3), which includes the major H3 and two minor variants, H3.3 and H3.4 (46, 47). N-terminal tails, are subject to numerous covalent post-translational modifications (PTMs)1 including acetylation, methylation, phosphorylation, ubiquitination, and citrullination (1C3). A combinatorial set of PTMs on one or more histones, deposited by histone-modifying enzymes, effectively serves to modulate numerous DNA pathways, including gene expression and replication as postulated in the histone code hypothesis (4, 5). Prominent among the PTMs is the reversible epigenetic mark, lysine methylation, present in mono-, di-, and trimethylation says. Methyl groups are added to the -amine of the lysyl residue by histone methyltransferases (HMTs) and removed by histone demethylases (6C9). Different lysine methylation says are often associated with differentsometimes even oppositebiological functions. For example, the monomethylation of histone residues H3K9, H3K27, and H4K20 is usually linked to active transcription, whereas their trimethylation says are associated with transcriptional repression (6, 10). The functional variation of the different methylation says is usually further underscored by the presence of divergent state-specific HMTs, such as SETDB1/SETDB2 (for H3K9Me1) and SUV39H1/SUV39H2 (for H3K9Me2 and H3K9Me3) (11), SETD8 (for H4K20Me1) (12), and SUV4C20 (for H4K20Me2 and H4K20Me3) (13). It is a major challenge in the field of epigenetics to unravel the mechanism regulating histone lysine methylation events, which are dynamically affected by many factors and implicated in various biological processes. The modifications of histone lysines are dynamic, and mono-, di-, and trimethylated residues are generally considered to be progressively methylated (14). Most HMTs for lysine methylation contain a conserved catalytic domain name, the SET (suppressor of variegation, enhancer of zeste, trithorax) domain name (15). NSC-41589 HMTs, in particular lysine methyltransferases, have been implicated in human diseases, including cancers (16). In the past decade, a large number NSC-41589 of HMTs have been recognized in a wide range of eukaryotic organisms, and they have been classified according to their sequence homology into subfamilies, whose users generally share the substrate specificity. trithorax related 5 (ATXR5) and ATXR6, the founding users of a recently recognized HMT subfamily, were first isolated as proliferating cell nuclear antigen interacting proteins in (17). Both ATXR5 and ATXR6 feature a divergent SET domain name (17), a herb homeodomain (PHD) finger that binds the altered histones (18C21), and a proliferating cell nuclear antigen interacting protein box that binds proliferating cell nuclear antigen (22). Homologues of ATXR5 and ATXR6 are found in plants but not in animals. The atxr5 atxr6 double mutant exhibits reduced H3K27Me1 levels (23), supporting the notion that ATXR5 and ATXR6 function as the H3K27 monomethylation-specific HMTs. trithorax related 1 (Txr1p) was recently identified as a putative HMT in the ciliate model organism (encoded by the gene) via homology to ATXR5 and ATXR6. Txr1p carries two PHD domains (PHD1 and PHD2), one proliferating cell nuclear antigen interacting protein box (QKLIEDYF), and one C-terminal SET domain name (Fig. 1), all of which are consistent with NSC-41589 its being a member of the ATXR5/ATXR6 subfamily of HMTs. In is expressed at significant levels (24, 25) and required for H3K27 di- and trimethylation in asexually dividing cells (observe below). Open in a separate windows Fig. 1. Domain name structure analysis of protein Txr1p. homologues ATXR5 and ATXR6. Identical and comparable residues are darkly and lightly shaded, respectively. Mass spectrometry (MS) has played an important role in the study of histone PTMs for the following reasons: (1) MS is usually capable of simultaneously monitoring multiple PTMs; and (2) it can identify and quantify known and unknown PTMs in histones that cannot be very easily determined via other analytical approaches such as S5mt micro-sequencing by Edman degradation or immunoblotting with modification site-specific antibodies (26C30). The quantification of histone PTMs can be achieved via a label-free strategy based on the relative intensities of extracted ion chromatograms of precursors (31) or, more accurately through stable isotope labeling techniques such NSC-41589 as SILAC or iTRAQ (32C35). However, the analysis of histone PTMs via LC/MS is particularly challenging because of the enormous quantity of isoforms generated by the combination of numerous densely deposited PTMs (29). The problem is usually further exacerbated by the basicity of histones, which, after trypsin digestion, generate peptides too small or hydrophilic to be effectively retained on reversed-phase HPLC columns and analyzed via MS. Chemical derivatization using propionic anhydride was launched in order to overcome some of these difficulties (36, 37). Briefly, the propionylated histones are only cleaved after arginyl residues when digested with trypsin and thus generate nicely sized, more hydrophobic peptides that can.

3), which includes the major H3 and two minor variants, H3