disease. We found that gene expression in the heart is altered more by diet than by sex or an inherited disease. We also found that the healthy male heart may be sensitized to dietary perturbations of gene expression in that it displays a gene expression profile more similar to diseased male and female hearts than to healthy female hearts. These observations may in part account for documented divergence in HCM phenotypes between males and females and between diets.

A transcriptional enhancer from the coding region of ADAMTS5.

BACKGROUND: The revelation that the human genome encodes only approximately 25,000 genes and thus cannot account for phenotypic complexity has been one of the biggest surprises in the post-genomic era. However, accumulating evidence suggests that transcriptional regulation may be in large part responsible for this observed mammalian complexity. Consequently, there has been a strong drive to locate cis-regulatory regions in mammalian genomes in order to understand the unifying principles governing these regions, including their genomic distribution. Although a number of systematic approaches have been developed, these all discount coding sequence. METHODOLOGY/PRINCIPAL FINDINGS: Using the computational tool PRI (Pattern-defined Regulatory Islands), which does not mask coding sequence, we identified a regulatory region associated with the gene ADAMTS5 that encompasses the entirety of the essential coding exon 2. We demonstrate through a combination of chromatin immunoprecipitation and reporter gene studies that this region can not only bind the myogenic transcription factors MYOD and myogenin and the E-protein HEB but can also function as a very strong myogenic transcriptional enhancer. CONCLUSIONS/SIGNIFICANCE: Thus, we report the identification and detailed characterization of an exonic enhancer. Ultimately, this leads to the interesting question of why evolution would be so parsimonious in the functional assignment of sequence.

Crystal structure of NFAT bound to the HIV-1 LTR tandem kappaB enhancer element.

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.

Identifying pattern-defined regulatory islands in mammalian genomes.

Identifying cis-regulatory regions in mammalian genomes is a key challenge toward understanding transcriptional regulation. However, identification and functional characterization of those regulatory elements governing differential gene expression has been hampered by the limited understanding of their organization and locations in genomes. We hypothesized that genes that are conserved across species will also display conservation at the level of their transcriptional regulation and that this will be reflected in the organization of cis-elements mediating this regulation. Using a computational approach, clusters of transcription factor binding sites that are absolutely conserved in order and in spacing across human, rat, and mouse genomes were identified. We term these regions pattern-defined regulatory islands (PRIs). We discovered that these sequences are frequently active sites of transcriptional regulation. These PRIs occur in approximately 1.1% of the half-billion base pairs covered in the search and are located mainly in noncoding regions of the genome. We show that the premise of PRIs can be used to identify previously known and novel cis-regulatory regions controlling genes regulated by myogenic differentiation. Thus, PRIs may represent a fundamental property of the architecture of cis-regulatory elements in mammalian genomes, and this feature can be exploited to pinpoint critical transcriptional regulatory elements governing cell type-specific gene expression.

Ubc9 expression is essential for myotube formation in C2C12.

Myogenic differentiation is a fundamental biological process that involves a hierarchical series of events that ultimately leads to muscle-specific gene expression and myofiber formation. Posttranslational modifications of the myogenic regulatory factors have been implicated as important regulatory mechanisms in this process. Here we investigate whether covalent protein modification by a small ubiquitin-like modifier (SUMO) that is known to affect transcription factor activity can impact muscle differentiation. We show that the overall load of sumoylated proteins present in myoblasts diminishes progressively throughout myogenesis. Interestingly, knockdown of the SUMO-conjugating enzyme, Ubc9, severely compromises C2C12 muscle cell terminal differentiation. However, it does not affect the expression, the localization and the activation of MyoD and myogenin. These novel results suggest that protein sumoylation plays a pivotal role in myoblast differentiation and is required to regulate the activity of key targets downstream of MyoD and myogenin.

SUMO-1 modification of MEF2A regulates its transcriptional activity.

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 method of mapping protein sumoylation sites by mass spectrometry using a modified small ubiquitin-like modifier 1 (SUMO-1) and a computational program.

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.