The combined experimental results presented here and elsewhere (8
) and phylogenetic analyses on KDM4A–E clearly show that they can be divided into two subclasses, KDM4A–C, which are highly conserved in all vertebrates, and demethylate both H3K9 and H3K36 and KDM4D/E, which only accept H3K9 substrates. KDM4A–C have conserved dual PHD and Tudor domains in addition to their JmjC domains, which are likely involved in “targeting” the catalytic domains (as precedented with PHF8 (30
)). The histone marks that are recognized by the Tudor and PHD domains are uncharacterized except for the JMJD2A Tudor domain, which is selective for H3K4me3 and H4K20me3 (55
). However, KDM4D–E do not possess such “additional” domains (); thus, it seems likely that at least some of the selectivity of the KDM4 subfamily residues arises from residues in or close to the catalytic domain. Selectivity arising from the catalytic domain may be relatively more important for those members (e.g.
KDM4D/E) that do not contain additional domains. The altered selectivity features of the generated KDM4A variants reveal that sequence specificity within the KMD4 subfamily is not achieved by differences in the methylammonium-binding pocket but by other enzyme-histone interactions. The dramatic decrease in activity toward H3K36 of the I71L variant and the complete loss for the N86H and I87H variants reveal that these residues are key determinants of the sequence specificity for H3K36. Because the Q88K and R309G variants still demethylated H3K36, including with bulk histones and in cells, these residues are not a major determinant for sequence specificity. Interestingly, the I71L variant showed the same extent of demethylation of H3K9 as wild-type KDM4A. Thus, the interaction between Ile-71 and the H3K36 substrate could be a promising target for sequence-specific inhibitors, which would only inhibit either Lys-9- or Lys-36-directed activity of KDM4A.
The yeast KDM4 orthologue Rph1 has been reported to demethylate both H3K9 and H3K36 in vitro
and in mammalian cells (54
). However, as yet there is no reported methyltransferase acting on H3K9 in budding yeast, and this mark is therefore probably unmethylated. The capacity of Rph1 to demethylate H3K9 was suggested to be a vestige of an H3K9 methylation system in budding yeast. Interestingly, human KDM4A–C have been reported to demethylate non-histone substrates in vitro
), and the identified substrates share sequence similarity to H3K9; the biological significance of these observations is unknown. Thus, we speculate that the activity of Rph1 for H3K9 may result from its putative activity for non-histone substrates in budding yeast or earlier organisms.
The roles of bivalent Nϵ
-lysine methylations in transcriptional activation and repression are established for H3K4me3 (activation) and H3K27me3 (repression) for several genes involved in development (59
). Related roles are emerging for H3K9me3 and H3K36me3 (35
); H3K36me3 accumulates in the 3′-region of active genes, whereas H3K9me3 has been found in the promoter region of repressed genes, as well as in the coding region of active genes (1
). The demethylation of H3K9me3/H3K36me3 by KDM4A is proposed to repress transcription (36
). However, a recent report implies that KDM4A activates genes in neural crest development by demethylating H3K9me3 at the Sox10
gene locus; no H3K36me3 was observed at the 3′-end of the Sox10
gene during active transcription (35
). Overall, these and other results imply that the interplay between different methylation states is complex and context-dependent. In this regard, the finding that the sequence selectivity (i.e.
Lys-36) can be altered by mutagenesis may also be useful in dissecting the roles of the KDM4 subfamily members and the relative importance of binding and catalytic domains in determining in vivo
Although the activity toward H3K9 decreased for most point substitutions, replacement of all five residues (I71L, N86H, I87K, Q88K, and R309G) resulted in a variant with similar activity toward H3K9 compared with wild-type KDM4A, implying that there is a synergistic effect between the substitutions and hence binding interactions. Although it cannot be ruled out that in vitro
studies of JmjC histone demethylases have consistently failed to identify additional stimulatory factors (e.g.
protein complex partners/other conditions for optimal activity), turnover numbers for the KDM4s are low compared with some other human 2-OG oxygenases, e.g.
γ-butyrobetaine hydroxylase (61
), and comparable with those reported for the hypoxia-inducible factor hydroxylases (62
). Given the central role proposed for the hypoxia-inducible factor hydroxylases in regulating the hypoxic response in all animals (63
), it is possible that the demethylase activities of the KDM4 histone demethylases have roles in directly regulating gene expression, perhaps in a redox-regulated manner. In this regard, it is interesting that expression of KDM4B is itself regulated in a hypoxia-inducible factor- and hypoxia-dependent manner (65
), suggesting multiple ways in which oxygen levels regulate histone methylation.