Protein-arginine Methyltransferase 1 (PRMT1) Methylates Ash2L, a Shared Component of Mammalian Histone H3K4 Methyltransferase Complexes

Abstract

Multiple enzymes and enzymatic complexes coordinately regulate the addition and removal of post-translational modifications on histone proteins. The oncoprotein Ash2L is a component of the mixed lineage leukemia (MLL) family members 1-4, Setd1A, and Setd1B mammalian histone H3K4 methyltransferase complexes and is essential to maintain global trimethylation of histone H3K4. However, regulation of these complexes at the level of expression and activity remains poorly understood. In this report, we demonstrate that Ash2L is methylated on arginine residues both in vitro and in cells. We found that both protein-arginine methyltransferases 1 and 5 methylate Arg-296 within Ash2L. These findings are the first to demonstrate that post-translational modifications occur on the Ash2L protein and provide a novel example of cross-talk between chromatinmodifying enzyme complexes.Changes in gene expression are brought about in part by dynamic regulation of chromatin structure and govern numerous cellular processes, including differentiation. In this manner, transcription of the developmental Hox gene cluster is temporally and spatially regulated by the opposing action of chromatin modifiers belonging to the Trithorax (Trx) 2 and Polycomb group (PcG) families (1, 2). Several Trx and PcG genes encode lysine methyltransferases that contain a suppressor of variegation, enhancer of zeste, and Trithorax (SET) domain and target histone proteins to activate or repress transcription, respectively. Genetic alterations of several Trx and PcG genes, including members of the mixed lineage leukemia (MLL) family, have been linked to oncogenesis in mammals and are especially prevalent in hematological malignancies (3-8).MLL, Setd1A, and Setd1B methyltransferase complexes catalyze mono-, di-, and trimethylation of histone H3K4 (9 -15). The absent, small, homeotic discs 2-like (Ash2L) protein functions at the molecular level along with WDR5 and RbBP5 to form a submodule of the Setd1A, Setd1B, and MLL methyltransferase complexes. Interestingly, the Ash2L, RbBP5, WDR5, DPY30 submodule was shown to possess an intrinsic methyltransferase activity toward histone H3K4 in vitro that is independent of MLL, although none of these proteins contain a catalytic SET domain (16). Furthermore, depletion of Ash2L in cell lines leads to decreased global levels of histone H3K4me3, whereas global levels of histone H3K4 methylation are unchanged in the absence of MLL (9, 17, 18). Likewise, MLL complexes reconstituted in vitro that lack Ash2L exhibit reduced di-and trimethyltransferase activity toward recombinant histone H3 (16,17,19). These results demonstrate that Ash2L is necessary to maintain histone H3K4 methylation levels in vivo, although the molecular mechanism whereby Ash2L promotes lysine di-and trimethylation remains unclear.Ash2 is a member of the Trx family and was identified in a screen for Drosophila mutants exhibiting imaginal disc abnormalities and late larval/early pupal lethality (20). Ash2L is ubiquitously expressed in humans with the highest lev...

Loss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs

“…The PRMT6 −/− MEF line was created by immortalizing MEFs from PRMT6-null embryos, according to standard 3T3 protocol20. PRMT5 stable knockdown HeLa cell lines were a gift from Sharon Dent21. All cell lines were maintained in Dulbecco modified Eagle medium containing 10% fetal bovine serum.…”

Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions

“…Thus, the singly methylated species was actually a mixture of peptides Table 1) were methylated by PRMT7 as described in Fig. 3 legend. A-H show PRMT7-catalyzed methylation products of H2B (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37) with monomethylation on different arginine residues, and Arg-29 and Arg-31 should be the major methylation sites on H2B. Additionally, the fragmentation pattern for the methylated H2B(23-37)R29K peptide is more consistent with the Arg-31 site of methylation (Fig.…”

“…Fig. 9A shows an example of the 5-charge state of H2B (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37) with an m/z of 388.03, which was deconvoluted to the monoisotopic mass of the peptide (1935.12 Da). After the reaction was catalyzed by PRMT7, Fig.…”

PRMT5 modulates the metabolic response to fasting signals

“…Recombinant H3 proteins (Active Motif) were methylated in vitro using purified PRMTs expressed either in bacteria as GST fusion proteins (GST-PRMT6, GST-PRMT7) or as myelocytomatosis (myc)-tagged proteins (myc-PRMT5) purified from HEK293T cells as described (24). Methylated H3 proteins were then isolated and incubated with GST-WDR5 proteins in NTP buffer (150 mM NaCl, 50 mM Tris·HCl, 0.5% Nonidet P-40) overnight.…”