The coactivator-associated arginine methyltransferase CARM1 is recruited by many different transcription factors as a positive regulator. To understand the mechanism by which CARM1 functions, we sought to isolate its substrates. We developed a small-pool screening approach for this purpose and identified CA150, SAP49, SmB, and U1C as splicing factors that are specifically methylated by CARM1. We further showed that CA150, a molecule that links transcription to splicing, interacts with the Tudor domain of the spinal muscular atrophy protein SMN in a CARM1-dependent fashion. Experiments with an exogenous splicing reporter and the endogenous CD44 gene revealed that CARM1 promotes exon skipping in an enzyme-dependent manner. The identification of splicing factors that are methylated by CARM1, and protein-protein interactions that are regulated by CARM1, strongly implicates this enzyme in the regulation of alternative splicing and points toward its involvement in spinal muscular atrophy pathogenesis.

Here we report the identification of small molecules that specifically inhibit protein arginine N-methyltransferase (PRMT) activity. PRMTs are a family of proteins that either monomethylate or dimethylate the guanidino nitrogen atoms of arginine side chains. This common post-translational modification is implicated in protein trafficking, signal transduction, and transcriptional regulation. Most methyltransferases use the methyl donor, S-adenosyl-L-methionine (AdoMet), as a cofactor. Current methyltransferase inhibitors display limited specificity, indiscriminately targeting all enzymes that use AdoMet. In this screen we have identified a primary compound, AMI-1, that specifically inhibits arginine, but not lysine, methyltransferase activity in vitro and does not compete for the AdoMet binding site. Furthermore, AMI-1 prevents in vivo arginine methylation of cellular proteins and can modulate nuclear receptor-regulated transcription from estrogen and androgen response elements, thus operating as a brake on certain hormone actions.

Histone tail post-translational modification results in changes in cellular processes, either by generating or blocking docking sites for histone code readers or by altering the higher order chromatin structure. H3K4me3 is known to mark the promoter regions of active transcription. Proteins bind H3K4 in a methyl-dependent manner and aid in the recruitment of histone-remodeling enzymes and transcriptional cofactors. The H3K4me3 binders harbor methyl-specific chromatin binding domains, including plant homeodomain, Chromo, and tudor domains. Structural analysis of the plant homeodomains present in effector proteins, as well as the WD40 repeats of WDR5, reveals critical contacts between residues in these domains and H3R2. The intimate contact between H3R2 and these domain types leads to the hypothesis that methylation of this arginine residue antagonizes the binding of effector proteins to the N-terminal tail of H3. Here we show that H3 tail binding effector proteins are indeed sensitive to H3R2 methylation and that PRMT6, not CARM1/PRMT4, is the primary methyltransferase acting on this site. We have tested the expression of a select group of H3K4 effector-regulated genes in PRMT6 knockdown cells and found that their levels are altered. Thus, PRMT6 methylates H3R2 and is a negative regulator of N-terminal H3 tail binding.The tight packing of DNA into chromatin creates a need for mechanisms to relax chromatin and expose DNA for transcription, replication, and DNA repair (1). One of the mechanisms used by the cell to access DNA is the post-translational modification of histone tails. Specifically, methylation of histone tails generates a docking site for effector proteins, which aid in the recruitment of other enzymes necessary for the function at hand. In general, methylation of histone residues lysines 4 and 36 on H3 are correlated with active gene regions, whereas methylation of lysines 9 and 27 on H3 is correlated with repressed gene regions, although exceptions exist (2). The domain types that bind histone tails include the Chromodomain, tudor domains, MBT domains, WD40 repeats, and PHD 5 fingers (3-5).Recently, two groups reported that select PHD fingers have the propensity to bind trimethyl lysine 4 on H3 (6, 7). The structures further showed important aromatic residues in the PHD that cage the methylated lysine but also revealed critical contacts made between the arginine at the second position of the H3 tail and the PHD (8, 9). During this same time, the WD40 domain of WDR5 was reported to complex with the H3 tail (10). The structure of the WD40 repeats of WDR5 revealed arginine 2 of H3, and not lysine 4, buried within the donut hole of the large domain (11,12). Specifically, four amino acids in WDR5 critically interact with arginine 2 (11). In addition, the tudor domains of JMJD2A also bind in an H3K4me3-dependent manner, and again, the H3R2 residue forms critical interactions with an Asp residue of one of the tudor domains (13). The analysis of the structures of these three different domain types bound to t...

The Cyclin E1 gene (CCNE1) is an ideal model to explore the mechanisms that control the transcription of cell cycle-regulated genes whose expression rises transiently before entry into S phase. E2F-dependent regulation of the CCNE1 promoter was shown to correlate with changes in the level of H3-K9 acetylation͞methyl-ation of nucleosomal histones positioned at the transcriptional start site region. Here we show that, upon growth stimulation, the same region is subject to variations of H3-R17 and H3-R26 methylation that correlate with the recruitment of coactivator-associated arginine methyltransferase 1 (CARM1) onto the CCNE1 and DHFR promoters. Accordingly, CARM1-deficient cells lack these modifications and present lowered levels and altered kinetics of CCNE1 and DHFR mRNA expression. Consistently, reporter gene assays demonstrate that CARM1 functions as a transcriptional coactivator for their E2F1͞DP1-stimulated expression. CARM1 recruitment at the CCNE1 gene requires activator E2Fs and ACTR, a member of the p160 coactivator family that is frequently overexpressed in human breast cancer. Finally, we show that grade-3 breast tumors present coelevated mRNA levels of ACTR and CARM1, along with their transcriptional target CCNE1. All together, our results indicate that CARM1 is an important regulator of the CCNE1 gene.ACTR ͉ CCNE1 ͉ histone ͉ arginine methylation ͉ breast tumor C yclin E1 (CCNE1) protein and mRNA levels are tightly regulated as an endpoint of several regulatory pathways that are critical for growth control and frequently altered in cancer cells (1, 2). CCNE1 gene transcription is undetectable in G 0 and G 1 phases of the cell cycle, whereas it rises sharply during a narrow window of time that precedes each entry into S phase. Several pieces of evidence suggest that the periodic association of activators E2Fs-and E2F-pocket protein complexes regulate CCNE1 gene expression (3-18). E2F complexes bound to this gene were found to recruit chromatin modifiers, including members of the SNF2-like helicase family, type I histone deacetylases, the acetyltransferase CBP͞p300, the lysine methyl transferase SUVAR39H1, and the protein arginine N-methyltransferase (PRMT) 5 (7, 9-14, 17, 18), suggesting that they foster periodic chromatin remodeling of the CCNE1 promoter region (11,12,14). Notably, repression of the CCNE1 gene in G 0 -G 1 correlates with the methylation of H3-K9 and H4-R3 on a single nucleosome positioned at the transcriptional start site (11)(12)(13)(14). Conversely, the late G 1 activation of the CCNE1 gene correlates with decreased H3-K9 methylation and with enhanced H3͞H4 acetylation of the same chromatin region (11)(12)(13)(14). Here, we reveal that this CCNE1 proximal promoter region is targeted by another histone arginine methyl-transferase, the type I enzyme PRMT4 [coactivator-associated arginine methyltransferase (CARM1)] (19-25). PRMT4͞CARM1 was initially described as a transcriptional coactivator of the p160 family of nuclear receptor-associated factors (Src-1͞NCoA1, GRIP1͞TIF2͞Src-2͞ NC...

ABSTRACT. Insect olfactory perception involves many aspects of insect life, and can directly or indirectly evoke either individual or group behaviors. Insect olfactory receptors and odorant-binding proteins (OBPs) are considered to be crucial to insect-specific and -sensitive olfaction. Although the mechanisms of interaction between OBPs or OBP/ligand complex with olfactory receptors are still not well understood, it has been shown that many OBPs contribute to insect olfactory perception at various levels. Some of these are numerous and divergent members in OBP family; expression in the olfactory organ at high concentration; a variety of combinational patterns between different OBPs and ligands, but exclusive affinity for one OBP to specific binding ligands; complicated interactions between OBP/ligand complex and transmembrane proteins (olfactory receptors or sensory neuron membrane proteins). First, we review OBPs' ligand-binding property based on OBP structural research and ligandbinding test; then, we review current progress around the points cited above OBP functions in insect peripheral olfactory reception to show the role of such proteins in insect olfactory signal transmission; finally, we discuss applications based on insect OBP research.