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.     

组蛋白赖氨酸特异性去甲基化酶1 抑制剂的研究进展

组蛋白赖氨酸特异性去甲基化酶1（LSD1）能够催化氧化去除组蛋白H3K4 和H3K9 的单、双甲基,该酶在多种恶性肿瘤组织 中高度表达,与肿瘤的发生发展密切相关,是一个新兴的肿瘤治疗靶标。综述LSD1 的结构、催化机制以及近年来LSD1 抑制剂的研究进展。     

A highly specific mechanism of histone H3-K4 recognition by histone demethylase LSD1

Human lysine-specific demethylase (LSD1) is a chromatin-modifying enzyme that specifically removes methyl groups from mono- and dimethylated Lys4 of histone H3 (H3-K4). We used a combination of in vivo and in vitro experiments to characterize the substrate specificity and recognition by LSD1. Biochemical assays on histone peptides show that essentially all epigenetic modifications on the 21 N-terminal amino acids of histone H3 cause a significant reduction in enzymatic activity. Replacement of Lys4 with Arg greatly enhances binding affinity, and a histone peptide incorporating this mutation has a strong inhibitory power. Conversely, a peptide bearing a trimethylated Lys4 is only a weak inhibitor of the enzyme. Rapid kinetics measurements evidence that the enzyme is efficiently reoxidized by molecular oxygen with a second-order rate constant of 9.6x10(3) M-1 s-1, and that the presence of the reaction product does not greatly influence the rate of flavin reoxidation. In vivo experiments provide a correlation between the in vitro inhibitory properties of the tested peptides and their ability of affecting endogenous LSD1 activity. Our results show that epigenetic modifications on histone H3 need to be removed before Lys4 demethylation can efficiently occur. The complex formed by LSD1 with histone deacetylases 1/2 may function as a "double-blade razor" that first eliminates the acetyl groups from acetylated Lys residues and then removes the methyl group from Lys4. We suggest that after H3-K4 demethylation, LSD1 recruits the forthcoming chromatin remodelers leading to the introduction of gene repression marks.     

H3K4me3 demethylation by the histone demethylase KDM5C/JARID1C promotes DNA replication origin firing

DNA replication is a tightly regulated process that initiates from multiple replication origins and leads to the faithful transmission of the genetic material. For proper DNA replication, the chromatin surrounding origins needs to be remodeled. However, remarkably little is known on which epigenetic changes are required to allow the firing of replication origins. Here, we show that the histone demethylase KDM5C/JARID1C is required for proper DNA replication at early origins. JARID1C dictates the assembly of the pre-initiation complex, driving the binding to chromatin of the pre-initiation proteins CDC45 and PCNA, through the demethylation of the histone mark H3K4me3. Fork activation and histone H4 acetylation, additional early events involved in DNA replication, are not affected by JARID1C downregulation. All together, these data point to a prominent role for JARID1C in a specific phase of DNA replication in mammalian cells, through its demethylase activity on H3K4me3.     

LSD1 regulates the balance between self-renewal and differentiation in human embryonic stem cells

We identify LSD1 (lysine-specific demethylase 1; also known as KDM1A and AOF2) as a key histone modifier that participates in the maintenance of pluripotency through the regulation of bivalent domains, a chromatin environment present at the regulatory regions of developmental genes that contains both H3K4 di/trimethylation and H3K27 trimethylation marks. LSD1 occupies the promoters of a subset of developmental genes that contain bivalent domains and are co-occupied by OCT4 and NANOG in human embryonic stem cells, where it controls the levels of H3K4 methylation through its demethylase activity. Thus, LSD1 has a role in maintaining the silencing of several developmental genes in human embryonic stem cells by regulating the critical balance between H3K4 and H3K27 methylation at their regulatory regions.     

Taking LSD 1 to a new high

Histone modifications mediate changes in gene expression by altering the underlying chromatin structure or by serving as a binding platform to recruit other proteins. One such modification, histone methylation, was thought to be irreversible until last year when Shi and co-workers broke new ground with their discovery of a lysine-specific histone demethylase (LSD 1). They showed that LSD 1, a nuclear amine oxidase homolog, is a bona fide histone H3 lysine 4 demethylase (Shi et al., 2004). Now, a new study from published in a recent issue of Molecular Cell, together with two studies recently published by and in Nature, reveal that LSD 1's specificity and activity is in fact regulated by associated protein cofactors.     

Lysine-specific demethylase 1-mediated demethylation of histone H3 lysine 9 contributes to interleukin 1β-induced microsomal prostaglandin E synthase 1 expression in human osteoarthritic chondrocytes

Introduction

Microsomal prostaglandin E synthase 1 (mPGES-1) catalyzes the terminal step in the biosynthesis of PGE₂, a critical mediator in the pathophysiology of osteoarthritis (OA). Histone methylation plays an important role in epigenetic gene regulation. In this study, we investigated the roles of histone H3 lysine 9 (H3K9) methylation in interleukin 1β (IL-1β)-induced mPGES-1 expression in human chondrocytes.

Methods

Chondrocytes were stimulated with IL-1β, and the expression of mPGES-1 mRNA was evaluated using real-time RT-PCR. H3K9 methylation and the recruitment of the histone demethylase lysine-specific demethylase 1 (LSD1) to the mPGES-1 promoter were evaluated using chromatin immunoprecipitation assays. The role of LSD1 was further evaluated using the pharmacological inhibitors tranylcypromine and pargyline and small interfering RNA (siRNA)-mediated gene silencing. The LSD1 level in cartilage was determined by RT-PCR and immunohistochemistry.

Results

The induction of mPGES-1 expression by IL-1β correlated with decreased levels of mono- and dimethylated H3K9 at the mPGES-1 promoter. These changes were concomitant with the recruitment of the histone demethylase LSD1. Treatment with tranylcypromine and pargyline, which are potent inhibitors of LSD1, prevented IL-1β-induced H3K9 demethylation at the mPGES-1 promoter and expression of mPGES-1. Consistently, LSD1 gene silencing with siRNA prevented IL-1β-induced H3K9 demethylation and mPGES-1 expression, suggesting that LSD1 mediates IL-1β-induced mPGES-1 expression via H3K9 demethylation. We show that the level of LSD1 was elevated in OA compared to normal cartilage.

Conclusion

These results indicate that H3K9 demethylation by LSD1 contributes to IL-1β-induced mPGES-1 expression and suggest that this pathway could be a potential target for pharmacological intervention in the treatment of OA and possibly other arthritic conditions.     

Overexpression of a histone H3K4 demethylase, JMJ15, accelerates flowering time in Arabidopsis

The methylation of histone 3 lysine 4 (H3K4) is essential for gene activation. Flowering Locus C (FLC), an important flowering repressor, quantitatively regulates flowering time in Arabidopsis and its expression level is coincident with H3K4 trimethylation (H3K4me3) dynamics. The methylation state of FLC chromatin is determined by the balance between methylation and demethylation, which is mediated by histone methyltransferases and demethylases, respectively. However, little is known about the role of histone demethylase(s) in FLC regulation. Here, we characterized the biochemical activity and biological function of a novel JmjC domain-containing H3K4 demethylase, JMJ15, in Arabidopsis. JMJ15, which is a member of the H3K4 demethylase JARID1 family, displayed H3K4me3 demethylase activity both in vitro and in vivo. The mutation of JMJ15 did not produce an obvious phenotype; however, overexpression JMJ15 resulted in an obvious early flowering phenotype, which was associated with the repression of FLC level and reduction in H3K4me3 at the FLC locus, resulting in increased FT expression. Our results suggest that JMJ15 is a novel H3K4 demethylase, involved in the control of flowering time by demethylating H3K4me3 at FLC chromatin when it was overexpressed in Arabidopsis. KEY MESSAGE: Overexpression of a histone H3K4 demethylase, JMJ15, represses FLC expression by decreasing its chromatin H3K4me3 level, thereby controlling flowering time in Arabidopsis.     

Genome-wide studies of histone demethylation catalysed by the fission yeast homologues of mammalian LSD1

In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO) domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex. However, we find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) and both up- and down-regulates expression of different groups of genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1(+) gene. Using hyper-geometric probability comparisons we uncover genetic links between lysine-specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. The data presented here demonstrate that in fission yeast the SWIRM/PAO domain proteins Swm1 and Swm2 are associated in complexes that can remove methyl groups from lysine 9 methylated histone H3. In vitro, we show that bacterially expressed Swm1 also possesses lysine 9 demethylase activity. In vivo, loss of Swm1 increases the global levels of both H3K9me2 and H3K4me2. A significant accumulation of H3K4me2 is observed at genes that are up-regulated in a swm1 deletion strain. In addition, H3K9me2 accumulates at some genes known to be direct Swm1/2 targets that are down-regulated in the swm1Delta strain. The in vivo data indicate that Swm1 acts in concert with the HDAC Clr6 and the chromatin remodeller Hrp1 to repress gene expression. In addition, our in vitro analyses suggest that the H3K9 demethylase activity requires an unidentified post-translational modification to allow it to act. Thus, our results highlight complex interactions between histone demethylase, deacetylase and chromatin remodelling activities in the regulation of gene expression.