The lack of methods for proteome-scale detection of arginine methylation restricts our knowledge of its relevance in physiological and pathological processes. Here we show that most tryptic peptides containing methylated arginine(s) are highly basic and hydrophilic. Consequently, they could be considerably enriched from total cell extracts by simple protocols using either one of strong cation exchange chromatography, isoelectric focusing, or hydrophilic interaction liquid chromatography, the latter being by far the most effective of all. These methods, coupled with heavy methyl-stable isotope labeling by amino acids in cell culture and mass spectrometry, enabled in T cells the identification of 249 arginine methylation sites in 131 proteins, including 190 new sites and 93 proteins not previously known to be arginine methylated. By extending considerably the number of known arginine methylation sites, our data reveal a novel proline-rich consensus motif and identify for the first time arginine methylation in proteins involved in cytoskeleton rearrangement at the immunological synapse and in endosomal trafficking.

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous disorder. All mendelian patterns of inheritance have been described. We identified a homozygous p.A335V mutation in the MED25 gene in an extended Costa Rican family with autosomal recessively inherited Charcot-Marie-Tooth neuropathy linked to the CMT2B2 locus in chromosome 19q13.3. MED25, also known as ARC92 and ACID1, is a subunit of the human activator-recruited cofactor (ARC), a family of large transcriptional coactivator complexes related to the yeast Mediator. MED25 was identified by virtue of functional association with the activator domains of multiple cellular and viral transcriptional activators. Its exact physiological function in transcriptional regulation remains obscure. The CMT2B2-associated missense amino acid substitution p.A335V is located in a proline-rich region with high affinity for SH3 domains of the Abelson type. The mutation causes a decrease in binding specificity leading to the recognition of a broader range of SH3 domain proteins. Furthermore, Med25 is coordinately expressed with Pmp22 gene dosage and expression in transgenic mice and rats. These results suggest a potential role of this protein in the molecular etiology of CMT2B2 and suggest a potential, more general role of MED25 in gene dosage sensitive peripheral neuropathy pathogenesis.

VP16 has been widely used to unravel the mechanisms underlying gene transcription. Much of the previous work has been conducted in reconstituted in vitro systems. Here we study the formation of transcription complexes at stable reporters under the control of an inducible Tet-VP16 activator in living cells. In this simplified model for gene activation VP16 recruits the general factors and the cofactors Mediator, GCN5, CBP, and PC4, within minutes to the promoter region. Activation is accompanied by only minor changes in histone acetylation and H3K4 methylation but induces a marked promoter-specific increase in H3K79 methylation. Mediated through contacts with VP16 several subunits of the cleavage and polyadenylation factor (CPSF/CstF) are concentrated at the promoter region. We provide in vitro and in vivo evidence that VP16 activates transcription through a specific MED25-associated Mediator, which is deficient in CDK8.

The estrogen-related receptor alpha (ERRalpha) is one of the first orphan nuclear receptors identified. Still, we know little about the mechanisms that regulate its expression and its activity. In this study, we show that the transcriptional coactivator PGC-1, which is implicated in the control of energy metabolism, regulates ERRalpha at two levels. First, PGC-1 induces the expression of ERRalpha. Consistent with this induction, levels of ERRalpha mRNA in vivo are highest in PGC-1 expressing tissues, such as heart, kidney, and muscle, and up-regulated in response to signals that induce PGC-1, such as exposure to cold. Second, PGC-1 interacts physically with ERRalpha and enables it to activate transcription. Strikingly, we find that PGC-1 converts ERRalpha from a factor with little or no transcriptional activity to a potent regulator of gene expression, suggesting that ERRalpha is not a constitutively active nuclear receptor but rather one that is regulated by protein ligands, such as PGC-1. Our findings suggest that the two proteins act in a common pathway to regulate processes relating to energy metabolism. In support of this hypothesis, adenovirus-mediated delivery of small interfering RNA for ERRalpha, or of PGC-1 mutants that interact selectively with different types of nuclear receptors, shows that PGC-1 can induce the fatty acid oxidation enzyme MCAD (medium-chain acyl-coenzyme A dehydrogenase) in an ERRalpha-dependent manner.