A crack in histone lysine methylation

Histone lysine methylation is regarded as a very stable modification with important functions in epigenetic gene control and for organizing chromatin domains. While more robust modifications of the chromatin template are essential to stabilize epigenetic information, there is now the first evidence for a histone lysine demethylase that reverts an activating methyl mark to the unmodified state (Shi et al., 2004 [this issue of Cell]).     

The many faces of histone lysine methylation

Diverse post-translational modifications of histone amino termini represent an important epigenetic mechanism for the organisation of chromatin structure and the regulation of gene activity. Within the past two years, great progress has been made in understanding the functional implications of histone methylation; in particular through the characterisation of histone methyltransferases that direct the site-specific methylation of, for example, lysine 9 and lysine 4 positions in the histone H3 amino terminus. All known histone methyltransferases of this type contain the evolutionarily conserved SET domain and appear to be able to stimulate either gene repression or gene activation. Methylation of H3 Lys9 and Lys4 has been visualised in native chromatin, indicating opposite roles in structuring repressive or accessible chromatin domains. For example, at the mating-type loci in Schizosaccharomyces pombe, at pericentric heterochromatin and at the inactive X chromosome in mammals, striking differences between these distinct marks have been observed. H3 Lys9 methylation is also important to direct additional epigenetic signals such as DNA methylation--for example, in Neurospora crassa and in Arabidopsis thaliana. Together, the available data strongly establish histone lysine methylation as a central modification for the epigenetic organisation of eukaryotic genomes.     

The epigenetic magic of histone lysine methylation

Epigenetic mechanisms control eukaryotic development beyond DNA-stored information. There are several pathways, including histone tail modifications, histone variant incorporation, nucleosome remodelling, DNA methylation and noncoding RNAs that together all contribute to the dynamic 'make-up' of chromatin under distinct developmental options. The histone tail modifications are most variable and over 50 marks have by now been mapped. While the majority of these modifications are transient, histone lysine methylation and, in particular, a histone lysine tri-methyl state has been regarded as a more robust signal, consistent with proposed roles to impart long-term epigenetic memory. Based on the paradigm of SET-domain histone lysine methyltransferases (HMTases) and chromo-domain adaptor proteins, and in conjunction with the Sir Hans Krebs Medal 2005, I describe here my personal view on the discovery of the first HMTase in 2000, and the subsequent advances on the biology of histone lysine methylation. This discovery has changed my scientific career and significantly contributed to a better understanding of epigenetic control, with important implications for heterochromatin formation, X inactivation, Polycomb group silencing and novel insights into stem cell research, nuclear reprogramming and cancer.     

Trimethylation of histone H3 lysine 4 impairs methylation of histone H3 lysine 9

Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4^me3) while heterochromatin contains the H3K9^me3 marks. The H3K9^me3 modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4^me3. In addition, we show that SETDB1 fails to methylate substrates containing the H3K4^me3 mark. Likewise, the functionally related H3K9 KMTs G9A, GLP, and SUV39H1 also fail to bind and to methylate H3K4^me3 substrates. Accordingly, we provide in vivo evidence that H3K9^me2-enriched histones are devoid of H3K4^me2/3 and that histones depleted of H3K4^me2/3 have elevated H3K9^me2/3. The correlation between the loss of interaction of these KMTs with H3K4^me3 and concomitant methylation impairment leads to the postulate that, at least these four KMTs, require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1α. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4^me3 and H3K9^me3. Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners.     

Chemical biology and pharmacology of histone lysine methylation inhibitors

Histone lysine methylation is a post-translational modification that plays a key role in the epigenetic regulation of a broad spectrum of biological processes. Moreover, the dysregulation of histone lysine methyltransferases (KMTs) has been implicated in the pathogenesis of several diseases particularly cancer. Due to their pathobiological importance, KMTs have garnered immense attention over the last decade as attractive therapeutic targets. These endeavors have culminated in tens of chemical probes that have been used to interrogate many aspects of histone lysine methylation. Besides, over a dozen inhibitors have been advanced to clinical trials, including the EZH2 inhibitor tazemetostat approved for the treatment of follicular lymphoma and advanced epithelioid sarcoma. In this Review, we highlight the chemical biology and pharmacology of KMT inhibitors and targeted protein degraders focusing on the clinical development of EZH1/2, DOT1L, Menin-MLL, and WDR5-MLL inhibitors. We also briefly discuss the pharmacologic targeting of other KMTs.     

Cancer-associated 2-oxoglutarate analogues modify histone methylation by inhibiting histone lysine demethylases

Histone lysine demethylases (KDMs) are 2-oxoglutarate-dependent dioxygenases (2-OGDDs) that regulate gene expression by altering chromatin structure. Their dysregulation has been associated with many cancers. We set out to study the catalytic and inhibitory properties of human KDM4A, KDM4B, KDM5B, KDM6A and KDM6B, aiming in particular to reveal which of these enzymes are targeted by cancer-associated 2-oxoglutarate (2-OG) analogues. We used affinity-purified insect cell-produced enzymes and synthetic peptides with trimethylated lysines as substrates for the in vitro enzyme activity assays. In addition, we treated breast cancer cell lines with cell-permeable forms of 2-OG analogues and studied their effects on the global histone methylation state. Our data show that KDMs have substrate specificity. Among the enzymes studied, KDM5B had the highest affinity for the peptide substrate but the lowest affinity for the 2-OG and the Fe^2 + cosubstrate/cofactors. R-2-hydroxyglutarate (R-2HG) was the most efficient inhibitor of KDM6A, KDM4A and KDM4B, followed by S-2HG. This finding was supported by accumulations of the histone H3K9me3 and H3K27me3 marks in cells treated with the cell-permeable forms of these compounds. KDM5B was especially resistant to inhibition by R-2HG, while citrate was the most efficient inhibitor of KDM6B. We conclude that KDM catalytic activity is susceptible to inhibition by tumorigenic 2-OG analogues and suggest that the inhibition of KDMs is involved in the disease mechanism of cancers in which these compounds accumulate, such as the isocitrate dehydrogenase mutations.     

Probe the function of histone lysine 36 methylation using histone H3 lysine 36 to methionine mutant transgene in mammalian cells

Chondroblastoma is a cartilaginous tumor that typically arises under 25 y of age (80%). Recent studies have identified a somatic and heterozygous mutation at the H3F3B gene in over 90% chondroblastoma cases, leading to a lysine 36 to methionine replacement (H3.3K36M). In human cells, H3F3B gene is one of 2 genes that encode identical H3.3 proteins. It is not known how H3.3K36M mutant proteins promote tumorigenesis. We and others have shown that, the levels of H3K36 di- and tri-methylation (H3K36me2/me3) are reduced dramatically in chondroblastomas and chondrocytes bearing the H3.3K36M mutation. Mechanistically, H3.3K36M mutant proteins inhibit enzymatic activity of some, but not all H3K36 methyltransferases. Chondrocytes harboring the same H3F3B mutation exhibited the cancer cell associated phenotypes. Here, we discuss the potential effects of H3.3K36M mutation on epigenomes including H3K36 and H3K27 methylation and cellular phenotypes. We suggest that H3.3K36M mutant proteins alter epigenomes of specific progenitor cells, which in turn lead to cellular transformation and tumorigenesis.     

DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis

We propose a model for heterochromatin assembly that links DNA methylation with histone methylation and DNA replication. The hypomethylated Arabidopsis mutants ddm1 and met1 were used to investigate the relationship between DNA methylation and chromatin organization. Both mutants show a reduction of heterochromatin due to dispersion of pericentromeric low-copy sequences away from heterochromatic chromocenters. DDM1 and MET1 control heterochromatin assembly at chromocenters by their influence on DNA maintenance (CpG) methylation and subsequent methylation of histone H3 lysine 9. In addition, DDM1 is required for deacetylation of histone H4 lysine 16. Analysis of F(1) hybrids between wild-type and hypomethylated mutants revealed that DNA methylation is epigenetically inherited and represents the genomic imprint that is required to maintain pericentromeric heterochromatin.     

Relationship between occupational aluminium exposure and histone lysine modification through methylation

BackgroundAluminium is an environmental neurotoxin to which human beings are extensively exposed. However, the molecular mechanism of aluminium toxicity remains unclear.MethodsThe changes in cognitive function of aluminum exposed workers under long-term occupational exposure were evaluated, and the relationship between cognitive changes, plasma memory related BDNF and EGR1 protein expression, and variations of epigenetic markers H3K4me3, H3K9me2, H3K27me3 expression levels in blood was explored.ResultsMMSE, DSFT, DST scores in cognitive function and the levels of plasma BDNF and EGR1 protein expression decreased with the increase of blood aluminum level. H3K4me3, H3K9me2, H3K27me3 expression levels in peripheral blood lymphocytes of aluminum exposed workers were statistically different (all P<0.05). H3K4me3, H3K9me2 and H3K27me3 expression levels in lymphocytes were correlated with blood aluminum level. BDNF, EGR1 protein level and H3K4me3, H3K9me2, H3K27me3 expression levels have different degrees of correlation. There was a linear regression relationship between plasma BDNF, H3K4me3 and H3K9me2. H3K9me2 had a greater effect on BDNF than H3K4me3. There is a linear regression relationship between EGR1, H3K4me3 and H3K27me3, and the influence of H3K4me3 on EGR1 is greater than that of H3K27me3 on EGR1.ConclusionAlummnum may regulate the expression of BDNF and EGR1 by regulating H3K4me3, H3K27me3 and H3K9me2, and affect the cognitive function of workers by affecting the expression of BDNF and EGR1.