- Arginine demethylation
- Lysine demethylation
- LSD1 mediates histone demethylation
- JmjC domain containing proteins and histone demethylation
Histone methylation at lysine and arginine residues has been linked to a number of cellular processes including DNA repair, replication, transcriptional activation and repression (Kouzarides, 2007). Arginine residues can accept one or two methyl groups, the latter in a symmetric or asymmetric conformation (Rme1, Rme2s, Rme2a). Lysine residues can be labelled with one, two or three methyl groups (Kme1, Kme2, Kme3). Histone methylation was regarded as a more permanent mark compared to other histone modifications such as acetylation or phosphorylation (Bannister et al., 2002). But with the discovery of novel histone demethylases it is now considered a more dynamic modification.
2. Arginine demethylation
It was proposed that reversal of arginine methylation might be catalyzed by deiminases (Bannister et al., 2002). Members of the peptidyl arginine deiminase family deiminate arginine residues by converting them into citrulline (Nakashima et al., 2002). PADI4 is a member of this family and localizes to the nucleus. Therefore it was hypothesized that it may deiminate histones (Cuthbert et al., 2004; Wang et al., 2004). Incubation of PADI4 with bulk histones results in an increase in citrullination on H3 and H4. Furthermore PADI4 can target arginines found within these histones in either the me1 or unmodified state (Figure 1).
Histone citrullination has been linked to estrogen regulated transcription of the pS2 promoter where gene activity is regulated in a cyclic fashion. After the initial increase in transcription, a decrease in arginine methylation is observed as RNA polymerase II drops away from the promoter (Bauer et al., 2002; Metivier et al., 2003). This correlates with an increase in both PADI4 recruitment and citrullination. Therefore, citrullination may antagonize arginine methylation. The removal of methyl groups from arginine may directly repress transcription, or the conversion may indicate that citrullination is a repressive modification. Arguably, PADI4 does not complete full demethylation as it converts methyl-arginine to citrulline rather than an unmodified arginine. Therefore further processing by histone replacement or aminotransferases will be needed for complete demethylation (Bannister et al., 2002).
3. Lysine demethylation
Levels of lysine methylation are known to change during processes such as transcriptional regulation. Therefore it was proposed that specific enzymatic activity might remove the methyl groups (Bannister et al., 2002). Indeed recent work has confirmed the existence of enzymatic demethylation and two separate mechanisms of lysine demethylation have been demonstrated (Figure 2). Amine oxidation by LSD1 and hydroxylation by JmjC-domain containing proteins are novel histone modifying enzymes that can remove methyl groups on lysines (Shi et al., 2004; Tsukada et al., 2006).
4. LSD1 mediates histone demethylation
LSD1 (BHC110) contains a SWIRM domain that has been identified in a number of chromatin associated proteins, and an FAD-dependent amine oxidase domain (Shi et al., 2004). LSD1 removes the methyl group using FAD as a cofactor releasing hydrogen peroxide (Figure 2a). LSD1 needs a protonated hydrogen to enable conversion to the imine intermediate, therefore it only demethylates me2 or me1 modified lysines.
LSD1 is associated with complexes that function as both transcriptional repressors and activators (Metzger et al., 2005; Shi et al., 2004). It demethylates H3K4me2/me1 when associated with the Co-REST complex at neuronal genes, or, H3K9me2/me1 when associated with the androgen receptor (AR) (Shi et al., 2004; Metzger et al., 2005).
LSD1 is also thought to function in the organization of higher-order chromatin structure by two different mechanisms (Lan et al., 2007; Rudolph et al., 2007). The LSD1 homologs in S. pombe (spLsd1/2 also known as SWIRM1/2) exhibit H3K9me demethylase activity and are associated with heterochromatin boundaries and euchromatic promoters (Lan et al., 2007; Nicolas et al., 2006). Loss of spLsd1 induces heterochromatic propagation beyond normal regions. In addition a decrease in gene transcription is observed at adjacent sites, which correlates with an increase in H3K9me. The Drosophila homolog Su(var)3-3 demethylates H3K4me but is also important for heterochromatin formation (Rudolph et al., 2007). Demethylation of H3K4me1 and me2 is needed for subsequent H3K9me and heterochromatin formation.
5. JmjC domain containing proteins and histone demethylation
The Jmj-C domain containing proteins can be defined into seven subfamilies according to sequence similarity within the JmjC domain, and the presence of other domains in the full-length protein (Table 1) (Klose et al., 2006a). The JmjC-domain-containing histone demethylase proteins (JHDM) use a mechanism similar to that of AlkB that demethylates damaged DNA (Figure 1b)(Tsukada et al., 2006). Unlike LSD1, the enzymatic reaction catalyzed by the Jmj-C domain containing proteins is compatible with demethylation of tri-methyl lysine.
JHDM1A was the first histone demethylase with a JmjC domain to be isolated and is a founding member of the JHDM1 family (Tsukada et al., 2006). The homolog in S. cerevisae is Jhd1, and together they demonstrate H3K36me2/me1 demethylase activity. In addition to the JmjC domain, JHDM1 family members contain CXXC zing-finger and F-box domains. The enzyme capable of reversing tri-methylation was still to be identified, therefore the search continued.
This led to the discovery of the JHDM3/JMJD2 subfamily of which JMJD2A/ JHDM3A, JMJD2B, JMJD2C and JMJD2D are members. They demethylate H3K9me and H3K36me in either the me2 or me3 state, but there is no evidence that they demethylate me1 (Cloos et al., 2006; Fodor et al., 2006; Klose et al., 2006b; Whetstine et al., 2006). Subfamily members contain a JmjC and JmjN domain that are both required for catalytic activity, and tudor domains. Ectopic expression of JMJD2B and JMJD2C markedly decreases H3K9me3 and me2 levels at heterochromatin, delocalizing HP1 (Cloos et al., 2006; Fodor et al., 2006). Furthermore, JMJD2A is able to bind H3K4me via its tudor domain which could act as a recruiting mechanism (Huang et al., 2006).
The JARID subfamily contains the members JARID1A (RBP2), JARID1B (PLU-1) , JARID1C (SMCX) and JARID1D (SMCY). They target H3K4me3/me2 for demethylation (Iwase et al., 2007; Klose et al., 2007; Lee et al., 2007; Yamane et al., 2007). JARID1B is overexpressed in cancer cells and is thought to mediate increased cellular proliferation by repressing the transcription of cell growth inhibitors (Yamane et al., 2007). JARID1D can bind H3K9me and coupled with its H3K4me3/me2 demethylating activity could establish a repressive chromatin environment (Iwase et al., 2007).
UTX and JMJD3 of the UTX/UTY sub-family have recently been identified as H3K27me3/me2 demethylases (Agger et al., 2007). At the HOXB1 promoter during differentiation, UTX reduces H3K27me3 levels to activate gene expression. Furthermore the association of UTX with the H3K4me3 histone methyltransferase MLL2 suggests a model by which the removal of the repressive mark is coupled to gene activation (Issaeva et al., 2007).
JHDM2A is a member of the JHDM2 subfamily. It has recently been shown to possess H3K9me2/me1 demethylase activity (Yamane et al., 2006). JHDM2A associates with the androgen receptor (AR) and upon hormone treatment contributes to AR-mediated gene activation. This is likely due to reducing H3K9me levels similar to the action of LSD1.
Other sub-families include PHF2/PHF8 and JmjC domain only. Family members have not been shown to possess histone demethylase activity as yet. In addition to enzymatic demethylation, it has been proposed that histone methylation could be reversed by histone replacement or clipping of the tail (Bannister et al., 2002). The identification of several histone demethylases has clearly demonstrated that histone methylation is a reversible mark.
(a) Deimination of mono-methyl (me1) arginine and (b) non-methylated arginine into citrulline
(a) LSD1 demethylates H3K4me2/me1 via an amine oxidation reaction using FAD as a cofactor. The imine intermediate is hydrolyzed to an unstable carbinolamine that spontaneously degrades to release formaldehyde. (b) The JHDM proteins use alpha ketoglutarate and iron (Fe) as cofactors to hydroxylate the methylated substrate. Fe(II) in the active site, activates a molecule of dioxygen to form a highly reactive oxoferryl (Fe(IV)=O) species to react with the methyl group. The resulting carbinolamine spontaneously degrades to release formaldehye.
Table 1 Enzymes that demethylate histones
The enzymes identified that demethylate histones, subsequent subfamilies and specific substrates.
|Enzymatic family||Subfamily||Enzyme(s)||Specific activity|
H3R2, R8, R17, R26 H4R3
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- Bannister, A. J., Schneider, R., and Kouzarides, T. (2002). Histone methylation: dynamic or static? Cell 109, 801-806.
- Bauer, U. M., Daujat, S., Nielsen, S. J., Nightingale, K., and Kouzarides, T. (2002). Methylation at arginine 17 of histone H3 is linked to gene activation. EMBO Rep 3, 39-44.
- Cloos, P. A., Christensen, J., Agger, K., Maiolica, A., Rappsilber, J., Antal, T., Hansen, K. H., and Helin, K. (2006). The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3. Nature 442, 307-311.
- Cuthbert, G. L., Daujat, S., Snowden, A. W., Erdjument-Bromage, H., Hagiwara, T., Yamada, M., Schneider, R., Gregory, P. D., Tempst, P., Bannister, A. J., and
Kouzarides, T. (2004). Histone deimination antagonizes arginine methylation. Cell 118, 545-553.
- Fodor, B. D., Kubicek, S., Yonezawa, M., O'Sullivan, R. J., Sengupta, R., Perez-Burgos, L., Opravil, S., Mechtler, K., Schotta, G., and Jenuwein, T. (2006). Jmjd2b antagonizes H3K9 trimethylation at pericentric heterochromatin in mammalian cells. Genes Dev 20, 1557-1562.
- Huang, Y., Fang, J., Bedford, M. T., Zhang, Y., and Xu, R. M. (2006). Recognition of histone H3 lysine-4 methylation by the double tudor domain of JMJD2A. Science 312, 748-751.
- Issaeva, I., Zonis, Y., Rozovskaia, T., Orlovsky, K., Croce, C. M., Nakamura, T., Mazo, A., Eisenbach, L., and Canaani, E. (2007). Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth. Mol Cell Biol 27, 1889-1903.
- Iwase, S., Lan, F., Bayliss, P., de la Torre-Ubieta, L., Huarte, M., Qi, H. H., Whetstine, J. R., Bonni, A., Roberts, T. M., and Shi, Y. (2007). The X-linked mental retardation gene SMCX/JARID1C defines a family of histone H3 lysine 4 demethylases. Cell 128, 1077-1088.
- Klose, R. J., Kallin, E. M., and Zhang, Y. (2006a). JmjC-domain-containing proteins and histone demethylation. Nat Rev Genet 7, 715-727.
- Klose, R. J., Yamane, K., Bae, Y., Zhang, D., Erdjument-Bromage, H., Tempst, P., Wong, J., and Zhang, Y. (2006b). The transcriptional repressor JHDM3A demethylates trimethyl histone H3 lysine 9 and lysine 36. Nature 442, 312-316.
- Klose, R. J., Yan, Q., Tothova, Z., Yamane, K., Erdjument-Bromage, H., Tempst, P., Gilliland, D. G., Zhang, Y., and Kaelin, W. G., Jr. (2007). The retinoblastoma binding protein RBP2 is an H3K4 demethylase. Cell 128, 889-900.
Kouzarides, T. (2007). Chromatin modifications and their function. Cell 128, 693-705.
- Lan, F., Zaratiegui, M., Villen, J., Vaughn, M. W., Verdel, A., Huarte, M., Shi, Y., Gygi, S. P., Moazed, D., Martienssen, R. A., and Shi, Y. (2007). S. pombe LSD1 homologs regulate heterochromatin propagation and euchromatic gene transcription. Mol Cell 26, 89-101.
- Lando, D., Peet, D. J., Gorman, J. J., Whelan, D. A., Whitelaw, M. L., and Bruick, R. K. (2002). FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. Genes Dev 16, 1466-1471.
- Lee, M. G., Norman, J., Shilatifard, A., and Shiekhattar, R. (2007). Physical and functional association of a trimethyl H3K4 demethylase and Ring6a/MBLR, a polycomb-like protein. Cell 128, 877-887.
- Metivier, R., Penot, G., Hubner, M. R., Reid, G., Brand, H., Kos, M., and Gannon, F. (2003). Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115, 751-763.
- Metzger, E., Wissmann, M., Yin, N., Muller, J. M., Schneider, R., Peters, A. H., Gunther, T., Buettner, R., and Schule, R. (2005). LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature 437, 436-439.
- Nakashima, K., Hagiwara, T., and Yamada, M. (2002). Nuclear localization of peptidylarginine deiminase V and histone deimination in granulocytes. J Biol Chem 277, 49562-49568.
- Nicolas, E., Lee, M. G., Hakimi, M. A., Cam, H. P., Grewal, S. I., and Shiekhattar, R. (2006). Fission yeast homologs of human histone H3 lysine 4 demethylase regulate a common set of genes with diverse functions. J Biol Chem 281, 35983-35988.
- Rudolph, T., Yonezawa, M., Lein, S., Heidrich, K., Kubicek, S., Schafer, C., Phalke, S., Walther, M., Schmidt, A., Jenuwein, T., and Reuter, G. (2007). Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3. Mol Cell 26, 103-115.
- Shi, Y., Lan, F., Matson, C., Mulligan, P., Whetstine, J. R., Cole, P. A., Casero, R. A., and Shi, Y. (2004). Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119, 941-953.
- Tsukada, Y., Fang, J., Erdjument-Bromage, H., Warren, M. E., Borchers, C. H., Tempst, P., and Zhang, Y. (2006). Histone demethylation by a family of JmjC domain-containing proteins. Nature 439, 811-816.
- Wang, Y., Wysocka, J., Sayegh, J., Lee, Y. H., Perlin, J. R., Leonelli, L., Sonbuchner, L. S., McDonald, C. H., Cook, R. G., Dou, Y., et al. (2004). Human PAD4 regulates histone arginine methylation levels via demethylimination. Science 306, 279-283.
- Whetstine, J. R., Nottke, A., Lan, F., Huarte, M., Smolikov, S., Chen, Z., Spooner, E., Li, E., Zhang, G., Colaiacovo, M., and Shi, Y. (2006). Reversal of histone lysine trimethylation by the JMJD2 family of histone demethylases. Cell 125, 467-481.
- Yamane, K., Tateishi, K., Klose, R. J., Fang, J., Fabrizio, L. A., Erdjument-Bromage, H., Taylor-Papadimitriou, J., Tempst, P., and Zhang, Y. (2007). PLU-1 is an H3K4 demethylase involved in transcriptional repression and breast cancer cell proliferation. Mol Cell 25, 801-812.
- Yamane, K., Toumazou, C., Tsukada, Y., Erdjument-Bromage, H., Tempst, P., Wong, J., and Zhang, Y. (2006). JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor. Cell 125, 483-495.