Methylation: lost in hydroxylation?

Abstract

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.

Figure 1

Predicted structure of Epe1 modelled on the structure of FIH. The three-dimensional-jury algorithm implemented on the meta-server http://BioInfo.PL/Meta/ was used to predict the fold of the Epe1 protein (Ginalski et al, 2003). All 11 algorithms found FIH to be the best hit. The consensus score of 145 is much greater than the cut-off value of 40, at which there is 90% confidence that the prediction has found the correct fold. The JmjC domain including the doublestranded β-helix (DSBH) is shown in cyan and the eight strands of the DSBH are numbered 1–8, corresponding to β-strands 8–15 of FIH, as defined by the PDBsum entry for 1h2k. The fold is viewed roughly in the direction of attack of the substrate, which would be positioned over the Fe²⁺ ion. The black dots represent insertions in Epe1 not present in FIH and the magenta-striped regions are deletions in Epe1 not present in FIH. Insertions or deletions are in surface loops, not within the secondary structure elements or the core of the molecule. Trimethyl-lysine can be modelled in our predicted structure; none of the methyl groups makes a steric clash in the model and all can be accommodated simultaneously.

Figure 2

Multiple sequence alignment of JmjC domains. The JmjC domains of several proteins were aligned using Clustal W. The position of the β-sheets from the crystal structure of Q9NMT6 (Homo sapiens FIH) are illustrated above the alignment. Positions with similarity scores greater than 0.5 are highlighted in yellow. The Fe(II) co-ordinating residues (predicted) are marked with a red asterisk. The 2-OG-Fe(II)-dependent dioxygenases bind Fe(II) using the consensus HXD/EXnH. These residues are conserved in most of the JmjC-domain sequences. A subgroup of JmjC domains, including Epe1 and the PHD-finger protein 2, use another Fe(II) co-ordinating residue, tyrosine, in place of the second histidine in this motif, which leads us to predict that Epe1 co-ordinates Fe(II) with His 297, Glu 299 and Tyr 370. The hairless and Jumonji proteins have cysteine and serine residues, respectively, instead of the first conserved histidine of the iron-binding motif. The Jumonji protein also has a valine residue instead of the second histidine so may be catalytically inactive. The 2-OG binding residues (predicted) are shown with green squares. In general, the 2-OG-Fe(II)-dependent dioxygenases use an arginine or lysine residue downstream of the iron-binding motif in strand 8 of the DSBH to interact with the 5-carboxylate of 2-OG. However, structural and sequence analysis of the JmjC-domain protein FIH demonstrates that FIH interacts with 2-OG in a different manner via a threonine (Thr 196) and a lysine (Lys 214) residue in strand 2 and strand 4 of the DSBH, respectively, and by an upstream tyrosine (Tyr 145) residue (Elkins et al, 2003). These 2-OG-interacting residues are conserved in many of the JmjC proteins including Epe1. Species abbreviations: at, Arabidopsis thaliana; br, Brachydanio rerio; ce, Caenorhabditis elegans; dd, Dictyostelium discoideum; dm, Drosophila melanogaster; hs, Homo sapiens; mm, Mus musculus; rn, Rattus norvegicus; sc, Saccharomyces cerevisiae; sp, Schizosaccharomyces pombe; um, Ustilago maydis; xl, Xenopus laevis.

Figure 3

Demethylation by hydroxylation. (A) Mechanism of demethylation of 3-methylcytosine by AlkB. (B) Proposed mechanism of demethylation of histones by Epe1 and/or other JmjC-domain proteins. The demethylation of monomethyl-lysine is illustrated for simplicity; however, di- or trimethylated residues could also be substrates.