Histone Demethylation by Hydroxylation: Chemistry in Action - ACS

Histone Demethylation by Hydroxylation: Chemistry in Action ..... R. D. (1974) Chromatin structure: a repeating unit of histones and DNA Science 184, ...
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Histone Demethylation by Hydroxylation: Chemistry in Action Jessica Schneider† and Ali Shilatifard†,‡,* †Department of Biochemistry, Saint Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, Missouri

63104, and ‡Saint Louis University Cancer Center, Saint Louis University School of Medicine, St. Louis, Missouri 63104

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ukaryotic cells wrap their chromosomal DNA around octamers of histones to form nucleosomes. This process is a crucial first step in compacting and packaging DNA into the nucleus. In addition to a role in DNA packaging, nucleosomal assembly and disassembly regulate accessibility of the transcriptional machinery to gene coding and regulatory regions. Electron microscopy studies have shown chromatin as a series of “beads on a string,” with the “string” as linker DNA and the “beads” as individual nucleosomes consisting of eight core histone proteins (two each of H3, H4, H2A, and H2B) (1, 2 ) (Figure 1a). The core histones are wrapped by 147 base pairs of DNA (1.65 turns around the histone octamer), forming the intact nucleosome (3 ). The amino termini of histone tails protrude away from the nucleosome and are therefore available for interactions with DNA and other proteins and many histone tail-modifying enzymes (3 ). The posttranslational modifications of histone tails so far include: acetylation, phosphorylation, ubiquitination, and methylation. Multiple modifications can decorate each histone tail, and some amino acids within the histone tail can be modified in several different ways (4, 5 ). In fact, the combinatorial effect of such histone tail modifications can serve to elicit a multitude of different responses. This “epigenetic regulation” denotes an inherited state of gene regulation that is independent of the genetic information encoded within DNA itself. Lysine or arginine residues within histones can be posttranslationally modified via the enzymatic addition of methyl groups from the donor S-adenosylmethionine (SAM) (6 ). A class of enzymes containing SET domains [this domain takes its name from the Drosophila proteins Su(var)3-9, Enhancer of zeste (E(z)), and trithorax (trx)] can catalyze methylation of the lysine residue of www.acschemicalbiolog y.o rg

A b s t r a c t Histone methylation plays an essential role in epigenetic regulation and has been thought to be an irreversible and stable modification of histones. However, several enzymes have recently been discovered to demethylate mono- and dimethylated lysine residues of histone H3 as well as monomethylated arginines via either amine oxidation or deimination, respectively. The JmjC domain-containing histone demethylase 1 (JHDM1), which is conserved from yeast to human, has been demonstrated to demethylate mono- and di- but not trimethylated H3 K36 via hydroxylation of the methyl moiety within the methylated lysine residue. This study broadens our understanding of different types of reaction mechanisms and cofactor requirements for a different category of histone demethyl­ ating machinery.

*To whom correspondence should be addressed. E-mail: [email protected].

Received for review January 26, 2006 and accepted February 21, 2006 Published online March 17, 2006 10.1021/cb600030b CCC: $33.50 © 2006 American Chemical Society

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a

b CARM1

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ARTKQTARKSTGGKAPRKQLASKAARKSAPSTGGVKKP .. ...DFKTDL...H3 K9

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K79

Dot1

PRMT1 Set7/Set8

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SGRGKGGKGLGKGGAKRHRKVLR ..GGVKRISGLIYEETRGVLKVFLE...H4

?

K20 K59

Figure 1. The histone tails. a) Nucleosomes are involved in processes ranging from DNA compaction to transcriptional regulation. They are also considered to be major carriers of epigenetically inherited information as well. As shown, the repeating nucleosomes with intervening “linker” DNA form the 10-nm fiber, known descriptively as “beads on a string”. The histone tails protrude away from each nucleosome and therefore are available for interactions with DNA and other proteins and many histone tail-modifying enzymes. b) The site of modification by methylation and the enzymatic machinery involved in histone tail methylation. The N-terminal amino acid sequences of histones H3 and H4 along with positions of specific methylation sites and the known enzymes required for such modifications are shown above.

histones on the ε-nitrogen (Figure 1b). The SET domaincontaining enzymes are specific in their substrate selection, unlike histone acetyltransferases. Therefore, methylation of each lysine residue within histones requires a specific SET domain-containing enzyme (4, 6). For example, the Set1 protein in yeast, which is found as a component of the macromolecular complex COMPASS, is specific for methylation of lysine 4 of Keywords histone H3 (Figure 1b) (7–9 ). Nucleosome: Fundamental repeating unit in chromosomes made up of DNA and histone Even its human counterpart, proteins. Nucleosomes are found in eukaryotic the MLL protein, exists in nuclei and appear as bead-like structures a similar macromolecular along the DNA when viewed by electron microscopy. Routinely referred to as the complex and has the same “beads on a string.” histone substrate selectivity Chromatin: Mass of genetic material located in as COMPASS (10, 11 ). Other the cell nucleus containing DNA and proteins that condenses to form chromosomes in a lysines within histones, such eukaryotic cell. as lysine 36 of histone H3, Epigenetic information: Heritable changes in require the catalytic activgene function not encoded by eukaryotic chromosomal DNA. ity of other SET domainHistone methylation: Posttranslational modificacontaining enzymes, such tions occurring on lysine or arginine residues as Set2 (Figure 1b) (12 ). within histone tails that carry epigenetic information. In addition to SET domaincontaining histone methyl76

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transferases, there are also non-SET domain-containing enzymes capable of methylating lysine residues within histones. The enzyme Dot1 (disruptor of telomeric silencing 1) is a histone lysine methyl­transferase that lacks the characteristic SET domain, and it has been shown to methylate the lysine residue of histone H3 on lysine 79 (Figure 1b) (13–15 ). Following the identification of enzymatic machinery capable of methylating the lysine residues within histone proteins, it was demonstrated that the methyl­ ation of histones is a stable and irreversible modification. The relatively high stability of lysine methylation compared to other reversible modifications such as phosphorylation and acetylation was in part attributed to the fact that the N-CH3 bond is thermodynamically highly stable. Furthermore, experimental studies demonstrated that once methylated, the lysine residues exhibit a half-life similar to that of unmodified histones (16, 17 ). For example, H3 K9 methylation is required for the regulation of epigenetic silencing and the maintenance of heterochromatin; therefore, one would expect this modification to be static rather than dynamic (18, 19 ). In addition to the importance of particular sites being methylated on histones, the state of methyl­ ation also plays an essential role, as lysine residues may be either mono-, di-, or trimethylated (20, 21 ). Transition from the tri- to dimethyl or the di- to monomethyl form may be required for the proper response to developmental or environmental signals. Therefore, regulation of the transition from fully methylated to partially or fully unmethylated lysine residues within histones is likely to be indispensable for the biological outcomes associated with histone methyl­ation; regulation of this process could be one of the main reasons for the presence of histone demethyl­ating machinery. Although histone methylation is considered stable, several different histone demethylating enzymes have recently been identified to reverse some, but not all, forms of histone methylation (Figure 2) (22 ). PADI4 gene product was discovered as a histone deiminase that antagonizes histone arginine methyl­ ation (23, 24 ). It was demonstrated that either free or monomethylated arginine can be cleaved at the guani­ dine C‑N bond (Figure 2a) by the arginine deiminase PADI4, generating the products citrulline and methylammonium. Although PADI4 is capable of deimination of free and monomethyl arginine, dimethyl­ation of arginines prevents deimination by PADI4. Although w w w. a c s c h e m i ca l biology.org

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+ HN

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+ H2 N

FADH2

NH3 + Methylammonium

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+ H 3N

FADH2

LSD1/BHC110

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CH3

Formaldehyde

H2O + FAD

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+ N H

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Lysine

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CH3 Succinate + CO2

CH3

Dioxygenase? α-ketogularate

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α-ketogularate

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+ H2 N

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+

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+ NH

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Hydroxylation Formaldehyde Dimethyl Hydroxylation lysine

C O

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H

C O

H

+

N H

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Hydroxylation Formaldehyde

O Lysine

Figure 2. Enzymatic mechanisms required to remove the methyl moiety from modified histones. a) It has been demonstrated that the arginine and monomethylated arginine can serve as substrates for protein arginine deiminase 4 (PADI4). PADI4 can reverse arginine methylation by cleaving the guanidino C-N bond via the deimination mechanism. The byproduct of such reaction is citrulline and methyl-ammonium. Lysine methylation can be reversed via either b) amine oxidation or c) direct hydroxylation of the methyl moiety. The byproducts of both reaction mechanisms in b and c are unmodified lysine and formaldehyde. Since the formation of an imine intermediate via transfer of two hydrogen atoms to FAD requires a protonated nitrogen, amine oxidation can only demethylate mono- and dimethylated lysine substrates. Since trimethylated lysine has been found in nature, it has been proposed that hydroxylation can represent an alternative demethylating mechanism. In this mechanism, a direct radical attack on the methyl-carbon by Fe(II) in α‑ketoglutarate-dependent dioxygenases such as the JmjC-domain containing proteins can lead to the formation of an unstable carbinolamine, which result in the generation of unmodified lysine and formaldehyde. It has been proposed that this reaction mechanism can be employed to demethylate trimethylated lysine residues.

PADI4 can prevent dimethylation of arginine residues, its discovery does not fully address how cells manage dimethylated arginine residues. Other possibilities include enzymatic machinery capable of demethylating dimethylated arginine or the involvement of histone replacement machinery. Amine oxidation was the first mechanism to be proposed for lysine demethylation (Figure 2b). The BHC110/LSD1 protein, a histone H3 K4 demethylase (25 ), is a riboflavin-binding protein, which is a member of a FAD-dependent enzyme family. BHC110/LSD1 is highly conserved in organisms ranging from Schizosaccharomyces pombe to human. The amine oxidase domain of this enzyme is found in its carboxylterminal end. This enzyme also contains a SWIRM domain, which is a protein-protein interaction domain found in several chromatin-associated proteins. The demethylation of mono- and dimethylated lysine 4 residue of histone H3 by BHC110/LSD1 is an oxidation reaction that requires the presence of the cofactor www.acschemicalbiolog y.o rg

flavin adenine dinucleotide (FAD). Formaldehyde and an unmodified lysine residue are the byproducts of the enzymatic reaction catalyzed by BHC110/LSD1 (25 ). Since the formation of an imine intermediate requires a protonated nitrogen, BHC110/LSD1 can only demethylate mono- and dimethylated lysine residues and not the trimethylated form. Biochemical investigations have demonstrated that BHC110/LSD1 can associate with the androgen receptor and can act as a coactivator for transcription (26 ). Surprisingly, the interaction of BHC110/LSD1 with the androgen receptor overlaps with the specific loss of H3 K9 methylation from the androgen-receptor DNA element, with little to no effect on H3 K4 methyl­ ation within the same region (26 ). Furthermore, in the presence of the androgen receptor but not its absence, affinity-purified BHC110/LSD1 can catalyze the demethyl­ation of H3 K9. This indicates that the specificity of BHC110/LSD1 towards its histone substrate can be modulated through its interaction with VOL.1 NO. 2 • ACS CHEMICAL BIOLO GY

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+ CO2

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N

CH3

N

H

CH2OH

N 2-OG + O2

O

dR

C O

H

+ N

O

dR

N

3-Methylcytosine

Figure 3. Demethylation via hydroxy­ lation. a) A mechanism for demethylation of 3-methylcytosine by AlkB has already been demonstrated. b) Allshire and colleagues have proposed that JmjC domain-containing proteins can function via the same mechanism to demethylate methylated lysine residues on proteins (32 ). A recent study supports a role for JmjC domaincontaining proteins in demethylation of mono-and di- but not trimethylated lysine residues on histones (34 ).

Cytosine

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CH3 Succinate JmjCdomain proteins

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Monomethyl lysine

other cofactors. In addition to the interaction with the androgen receptor, BHC110/LSD1 has been shown to exist in a large macromolecular complex containing the CoREST corepressor complex. Association of BHC110/LSD1 within the CoREST complex can increase the demethylation of histone H3 lysine 4 (H3K4) by nearly 5‑fold as compared to that of recombinant BHC110/LSD1 alone (27, 28 ). More importantly, recombinant BHC110/LSD1 is unable to demethylate H3 K4 on nucleosomes. However, within its complex, nucleosomes are readily demethylated by BHC110/LSD1 (27, 28 ). On the basis of its biochemical interaction with the CoREST complex and other biological studies, it has been proposed that BHC110/LSD1, within the CoREST complex, is recruited to genes containing the REST-responsive element to participate in gene silencing by demethylating K4 mono- and dimethylated histone H3 within the REST-responsive repressor element. Although BHC110/LSD1 is capable of demethylating both K4 and K9 of histone H3, several observations have suggested the possibility for the presence of additional demethylases that employ other chemical mechanisms to demethylate lysine residues within proteins. Such observations include that (a) it is chemically impossible for BHC110/LSD1 to demethylate trimethylated H3 K4, but trimethylated K4 exists from yeast to human; (b) the number of homologues of BHC110/LSD1 are limited within a given organism relative to the large number of modified histone residues; and (c) BHC110/LSD1 homologues do not exist in S. cerevisiae, but H3 K4 can be mono-, di-, and trimethylated by COMPASS in vivo (8, 21, 29). In theory, the mono-, di-, and trimethylated lysine residues can be demethylated by hydroxylation of 78

O

dR

H N H

O

C O

H

+ N H

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Lysine

the methyl group. This reaction could be catalyzed by dioxygenases that use Fe(II) in their catalytic center, resulting in hydroxylation of the methyl group and subsequent demethylation (Figure 3). Such an observation has already been reported for the DNA repair demethyl­ ase AlkB (Figure 3a) (30, 31 ). AlkB is a 2‑oxyglutarate (2OG)-Fe(II)-dependent dioxygenase, which uses Fe(II) at its active site in order to activate a molecule of dioxygene to form a highly reactive oxoferryl species and to hydroxylate the methyl group of certain forms of damage-induced DNA methylation (30, 31 ). This oxidized product is highly unstable and can be readily released as formaldehyde, resulting in the release of the methyl group from DNA. Recently, it was suggested by Allshire and colleagues (32 ) that Epe1 (33 ), which is genetically required for the integrity of heterochromatin in Schizosaccharomyces pombe, could be a histone demethylase functioning via hydroxylation (Figure 3b). Allshire and colleagues have modeled Epe1 onto the known structure of the factor inhibiting hypoxia inducible factor, which belongs to the 2-OG-Fe(II)-dependent dioxygenase family (32 ). This family of enzymes can catalyze two electron oxidations using iron in their catalytic core and 2-OG as cosubstrate. Furthermore, sequence alignment of Epe1 and its related family members, the JmjC family, show that Epe1 and many of the JmjC-domain proteins share distinctive features of 2-OG-Fe(II)-dependent dioxygenases. Allshire and colleagues have therefore suggested that JmjC-domain proteins may function as protein demethylases capable of demethylating mono-, di- and trimethylated lysine residues within proteins. Indeed, a recent study by Tsukada et al. (34 ) demonstrated that JHDM1 (JmjC domain-containing histone demethylase 1), a protein w w w. a c s c h e m i ca l biology.org

conserved from yeast to human, specifically demethylates mono- or dimethylated histone H3 yet is not capable of demethylating trimethylated nucleosomal substrates either in vitro or in vivo. In search for enzymatic activity capable of demethylating nucleosomal substrates, Tsukada et al. (34 ) identified an activity in HeLa cell nuclear extracts capable of demethylating radio-labelled nucleosomal substrates methylated on K36 of histone H3. This enzymatic assay was set up to detect the release of radioactive formaldehyde from K36 methylated histone H3, which was enzymatically synthesized using Set2 and radioactive SAM. To increase the sensitivity of the assay, the radioactive formaldehyde was converted to radioactive 3,5-diacethyl-1,4-dihydro­ lutidine to facilitate its extraction in the organic phase away from the substrate. Using this assay, Tsukada et al. (34 ) have identified the F-box and leucine-rich repeat protein 11 (FBXL11) as the enzyme capable of demethylating K36 methylated histone H3. FBXL11 was identified in a bioinformatic search for F-box containing proteins (35 ). In addition to the F-box domain, the FBXL11 contains three leucine-rich repeats at its C‑terminal domain, CxxC and PHD domains at its center, and a JmjC domain towards its N-terminal domain. Mutational analyses of FBXL11 indicated that its JmjC domain is required for the demethylase activity associated with this protein and that the deletion of other domains such as the CxxC, PHD, and leucine-rich domains partially impair its activity. In their report, Allshire and colleagues predicted that amino acids required for coordinating Fe(II) within the dioxy­genases should be required for demethylase activity (32 ). Indeed, a single point mutation of histidine 212 within FBXL11, which is highly conserved among FBXL11 orthologues and is predicted to coordinate Fe(II) in the catalytic center of the enzyme, abolishes the demethylase activity associated with this enzyme. On the basis of the demethylase activity associated with FBXL11, Tsukada et al. (34 ) have renamed this protein JHDM1, which will be used to describe this enzyme henceforth. To determine the in vitro specificity of JHDM1, Tsukada et al. (34 ) tested the enzymatic activity of recombinant JHDM1 towards several methylated nucleosomal substrates differentially methylated on K4, K9, K27, K36, and K79 of histone H3, as well as K20 and R3 of histone H4. This study indicated that JHDM1 preferentially demethylates dimethylated www.acschemicalbiolog y.o rg

H3 K36 and is not able to demethylate trimethylated H3 K36. Furthermore, analysis of the consequence of JHDM1 overexpression in 293 T-cells indicated that this enzyme is capable of demethylating dimethylated H3 K36 in vivo as well. The study by Tsukada et al. (34) supports the proposed model that JmjC domain-containing proteins demethylate methylated lysine residues within histones via hydroxylation. An advantage of this reaction mechanism is that all forms of methylated lysine residues (mono-, di-, and trimethylated forms) can be demethylated. Enzymatic analyses of JHDM1 indicate that this enzyme is not capable of demethylating trimethylated nucleosomal substrates. This observation can be explained in several ways. First, JHDM1 may require the association with other factors to be able to demethylate trimethylated lysine residues. Regulation of the catalytic activity of an enzyme by its interacting factors within a complex is not unprecedented in chromatin biology. For example, several components of COMPASS, Cps60 and Cps40, are required for histone H3 K4 trimethylation by Set1/COMPASS (21 ). Therefore, it is also possible that interaction of other factors with JHDM1 regulates its enzymatic activity. A second possibility is that the activity of JHDM1 may be regulated via direct post­transcriptional modification of JHDM1. Since trimethylation for several different sites on H3 appears to have different patterns of localization than the corresponding mono- or dimethyl form, it is possible that JHDM1 demethylase activity may be regulated towards these sites. A third possibility is that demethylation of trimethylated histones Keywords Histone methyltransferases: Factors/proteins may require the presence of that transfer methyl groups from donor other modifications on the S-adenosylmethionine (SAM) to lysine or same or other histones. An arginine residues on histones. SET-domain: (Drosophila Su(var)3-9, Enhancer example for such mechanism of zeste (E(z)), and trithorax (trx)). A domain was shown by the activity found within a class of histone methyltransof Rad6/Bre1 in histone ferase that catalyzes lysine residue methylation of histones on the ε-nitrogen. H2B monoubiquitination COMPASS: (Complex Proteins Associated with and signaling for histone H3 Set1). A SET domain-containing complex methylation by COMPASS capable of mono- di- and tri-methylating lysine 4 of histone H3. Its human homo(29, 36, 37 ). A fourth poslogue, the MLL complex, is also found in a sibility is the catalytic pocket COMPASS-like complex capable of methylatof JHDM1 may be too small ing lysine 4 of histone H3. Histone demethylases: Factors/proteins that to accommodate trimethyl­ remove the methyl moiety from methylated ated histones, and other JmjC lysine or arginine residues on histones. domain-containing proteins VOL.1 NO. 2 • ACS CHEMICAL BIOLO GY

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JmjC domain-containing pro­teins demethylate methylated lysine residues within histones via hydroxylation

could function in this process. Finally, it is also feasible to consider that once lysine residues within histones are trimethylated, such marks are permanent and irreversible. Recent studies reported by Tsukada et al. (34 ) extend our knowledge regarding the mechanism of posttranslational modification of histones by methylation. This study demonstrated the existence of reaction mechanisms and cofactor requirements fundamentally different from previously reported histone demethylases. Because JmjC domain-containing proteins are conserved from yeast to human and large numbers of JmjC homologues exist within an organism, this class

of enzymes could be involved in modulating a diverse range of existing histone and protein modifications via methylation. Future genetic and biological studies aiming to define the role of JmjC domain-containing proteins in the regulation of gene expression should shed further light on the role of the JmjC superfamily in pathways involving lysine methylation in histones and other proteins.

References

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Acknowledgment: The authors would like to thank Kristy Wendt and Adam Wood for editorial assistance and critical reading of this review. The work in A.S.’s laboratory is supported by grants from the National Institutes of Health (2R01CA089455 and 1R01GM069905) and the American Cancer Society. A.S. is a Scholar of the Leukemia and Lymphoma Society.

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