In Vitro and in Vivo Analyses of a Phe/Tyr Switch Controlling Product Specificity of Histone Lysine Methyltransferases

Abstract

The functional significance of mono-, di-, and trimethylation of lysine residues within histone proteins is under investigation. Evidence from several model organisms suggests that different methylated states of H3 Lys 9 (H3K9) are generated by specific histone methyltransferases (MTases) to mark distinct types of silent chromatin. Sequence alignment of all histone lysine MTases with known product specificity suggested that a key residue in the active site determines how many methyl groups they add. We examined this possibility both in vitro and in vivo and found that a Phe at the position equivalent to Phe 281 of Neurospora crassa DIM-5 or Phe 1205 of human G9a allows the enzyme to perform di and tri-methylation, whereas a Tyr at this position is restrictive, inhibiting tri-methylation and thus yielding a mono-or di-MTase. Phe to Tyr mutants of both DIM-5 and G9a restrict product specificity in vitro and in vivo without compromising overall catalysis. These mutants were employed to probe the biological significance of mono-, di-, and tri-methylation of H3K9 in both mouse embryonic stem cells and N. crassa. G9a F1205Y, when expressed in G9a (؊/؊) embryonic stem cells, rescued only H3K9 mono-methylation, but not di-methylation, to wild-type levels yet silenced Mage-a gene expression. When expressed in dim-5 strains, DIM-5 F281Y generated significant levels of mono-and di-H3K9 methylation (which are not observed in wild type Neurospora) as well as tri-methyl H3K9. The altered DIM-5 rescued the growth defect characteristic of dim-5 N. crassa but did not fully rescue the gross DNA hypomethylation of dim-5 strains.Histones are subject to diverse post-translational modifications including acetylation, phosphorylation, ubiquitination, methylation, and sumoylation. Evidence accumulated over the past few years suggests that such modifications constitute a "histone code" that directs a variety of processes involving chromatin (1, 2). Considering just methylation of lysines, there are at least six modification sites (Lys 4 , Lys 9 , Lys 27 , Lys 36 , and Lys 79 of histone H3 and Lys 20 of histone H4), and in principle each site can have zero, one, two, or three methyl groups. It has been suggested that methylation at these sites, in combination with other nearby modifications, generates "modification cassettes" (3), yielding distinct patterns on chromatin for signaling downstream events (reviewed in Refs. 3 and 4).With only one known exception, histone lysine methyltransferases (HKMTs) 1 contain a SET domain of ϳ130 amino acids. SET proteins can be grouped into families according to the sequences surrounding this distinctive domain (5, 6) (see Table I). In this study we focus on two members of the SUV family that methylate Lys 9 of histone H3, G9a characterized in mammals and DIM-5 characterized in the filamentous fungus Neurospora crassa. DIM-5 provided the first evidence that histone methylation can direct DNA methylation (7). DIM-5 normally generates primarily tri-methyl-Lys 9 on histone H3 (8, 9). G9a is essential for...

Regulation of chromatin by histone modifications

“…Elegant mutagenesis studies have shown that mutagenesis of DIM5 F281 to Y converts the enzyme to a monomethyltransferase, whereas the reciprocal mutation in SET7/9 (Y305 to F) creates an enzyme capable of trimethylating its substrate [23]. More generally speaking, it seems that the aromatic determinant (Y or F) is a mechanism widely employed by SET domain-containing HKMTs to control the degree of methylation [24,25].…”

Protein lysine methyltransferase G9a acts on non-histone targets

“…In vitro G9a generates mainly H3K9me1 and H3K9me2 (ref. 8), as well as H3K9me3 after long incubation 9 . In contrast to the Dim-5 and Suv39H1 H3K9 methyltransferases, G9a methylates not only H3K9 but also H3K27 (ref.…”

Structural dynamics of protein lysine methylation and demethylation

“…The mutation did not affect the catalytic activity of G9a; however, the reaction by F1205Y stalled at the monomethyl stage [56]. Thus, the F1205Y mutation changed the product specificity of G9a from a fast mono/di-MTase with a slow tri-MTase activity to a predominantly mono-MTase without affecting overall catalytic activity, analogous to the F281Y mutation for DIM-5.…”

“…Sequence alignment including HKMTs with known product specificity suggests that the tyrosine/phenlyalanine switch rule may be generalized (see Table 1 of reference [56]). Both Arabidopsis KYP and SUVH6 have a tyrosine at the switch position and are primarily monoMTases [57].…”