A second catalytic domain in the Elp3 histone acetyltransferases: a ...

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sequences. J. Mol. Biol. 287, 797–815 15 Moniaux, N. et al. (2000) Alternative splicing generates a family of putative secreted and membrane-associated MUC4 mucins. Eur. J. Biochem. 267, 4536–4544 16 Choundhury, A. et al. (2000) Human MUC4 mucin cDNA and its variants in pancreatic carcinoma. J. Biochem. 128, 233–243 17 Choundhury, A. et al. (2001) Alternate splicing at the 3′-end of the human pancreatic

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tumor-associated mucin MUC4 cDNA. Teratog. Carcinog. Mutagen. 21, 83–96 18 Moniaux, N. et al. (1999) Complete sequence of the human mucin MUC4: a putative cell membrane-associated mucin. Biochem. J. 338, 325–333 19 Hynes, R.O. et al. (2000) The evolution of cell adhesion. J. Cell Biol. 150, F89–F96 20 Eddy, S.R. (1998) Profile Hidden Markov models. Bioinformatics 14, 755–763

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Francesca D. Ciccarelli* Tobias Doerks Peer Bork European Molecular Biology Laboratory, 69012 Heidelberg, Meyerhofstr. 1, Max-Delbrueck-Centrum, PO Box 740238, D-13092 Berlin, Germany. *e-mail: [email protected]

A second catalytic domain in the Elp3 histone acetyltransferases: a candidate for histone demethylase activity? Yurii Chinenov A new subfamily of two-domain histone acetyltransferases (HATs) related to Elp3 has been identified. In addition to a HAT domain in the C terminus, these proteins have an N-terminal domain similar to the catalytic domain of S-adenosylmethionine radical enzymes. Two-domain organization is preserved in evolution, suggesting that both enzymatic activities are functionally or mechanistically coupled and directed towards highly conserved substrates. The functional implications of this similarity and a possible role for Elp3-related proteins as histone demethylases are discussed.

Histone N-terminal modifications affect gene expression by altering the repertoire of chromatin-associated proteins and, consequently, the compaction state of the chromatin. Methylation of specific residues in histones correlates with the persistently repressed state of heterochromatin [1–4]. The relative stability of this modification led to the hypothesis that certain methyl groups in histones provide a long-lasting epigenetic mark committing the chromatin to a specific transcriptional state [5,6]. However, recently reported transient repression of euchromatic genes associated with histone methylation suggests that histone methylation is less permanent than previously thought and that mechanisms of regulated removal of methyl groups must exist [7,8]. Several mechanisms, including specific degradation of methylated histones or enzymatic demethylation, have been proposed. The histone demethylase activity was described almost 30 years ago [9], but the protein that is responsible for http://tibs.trends.com

this activity has not yet been identified. Here, I report the sequence similarities of the yeast histone-acetyltransferase (HAT) Elp3 to an enzyme superfamily that uses S-adenosylmethionine (SAM) in radical reactions. I also discuss the possible role of Elp3 as a histone demethylase. The similarity between Elp3-related HATs and SAM radical enzymes

Screening for proteins similar to oxidases involved in heme biosynthesis revealed an extremely well-conserved group related to Elp3 [10]. All members of this group contain the C-terminal HAT domain and a separate, previously unreported region similar to the catalytic domain of both bacterial anaerobic coproporphyrinogen III oxidases (COPIII) and other proteins belonging to the SAM radical family. This similarity implies that, in addition to HAT activity, Elp3-related proteins might possess a second enzymatic activity. The conserved two-domain organization of Elp3-related proteins (Figs 1,2a) and high degree of sequence identity (81% in COPIII domain between human and yeast compared with 64% for cytochrome c and 81% for histone H2B) suggest that both enzymatic activities are functionally or mechanistically coupled and that the substrate or substrates of these enzymes are also well conserved. As twodomain Elp3-related proteins have been found in both Eukaryota and Archaea but not in Bacteria, the conserved substrates of Elp3 enzymatic activities are probably specific for these two kingdoms and absent from Bacteria. Anaerobic COPIII belongs to the SAM radical enzyme family, members of which use SAM in a wide range of reactions

including oxidation, oxidative cyclization, N- and P-methylation, isomerization and protein radical formation [11,12]. A conserved glycine-rich region in both Elp3 and SAM radical enzymes (Fig. 1, marked by a blue bar) is similar to the motif 1 in several SAM-dependent methyltransferases [13] and might be involved in SAM binding. Both Elp3-related proteins and SAM radical enzymes contain several conserved acidic and glycine residues reminiscent of motifs II and III of SAM-dependent methyltransferases. Coproporphyrinogen III oxidase catalyses the removal of the carboxyl group and two hydrogens of propionic groups in coproporphyrinogen III to form vinyl groups of protoporphyrinogen IX during haem biosynthesis [14]. In the context of chromatin, the COPIII-related domain of Elp3 could catalyse the modification of DNA, histones or other chromatin-associated proteins. Similarity to COPIII suggests that Elp3-related proteins catalyse a reaction involving elimination of a single-carbon fragment. Thus, it is possible that Elp3-related proteins are involved in demethylation of DNA or histones. As Elp3-related proteins have been found in organisms in which DNA methylation has not been detected (e.g. yeast, Caenorhabditis elegans), methylated DNA is a less likely substrate. Putative catalytic mechanism for histone demethylation

Similar to SAM radical enzymes, a histone demethylation reaction might be initiated by scission of SAM into methionine and 5′-deoxyadenosyl radical that is involved in further catalytic steps. In SAM radical

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GI_12654795 GI_12848586 GI_7295707 GI_7511380 GI_15240655 GI_6325171 GI_14324713 GI_13813635 GI_14521922 GI_7427732 GI_3122200 GI_3122198 GI_115001 GI_2500056 GI_2499573 GI_1825534 GI_731019 GI_116285

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GIAVVAVMCKPHRCPHISFTGNICVYCP(16)EPTSMRAIRARYDPFLQT--RHRIEQLKQLGHSVDKVEFIVMGGTFMA GIAVVAVMCKPHRCPHISFTGNICVYCP(16)EPTSMRAIRARYDPFLQT--RHRIEQLKQLGHSVDKVEFIVMGGTFMA GIAVVAVMCKPHRCPHINMTGNICVYCP(16)EPTSMRAIRSRYDPFLQT--RHRVEQLKQLGHSVDKVEFIVMGGTFMC GIAVVAVMSKPHRCPHINFTGNICVYCP(16)EPTSMRAIRARYNPYLQT--RGRLNQLMQLGHSVDKVEFIVMGGTFMS GIAVVAVMSKPHRCPHIATTGNICVYCP(16)EPTSMRAIRARYNPYVQA--RSRIDQLKRLGHSVDKVEFILMGGTFMS GIAVVAVMCKPHRCPHIAYTGNICVYCP(16)EPTSMRAIRARYDPYEQA--RGRVEQLKQLGHSIDKVEYVLMGGTFMS GVSVVAAMTSPDRCPH-----GKCIFCP(14)EPAALRGRNNRYDPYMET--FSRIKQLETIGHDTSKIDLIVMGGTFTA GVTIVSIMTHPHSCPH-----GKCVFCP(14)EPTLMRAIENDYDPFHQV--QSRLRQYVENGHTPSKVELIIMGGTFLS GVAVVAMMTKPFPCPH-----GRCIYCP(14)EPSALRAAQYGYHPYIIM--MARLKQLYDIGHPIDKVEVIIQGGTFPA MKSAYIHIP-FCEH------ICHYCD-----FNKYFIQSQPVDEYLNALEQEMINTIAKTGQPDLKTIF-IGGGTPTS GASASLYLHVP-FCRE------MCWYCG----CHTQIVRRDDLIAAYQRTLRSEIALVAETIGRRIKVEHIHFGGGTPTI PMPLSLYTHLP-FCRS------ACYFCA----CSVIYTSLEEKKIRYISYLKKELALLKNAMDTNREVAQFHYGGGTPTF VKLNMIMNAKSGYCPE------DCGYCS-----QSSKSTAPIEKYPFIT--KEEILAGAKRAFENKIGTYCIVASGRGPT DGPGIRTVVFFQGCGL------RCSYCH-------NPDTWNMAGGKELTAEELLKKLLRFKPYFDRSGGGVTFSGGEVLL

Protein-L-isoaspartate O-methyltransferase Mycolic acid methyltransferase Thioether S-methyltransferase Chemotaxis protein methyltransferase

69 64 50 112

eWVGldkgmrvleIGGGTGY gKLGlepgmtlldVGCGWGS fSTGgvggdvlidIGSGPTI rRHGEyrvwsaaasTGEEPY

78 83 69 131

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LPEEYRDYFIRNLHDALS(15)RSLTKCIGITIETRPDYCMKRHISDMLTYGCTRLEIGVQSVYEDVARDTNRGHTVKAV LPEEYRDYFIRSLHDALS(15)RSFTKCIGITIETRPDYCMKRHLSDMLTYGCTRLEIGVQSVYEDVARDTNRGHTVKAV LPEEYRDYFIRNLHDALS(15)KSRTKCIGITIETRPDYCLKRHISDMLSYGCTRLEIGVQSVYEDVARDTNRGHTVRAV LPEDYRDFFIRNLHDALS(15)RSKMKCIGITIETRPDYCLPRHLNDMLLYGCTRLEIGVQSTYEDVARDTNRGHTVKSV LPAEYRDFFIRNLHDALS(15)HSATKCIGMTIETRPDYCLGPHLRQMLIYGCTRLEIGVQSTYEDVARDTNRGHTVAAV LPKEYREDFIVKLHNALS(15)QSLTKCVGITIETRPDYCTQTHLDDMLKYGCTRLEIGVQSLYEDVARDTNRGHTVRSV RPVDYQNSFIKGCLDAMN(15)TSEHRCIGLTVETKPDWFFEKEIDDALNYGTTKVELGVQNINDRILRINNRGHTVEDI LPIDYQDWFVTYALEAMN(21)KAEVRCVGMTIETKPDWAKEWHADQMLRLGATKVELGVQTVYDDILKFKNRGHTVKDS VDLDYQEWFIKCAFKAMN(56)KAKVRMVGLTIETRPDWAFERQIDRMLSFGTTRVELGVQTIFNFIHERTKRGHGVEEI LSEEQLKKLMDMINRVL-----KPSSDLSEFAVDANPDDLSAEKLKILKEAGVNRLSFGVQTFEDDLLEKIGRVHKQKDV MAPEAFAELMAAMRQAFF------VLPSAEIAVEIDPRTLTADMVEAMRLSGVNRASLGVQSFDPIVQRAINRIQSFEQT FSPIQLDEITQSIQEVFP-----NFSKDIEMSCEIDPRHFTKEHMQTLFDRGFNRLSFGVQDFDFEVQKAIHRIQPFEMV RKDVNVVSEAVEEIKAK-----------YGLKVCACLGLLKEEQAQQLKEAGVDRYNHNLNTSERHHSYITTTHTYEDRV QPEFLIDILKLCKEQGIHTAIDTAGYGYGNYEEILKHTDLVLLDIKHVDDDGYKCIT-GKGKRGFDDFLKAVENIGVKVW

GI_12654795 GI_12848586 GI_7295707 GI_7511380 GI_15240655 GI_6325171 GI_14324713 GI_13813635 GI_14521922 GI_7427732 GI_3122200 GI_3122198 GI_115001 GI_2500056

CESFHLAKDSGFK-VVAHMMPDLPNVGLERDIEQFTEYFENPAFRPDGLKIYPTLV CESFHLAKDSGFK-VVTHMMPDLPNVGLERDIEQFTEYFENPAFRPDGLKLYPTLV CESFQLGKDAGYK-IVTHMMPDLPNVDFERDIEQFIEYFENPAFRSDGLKIYPTLV CETFHMAKDTGYK-VVIHMMPDLPNVGLERDKEQFLELFESPAFRPDGLKLYPTLV ADCFCLAKDAGFK-VVAHMMPDLPNVGVERDMESFKEFFESPSFRADGLKIYPTLV CETFAVSKDAGYK-VVSHMMPDLPNVGMERDIEQFKEYFENPDFRTDGLKIYPTLV ARSTQLARDAGLK-IVYHIMPGMYGSSPSLDRESFLLMVNDERFKPDMLKIYPTLV IESTRILKDSGFK-VVYHVMLGLPKSDPDKDLEAFKTIFSDPNFRPDMLKIYPTLV IKATQLLKDAGLK-INYHIMPGLPGSNFERDLYTFKTIFEDPRFRPDMLKIYPTLV FTSFERAREIGFENISLDLMFGLPGQTLKHLEHSINTALS---LDAEHYSVYSLIV AAVVDMLRHAGIAGINFDLIYGLPHQTVASCLDTVRRSLL---LAPDRFSVFGYAH QESVKLARDYGIKSINFDLIYGLPNQTKEGFLKTLEWVLK---LDPDRLAVFNYAH -NTVEVVKKHGISPCSGAII-GMKETKMDVVEIARALHQ----LDADSIPVNFLHA IRHVIVPTLTDSKENIRKLANII---KNIRNVEKVELLP----YHTLGINKYEKLN

322 322 324 325 334 331 312 247 343 193 240 246 234 210

Elp3 H.sapiens M.musculus D.melanogaster C.elegans A.thaliana Elp3 S.cerevisiae T.volcanium S.solfataricus P.abyssi HemN B.subtilis HemN B.japonicum HemN H.pylori BioB B.sphaericus PflA C.pasteurianum Ti BS

Fig. 1. Sequence similarity of two-domain Elp3-related histone acetyltransferases (HATs) and S-adenosylmethionine (SAM)-radical enzymes. A PSI–BLAST [18] search (E value = 0.001) of the NCBI non-redundant protein database with the N-terminal portion of yeast HAT Elp3 revealed similarity to bacterial oxygen-independent coproporphyrinogen III oxidases encoded by the HemN gene after the second iteration. The representative sequences of HemN proteins from Bacillus subtilis (GI: 7427732, E = 1e−23), Bradyrhizobium japonicum (GI: 3122200, E = 2e−10) and Helicobacter pylori (GI: 3122198, E = 2e−14) are shown alongside with Elp3-related proteins from several eukaryotic (red) and archaeal (green) species. An additional search against an expressed sequence tag (EST) database revealed two ESTs from Xenopus laevis (GI: 7398483) and rat (GI: 11660948) (not shown). An initial alignment that included several Elp3-related proteins and bacterial HemN-related proteins was used for further searches using iterative algorithm implemented in the program PROBE [19]. More than 600 proteins identified represented most of the SAM radical enzyme superfamily described by Sofia et al. [12]. Conserved amino acids (80%) are coloured according to the following scheme: white letters on dark blue background represent hydrophobic amino acids; violet, small; red, polar; teal, tiny; green, aliphatic. Positively charged amino acids are shown in blue (big amino acids in dark blue, and Ser and Thr in light blue), negatively charged in red. Positionally conserved amino acids with the frequency >80% are shown in blue on a yellow background. The left column represents GenBank identifiers for each protein; the right column indicates amino acid positions for each protein. The region involved in the formation of an FeS cluster in SAM radical enzymes is marked by a red bar above the alignment. The blue bar above the alignment marks the region similar to motif I that is characteristic of SAM-dependent methyltransferases [13]. The sequences of the motif I in several SAM-dependent methyltransferases are shown on the bottom of the alignment. Conserved acidic residues are marked by red circles. The multiple sequence alignment (alignment number ALIGN_000288) has been deposited with the European Bioinformatics Institute (ftp://ftp.ebi.ac.uk/pub/databases/embl/align/ALIGN_000288.dat).

enzymes, an electron required for this reaction (Fig. 2b, step 1) is donated by a http://tibs.trends.com

reduced form of an FeS cluster (Fig. 1, marked by a red bar on the top of the

alignment). A cysteine-rich motif, MCxxHxCxxxxxxxxxCxYCP, conserved in Elp3-related proteins, could be involved in the formation of an FeS cluster. Although it is possible that this motif represents a zinc finger, neither sequence- nor pattern-based searches yielded any proteins with characterized zinc fingers. 5′-Deoxyadenosyl radical abstracts a proton from the methyl group of an N-methyllysine or N-methyl-arginine, yielding deoxyadenosine and aminium cation radical (Fig. 2b, step 2). This intermediate is unstable and is hydrolysed, producing an unmethylated residue and formaldehyde (Fig. 2b, step 3). A similar reaction pathway proceeding through the formation of an aminium cation has recently been proposed for an oxidative N-demethylation of trimethylamine catalysed by

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H2N CH3

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Fig. 2. (a) Domain structure of Elp3-related proteins from various organisms. The COPIII oxidase-related domain is shown in green, and the HAT domain in blue. Numbers indicate amino acid portions in each protein. (b) Hypothetical mechanism of N-methyl demethylation. The proposed mechanism of histone demethylation includes three main steps. The first catalytic step is common for all studied SAM radical enzymes and leads to formation of a reactive 5′-deoxyadenosyl radical. An electron required for SAM cleavage is supplied by a reduced form of the FeS cluster [4Fe4S]+ which, in Elp3-related proteins, might be formed by cysteine residues 9, 14, 24 and 27. In the second step, the 5′-deoxyadenosyl radical initiates a radical reaction by abstracting a proton from the N-methyl group of methyllysine or methyl-arginine to form a radical intermediate, which rearranges into an aminium cation radical intermediate in step 3. Hydrolysis of intermediate 3 produces an unmethylated residue and formaldehyde. In a concomitant reaction, the oxidized FeS cluster [4Fe4S]2+ could be regenerated to produce a reduced [4Fe4S]+ form. Alternatively, external reducing equivalents in the form of NADH or NADPH could regenerate an FeS cluster. Unmethylated lysine residues could be further acetylated by Elp3 HAT activity. Abbreviations: COPIII, coproporphyrinogen III oxidase; HAT, histone acetyltransferase; SAM, S-adenosylmethionine.

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O

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GI_14521922 P. abyssi

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trimethylamine dehydrogenase [15]. Formaldehyde could be further detoxified by glutathione-dependent formaldehyde dehydrogenase [16]. Methylation of lysine or arginine residues in histones correlates with either activation or repression of transcription and apparently depends on a specific residue and modification state of other residues (reviewed in Refs [5,6]). Elp3 has been isolated as a part of a larger sixsubunit complex specifically associated with elongating RNA polymerase II [10,17]. Thus, both enzymatic activities of Elp3 probably exert an overall positive effect on transcription. The specificity of Elp3 demethylase activity might depend on the specificity of the Elp3 HAT domain or, alternatively, on the relative position in the multiprotein complex associated with RNA polymerase. The similarity reported here suggests an intriguing possibility that Elp3-related proteins constitute a new group of HATs with dual enzymatic activity. Further experimental studies are required to establish a precise nature of this activity. Sequence analysis outlines this activity in broad terms and provides a platform for further experimental analysis.

A

HO 5'-Deoxyadenosyl radical

N-Methyl lysine residue

GI_15240655 A. thaliana

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GI_7295707 D. melanogaster

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Acknowledgements /

I thank Mensur Dlakic for helpful comments and Martin Tompson, Ekaterina Pechenkina and Nirmala Rajaram for critical reading of the manuscript. References 1 Bannister, A.J. et al. (2001) Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410, 120–124 2 Nakayama, J. et al. (2001) Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292, 110–113 3 Lachner, M. et al. (2001) Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410, 116–120 4 Noma, K. et al. (2001) Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries. Science 293, 1150–1155 5 Jenuwein, T. and Allis, C.D. (2001) Translating the histone code. Science 293, 1074–1080 6 Rice, J.C. and Allis, C.D. (2001) Histone methylation versus histone acetylation: new insights into epigenetic regulation. Curr. Opin. Cell Biol. 13, 263–273 7 Nielsen, S.J. et al. (2001) Rb targets histone H3 methylation and HP1 to promoters. Nature 412, 561–565 8 Hwang, K.K. et al. (2001) Transcriptional repression of euchromatic genes by Drosophila heterochromatin protein 1 and histone modifiers. Proc. Natl. Acad. Sci. U. S. A. 98, 11423–11427 9 Paik, W.K. and Kim, S. (1973) Enzymatic demethylation of calf thymus histones. Biochem. Biophys. Res. Commun. 51, 781–788

CH2O Ti BS

10 Wittschieben, B.O. et al. (1999) A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme. Mol. Cell 4, 123–128 11 Frey, P.A. (2001) Radical mechanisms of enzymatic catalysis. Annu. Rev. Biochem. 70, 121–148 12 Sofia, H.J. et al. (2001) Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucleic Acids Res. 29, 1097–1106 13 Kagan, R.M. and Clarke, S. (1994) Widespread occurrence of three sequence motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes. Arch. Biochem. Biophys. 310, 417–427 14 Akhtar, M. (1994) The modification of acetate and propionate side chains during the biosynthesis of haem and chlorophylls: mechanistic and stereochemical studies. Ciba Found. Symp. 180, 131–151 15 Jang, M.H. et al. (1999) The reaction of trimethylamine dehydrogenase with trimethylamine. J. Biol. Chem. 274, 13147–13154 16 Hur, M.W. and Edenberg, H.J. (1992) Cloning and characterization of the ADH5 gene encoding human alcohol dehydrogenase 5, formaldehyde dehydrogenase. Gene 121, 305–311 17 Winkler, G.S. et al. (2001) RNA polymerase II elongator holoenzyme is composed of two discrete subcomplexes. J. Biol. Chem. 276, 32743–32749 18 Altschul, S.F. et al. (1997) Gapped BLAST and PSI–BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 19 Neuwald, A.F. et al. (1997) Extracting protein alignment models from the sequence database. Nucleic Acids Res. 25, 1665–1677

Yurii Chinenov Howard Hughes Medical Institute, University of Michigan Medical Center, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0650, USA. e-mail: [email protected]