The identification of demethylase enzymes has revealed that histone methylation can be dynamically regulated in a manner similar to that of histone acetylation and phosphorylation. In S. cerevisiae
, the enzymes that place histone methylation marks are well characterized and coordinate mainly the addition of these modifications during the process of active transcription (25
). Previously, only one histone demethylase enzyme, Jhd1, was identified in budding yeast. Jhd1 is a JmjC-domain-containing protein that catalyzes the demethylation of H3K36me2 and H3K36me1 modification states (36
). Given that Jhd1 does not target H3K36me3 in yeast, it remained possible that this methylation state was irreversible.
Here, we identify Rph1 as being a histone demethylase with activity towards histone H3K36me3 and H3K36me2 modification states. Deletion of RPH1 does not affect global histone H3K36 methylation profiles, and deletion strains are viable, displaying no obvious morphological or cellular defects. This observation is not surprising given that a deletion of SET2, the sole H3K36 methyltransferase in budding yeast, causes no obvious cellular defects and has subtle effects on gene expression. The overexpression of Rph1 leads to a cellular growth defect, but this property appears to be independent of H3K36 demethylase activity and instead relies on the C-terminal ZF DNA binding domain. It remains possible that the growth defect in Rph1-overexpressing cells is due to demethylase-independent repression of growth-related genes through the ZF DNA binding domain. The overexpression of the Rph1 JmjN/JmjC domains alone is sufficient to mediate the demethylation of H3K36, verifying that this portion of the protein is catalytically competent in vivo. In contrast to many other chromatin-modifying enzymes, Rph1 does not stably associate with other proteins but instead forms a homogenous complex comprised of four Rph1 subunits. Often, chromatin remodeling complexes rely on associated protein factors for enzyme targeting, but the fact that Rph1 has an intrinsic DNA binding domain may alleviate the requirement for genomic targeting by auxiliary protein factors in some instances. Removal of the ZF relieves growth defects in cells overexpressing Rph1, supporting the argument that this domain contributes to protein function and perhaps genomic targeting in vivo. Additional functional analyses will be required to define specific genomic targets of Rph1 and to understand how Rph1-mediated demethylation contributes to transcriptional regulation by Rph1.
The two characterized budding yeast histone demethylase enzymes, Jhd1 and Rph1, both target H3K36 methylation. Two of the three remaining JmjC-domain-containing proteins, Gis1 and Ecm5, have mutations in cofactor binding residues that ablate demethylase activity (Y. Tsukada, K. E. Gardner, and Y. Zhang, unpublished data). The remaining protein, Yjr119C, is an H3K4 demethylase that catalyzes the removal of the H3K4me3 modification state (our unpublished data). Therefore, it appears that JmjC-domain-containing proteins in budding yeast target the removal of H3K4 and H3K36 methylation but not H3K79 methylation. H3K4 and H3K36 methylation are placed by SET domain-containing histone methyltransferases. In contrast, H3K79 methylation is catalyzed by DOT1, which does not have a SET domain. The inability of JmjC-domain-containing proteins to remove H3K79 methylation strikingly parallels the fact that a unique enzyme is required to place this modification. Perhaps H3K79 methylation is also removed by a novel class of demethylase enzymes with unique enzymatic properties. Further biochemical and genetic analyses of H3K79 methylation in budding yeast will be instrumental in determining whether this modification is dynamically regulated and provide insight into potentially novel enzymes involved in metabolizing this modification.
The JmjC-domain-containing histone demethylase enzymes characterized thus far have a very defined substrate specificity towards the lysine modification site and state. The catalytic domain of Rph1 is homologous to the mammalian JHMD3/JMJD2 enzymes, which target both H3K36 and H3K9 demethylation. The capacity of mammalian enzymes to target H3K9 methylation, a modification which is absent from budding yeast chromatin, may have adaptively evolved in the presence of enzymes that place this modification. Surprisingly, the characterization of Rph1 substrate specificity revealed that Rph1 is also capable of demethylating H3K9 in vitro as well as on mammalian chromatin in vivo. This property of Rph1 is not simply due to promiscuous substrate specificity, as Rph1 does not affect other yeast or mammalian histone methylation sites. The capacity of Rph1 to demethylate this modification suggests that an H3K9 methylation system may have once existed in budding yeast. Despite the fact that H3K9 methylation is no longer found in budding yeast chromatin, the enzymatic activity of Rph1 towards this modification may have been inadvertently retained due to its bifunctional requirement as a regulator of H3K36 methylation. Other components of the H3K9 methylation system, including the H3K9 methyltransferase, may have been lost or become functionally inactive.
No SET-domain-containing protein has been shown to modify H3K9 in budding yeast. The SET-domain-containing protein Set3 is a structurally integral component of a high-molecular-weight histone deacetylase complex (30
) that, much like Set2, is targeted to the body of active genes, where it regulates chromatin modification (39
). Deletion of Set2 in a strain lacking any component of the Set3 complex results in synthetic growth defects, suggesting that these factors contribute to similar processes (18
). It has recently been demonstrated that in addition to H3K36 methylation, H3K9 methylation is targeted to the body of actively transcribed genes in mammalian cells (37
), and at least one mammalian histone deacetylase complex also contains H3K9 methyltransferase activity (33
). No histone methyltransferase activity has been identified for the budding yeast Set3 complex, and residues within the SET domain that are required for methyltransferase activity are substituted. The role of this complex in the transcribed regions of yeast genes raises the possibility that Set3 may have once played a role analogous to that of the methyltransferases that place H3K9 methylation in the body of mammalian genes. During the evolution of the yeast chromatin modification system, a loss of selective pressure for H3K9 methylation could have potentially allowed components of this system to functionally deteriorate, while an intact H3K9 methylation system in higher eukaryotes was retained. Perhaps Set3 remains as a relic of this modification system due to its essential structural role in the assembly of the Set3 protein complex and its role in histone deacetylation. It will be interesting to determine whether the SET domain of Set3 can be replaced with the SET domain from an active H3K9 methyltransferase to recapitulate H3K9 methylation profiles in budding yeast that are found in the body of transcribed genes in mammals. The revelation that Rph1 can demethylate H3K9 provides the first evidence for the possibility of an extinct H3K9 methylation system in budding yeast and suggests that Rph1 may represent a functional vestige of this system.