The p160 family of coactivators, SRC-1, GRIP1/TIF2, and p/CIP, mediate transcriptional activation by nuclear hormone receptors. Coactivator-associated arginine methyltransferase 1 (CARM1), a previously unidentified protein that binds to the carboxyl-terminal region of p160 coactivators, enhanced transcriptional activation by nuclear receptors, but only when GRIP1 or SRC-1a was coexpressed. Thus, CARM1 functions as a secondary coactivator through its association with p160 coactivators. CARM1 can methylate histone H3 in vitro, and a mutation in the putative S-adenosylmethionine binding domain of CARM1 substantially reduced both methyltransferase and coactivator activities. Thus, coactivator-mediated methylation of proteins in the transcription machinery may contribute to transcriptional regulation.

Posttranslational modifications of histone amino termini play an important role in modulating chromatin structure and function. Lysine methylation of histones has been well documented, and recently this modification has been linked to cellular processes involving gene transcription and heterochromatin assembly. However, the existence of arginine methylation on histones has remained unclear. Recent discoveries of protein arginine methyltransferases, CARM1 and PRMT1, as transcriptional coactivators for nuclear receptors suggest that histones may be physiological targets of these enzymes as part of a poorly defined transcriptional activation pathway. Here we show by using mass spectrometry that histone H4, isolated from asynchronously growing human 293T cells, is methylated at arginine 3 (Arg-3) in vivo. In support, a novel antibody directed against histone H4 methylated at Arg-3 independently demonstrates the in vivo occurrence of this modification and reveals that H4 Arg-3 methylation is highly conserved throughout eukaryotes. Finally, we show that PRMT1 is the major, if not exclusive, H4 Arg-3 methyltransfase in human 293T cells. These findings suggest a role for arginine methylation of histones in the transcription process.

Nuclear receptors (NRs) activate gene transcription by binding to specific enhancer elements and recruiting coactivators of the p160 family to promoters of target genes. The p160 coactivators in turn enhance transcription by recruiting secondary coactivators, including histone acetyltransferases such as CREB-binding protein (CBP) and p300/CBP-associated factor (p/CAF), as well as the recently identified protein methyltransferase, coactivator-associated arginine methyltransferase 1 (CARM1). In the current study, protein arginine methyltransferase 1 (PRMT1), another arginine-specific protein methyltransferase that shares a region of high homology with CARM1, was also found to act as a coactivator for NRs. PRMT1, like CARM1, bound to the C-terminal AD2 activation domain of p160 coactivators and thereby enhanced the activity of NRs in transient transfection assays. The shape of the graphs of reporter gene activity versus the amounts of CARM1 or PRMT1 expression vector indicated a cooperative relationship between coactivator concentration and activity. Moreover, CARM1 and PRMT1 acted in a synergistic manner to enhance reporter gene activation by both hormonedependent and orphan NRs. The synergy was most evident at low levels of transfected NR expression vectors, where activation of reporter genes was almost completely dependent on the presence of NR and all three exogenously supplied coactivators, i.e. GRIP1, CARM1, and PRMT1. In contrast, with the higher levels of NR expression vectors typically used in transient transfection assays, NR activity was much less dependent on the combination of coactivators, suggesting that target gene activation occurs by different mechanisms at high versus low cellular concentrations of NR. Because multiple coactivators are presumably required to mediate transcriptional activation of native genes in vivo, the low-NR conditions may provide a more physiologically relevant assay for coactivator function.

Hormone-activated nuclear receptors (NR) bind to specific regulatory DNA elements associated with their target genes and recruit coactivator proteins to remodel chromatin structure, recruit RNA polymerase, and activate transcription. The p160 coactivators (e.g., SRC-1, GRIP1, and ACTR) bind directly to activated NR and can recruit a variety of secondary coactivators. We have established a transient-transfection assay system under which the activity of various NR is highly or completely dependent on synergistic cooperation among three classes of coactivators: a p160 coactivator, the protein methyltransferase CARM1, and any of the three protein acetyltransferases, p300, CBP, or p/CAF. The three-coactivator functional synergy was only observed when low levels of NR were expressed and was highly or completely dependent on the methyltransferase activity of CARM1 and the acetyltransferase activity of p/CAF, but not the acetyltransferase activity of p300. Other members of the protein arginine methyltransferase family, which methylate different protein substrates than CARM1, could not substitute for CARM1 to act synergistically with p300 or p/CAF. A ternary complex of GRIP1, CARM1, and p300 or CBP was demonstrated in cultured mammalian cells, supporting a physiological role for the observed synergy. The transfection assay described here is a valuable new tool for investigating the mechanism of coactivator function and demonstrates the importance of multiple coactivators, including CARM1 and its specific protein methyltransferase activity, in transcriptional activation.

The androgen receptor (AR) binds to and activates transcription of specific genes in response to its cognate steroid hormone, dihydrotestosterone. Transcriptional activation by the DNA-bound AR is accomplished with the help of a variety of coactivator proteins. For example, the p160 coactivators bind directly to AR and recruit additional coactivators such as the histone acetyltransferase p300 and the histone methyltransferase CARM1. The current study tested whether CARM1 can cooperate with other types of coactivator proteins. Recently it was shown that ␤-catenin can also bind directly to and serve as a coactivator for AR. Here it is shown that CARM1 binds to ␤-catenin and can function in synergy with ␤-catenin and p300 as coactivators for AR. The methyltransferase activity of CARM1 is important for its synergistic coactivator function with ␤-catenin. The synergistic coactivator function of ␤-catenin and CARM1 is not restricted to steroid receptors because these two coactivators can also act synergistically with another type of DNA binding transcriptional activator, LEF-1/TCF-4.␤-catenin is a critical component of at least two signaling pathways, one that regulates cell-cell adhesion and organization of the actin cytoskeleton, and another that mediates Wnt/ Wingless (Wg) 1 signaling (1-3). ␤-catenin protein levels are usually maintained at a low level through active degradation. The glycogen synthase kinase complex constitutively phosphorylates ␤-catenin, targeting it for ubiquitination and degradation by proteasomes. Activation of the Wnt/Wg pathway leads to inhibition of glycogen synthase kinase activity and thus stabilizes ␤-catenin protein. As its level increases some ␤-catenin accumulates in the nucleus, where it binds to and enhances transcriptional activation by at least two different classes of enhancer element-binding transcriptional activator proteins: the lymphoid enhancer factor/T-cell factor (LEF/TCF) proteins (4, 5) and nuclear receptors such as the androgen receptor (AR) and retinoic acid receptor (6, 7). Thus, at the distal end of the Wnt/Wg and nuclear receptor signaling pathways ␤-catenin can act as a transcriptional coactivator protein.Transcriptional coactivators are a diverse group of proteins that are recruited to a specific promoter by interaction with specific enhancer-binding proteins (2,8,9). Coactivators participate in chromatin remodeling and recruitment of RNA polymerase II to the promoter. Many coactivators function as complexes, in which one component (hereafter, a primary coactivator) anchors the complex to the enhancer-binding protein, while other components (hereafter, secondary coactivators) mediate the transcriptional activation process (10, 11). Binding of ␤-catenin to LEF/TCF causes displacement of corepressor complexes containing histone deacetylases (5). In addition, ␤-catenin also recruits and acts synergistically as a coactivator with p300 or CBP (2, 12-14). These two related proteins acetylate histones and interact with components of the basal transcription machinery, su...