Identification of histone demethylases in Saccharomyces cerevisiae

Based on the prediction that histone lysine demethylases may contain the JmjC domain, we examined the methylation patterns of five knock-out strains (ecm5Delta, gis1Delta, rph1Delta, jhd1Delta, and jhd2Delta (yjr119cDelta)) of Saccharomyces cerevisiae. Mass spectrometry (MS) analyses of histone H3 showed increased modifications in all mutants except ecm5Delta. High-resolution MS was used to unequivocally differentiate trimethylation from acetylation in various tryptic fragments. The relative abundance of specific fragments indicated that histones K36me3 and K4me3 accumulate in rph1Delta and jhd2Delta strains, respectively, whereas both histone K36me2 and K36me accumulate in gis1Delta and jhd1Delta strains. Analyses performed with strains overexpressing the JmjC proteins yielded changes in methylation patterns that were the reverse of those obtained in the complementary knock-out strains. In vitro enzymatic assays confirmed that the JmjC domain of Rph1 specifically demethylates K36me3 primarily and K36me2 secondarily. Overexpression of RPH1 generated a growth defect in response to UV irradiation. The demethylase activity of Rph1 is responsible for the phenotype. Collectively, in addition to Jhd1, our results identified three novel JmjC domain-containing histone demethylases and their sites of action in budding yeast S. cerevisiae. Furthermore, the methodology described here will be useful for identifying histone demethylases and their target sites in other organisms.     

Methylation: lost in hydroxylation?

Methylation of histone tails is a key determinant in forming active and silent states of chromatin. Histone methylation was regarded as irreversible until the recent identification of a lysine-specific histone demethylase (LSD1), which acts specifically on mono- and dimethylated histone H3 lysine 4. Here, we propose that the fission yeast protein Epe1 is a putative histone demethylase that could act by oxidative demethylation. Epe1 modulates the stability of silent chromatin and contains a JmjC domain. The Epe1 protein can be modelled onto the structure of the 2-oxoglutarate-Fe(II)-dependent dioxygenase, factor inhibiting hypoxia inducible factor (FIH), which is a protein hydroxylase that also contains a JmjC domain. Thus, Epe1 and certain other chromatin-associated JmjC-domain proteins may be protein hydroxylases that catalyse a novel histone modification. Another intriguing possibility is that, by hydroxylating the methyl groups, Epe1 and certain other JmjC-domain proteins may be able to demethylate mono-, di- or trimethylated histones.     

A plant‐specific histone H3 lysine 4 demethylase represses the floral transition in Arabidopsis

Histone demethylation regulates chromatin structure and gene expression, and is catalyzed by various histone demethylases. Trimethylation of histone H3 at lysine 4 (H3K4) is coupled to active gene expression; trimethyl H3K4 is demethylated by Jumonj C (JmjC) domain‐containing demethylases in mammals. Here we report that a plant‐specific JmjC domain‐containing protein known as PKDM7B (At4g20400) demethylates trimethyl H3K4. PKDM7B mediates H3K4 demethylation in a key floral promoter, FLOWERING LOCUS T (FT), and an FT homolog, TWIN SISTER OF FT (TSF), and represses their expression to inhibit the floral transition in Arabidopsis. Our findings suggest that there are at least two distinct sub‐families of JmjC domain‐containing demethylases that demethylate the active trimethyl H3K4 mark in eukaryotic genes, and reveal a plant‐specific JmjC domain enzyme capable of H3K4 demethylation.     

Immediate chromatin immunoprecipitation and on-bead quantitative PCR analysis: a versatile and rapid ChIP procedure

Genome-wide chromatin immunoprecipitation (ChIP) studies have brought significant insight into the genomic localization of chromatin-associated proteins and histone modifications. The large amount of data generated by these analyses, however, require approaches that enable rapid validation and analysis of biological relevance. Furthermore, there are still protein and modification targets that are difficult to detect using standard ChIP methods. To address these issues, we developed an immediate chromatin immunoprecipitation procedure which we call ZipChip. ZipChip significantly reduces the time and increases sensitivity allowing for rapid screening of multiple loci. Here we describe how ZipChIP enables detection of histone modifications (H3K4 mono- and trimethylation) and two yeast histone demethylases, Jhd2 and Rph1, which were previously difficult to detect using standard methods. Furthermore, we demonstrate the versatility of ZipChIP by analyzing the enrichment of the histone deacetylase Sir2 at heterochromatin in yeast and enrichment of the chromatin remodeler, PICKLE, at euchromatin in Arabidopsis thaliana.     

Catalytic and Functional Roles of Conserved Amino Acids in the SET Domain of the S. cerevisiae Lysine Methyltransferase Set1

In S. cerevisiae, the lysine methyltransferase Set1 is a member of the multiprotein complex COMPASS. Set1 catalyzes mono-, di- and trimethylation of the fourth residue, lysine 4, of histone H3 using methyl groups from S-adenosylmethionine, and requires a subset of COMPASS proteins for this activity. The methylation activity of COMPASS regulates gene expression and chromosome segregation in vivo. To improve understanding of the catalytic mechanism of Set1, single amino acid substitutions were made within the SET domain. These Set1 mutants were evaluated in vivo by determining the levels of K4-methylated H3, assaying the strength of gene silencing at the rDNA and using a genetic assessment of kinetochore function as a proxy for defects in Dam1 methylation. The findings indicate that no single conserved active site base is required for H3K4 methylation by Set1. Instead, our data suggest that a number of aromatic residues in the SET domain contribute to the formation of an active site that facilitates substrate binding and dictates product specificity. Further, the results suggest that the attributes of Set1 required for trimethylation of histone H3 are those required for Pol II gene silencing at the rDNA and kinetochore function.     

Partitioning and plasticity of repressive histone methylation states in mammalian chromatin

Methylation of position-specific lysine residues in histone N termini is a central modification for regulating epigenetic transitions in chromatin. Each methylatable lysine residue can exist in a mono-, di-, or trimethylated state, thereby extending the indexing potential of this particular modification. Here, we examine all possible methylation states for histone H3 lysine 9 (H3-K9) and lysine 27 (H3-K27) in mammalian chromatin. Using highly specific antibodies together with quantitative mass spectrometry, we demonstrate that pericentric heterochromatin is selectively enriched for H3-K27 monomethylation and H3-K9 trimethylation. This heterochromatic methylation profile is dependent on the Suv39h histone methyltransferases (HMTases) but independent of the euchromatic G9a HMTase. In Suv39h double null cells, pericentric heterochromatin is converted to alternative methylation imprints and accumulates H3-K27 trimethylation and H3-K9 monomethylation. Our data underscore the selective presence of distinct histone lysine methylation states in partitioning chromosomal subdomains but also reveal a surprising plasticity in propagating methylation patterns in eukaryotic chromatin.     

Molecular regulation of histone H3 trimethylation by COMPASS and the regulation of gene expression

The Set1-containing complex COMPASS, which is the yeast homolog of the human MLL complex, is required for mono-, di-, and trimethylation of lysine 4 of histone H3. We have performed a comparative global proteomic screen to better define the role of COMPASS in histone trimethylation. We report that both Cps60 and Cps40 components of COMPASS are required for proper histone H3 trimethylation, but not for proper regulation of telomere-associated gene silencing. Purified COMPASS lacking Cps60 can mono- and dimethylate but is not capable of trimethylating H3(K4). Chromatin immunoprecipitation (ChIP) studies indicate that the loss subunits of COMPASS required for histone trimethylation do not affect the localization of Set1 to chromatin for the genes tested. Collectively, our results suggest a molecular requirement for several components of COMPASS for proper histone H3 trimethylation and regulation of telomere-associated gene expression, indicating multiple roles for different forms of histone methylation by COMPASS.     

Structural Basis of a Histone H3 Lysine 4 Demethylase Required for Stem Elongation in Rice

Histone lysine methylation is an important epigenetic modification in regulating chromatin structure and gene expression. Histone H3 lysine 4 methylation (H3K4me), which can be in a mono-, di-, or trimethylated state, has been shown to play an important role in gene expression involved in plant developmental control and stress adaptation. However, the resetting mechanism of this epigenetic modification is not yet fully understood. In this work, we identified a JmjC domain-containing protein, JMJ703, as a histone lysine demethylase that specifically reverses all three forms of H3K4me in rice. Loss-of-function mutation of the gene affected stem elongation and plant growth, which may be related to increased expression of cytokinin oxidase genes in the mutant. Analysis of crystal structure of the catalytic core domain (c-JMJ703) of the protein revealed a general structural similarity with mammalian and yeast JMJD2 proteins that are H3K9 and H3K36 demethylases. However, several specific features were observed in the structure of c-JMJ703. Key residues that interact with cofactors Fe(II) and N-oxalylglycine and the methylated H3K4 substrate peptide were identified and were shown to be essential for the demethylase activity in vivo. Several key residues are specifically conserved in known H3K4 demethylases, suggesting that they may be involved in the specificity for H3K4 demethylation.