Nuclear DNA in eukaryotic cells is assembled into the hierarchical chromatin 
structure via a process that is dynamically affected by the combinatorial set of 
post-translational modifications (PTMs) of histones in a dynamic manner 
responsive to physiological and environmental changes. The precise 
quantification of these complex modifications is challenging. Here we present a 
robust MS-based quantitative proteomics method for studying histone PTMs using 
(15)N metabolically labeled histones as the internal reference. Using this 
approach, we identified Tetrahymena trithorax related 1 (Txr1p) as a histone 
methyltransferase in Tetrahymena thermophila and characterized the relationships 
of the Txr1p and Ezl2p methyltransferases to histone H3 modification. We 
identified 32 PTMs in more than 60 tryptic peptides from histone H3 of the 
ciliate model organism Tetrahymena thermophila, and we quantified them (average 
coefficient of variation: 13%). We examined perturbations to histone 
modification patterns in two knockout strains of SET-domain-containing histone 
methyltransferases (HMT). Knockout of TXR1 led to progressively decreased mono-, 
di-, and tri-methylation of H3K27 and apparent reduced monomethylation of H3K36 
in vivo. In contrast, EZL2 knockout resulted in dramatic reductions in both di- 
and tri-methylation of H3K27 in vivo, whereas the levels of monomethylation of 
H3K27 increased significantly. This buildup of monomethyl H3K27 is consistent 
with its role as a substrate for Ezl2p. These results were validated via 
immunoblotting using modification site-specific antibodies. Taken together, our 
studies define Txr1p as an H3K27 monomethylation-specific HMT that facilitates 
the buildup of H3K27 di- and trimethylation by the canonical H3K27-specific HMT, 
Ezl2p. Our studies also delineate some of the interdependences between various 
H3 modifications, as compensatory increases in monomethylation at H3K4, H3K23, 
and H3K56 were also observed for both TXR1 and ELZ2 mutants.