A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading
Residue-specific posttranslational modifications of histones play essential roles in every aspect of DNA metabolism such as transcription , DNA replication , recombination , and DNA damage repair ( Lee et al . , 2010 ) . Among these modifications , lysine methylation is the most intriguing due to its remarkable specificity ( Greer and Shi , 2012 ) . For example , methylation of histone H3 lysine 9 ( H3K9me ) is critical for the assembly of constitutive heterochromatin at repetitive DNA elements , and H3K27me is essential for the formation of facultative heterochromatin at developmentally regulated genes . The methylation is controlled by specific histone methyltransferases and histone demethylases , mutations of which have been associated with many human diseases ( Greer and Shi , 2012; Herz et al . , 2013 ) . In comparison , histone genes are not prime suspects for disease-associated mutations , as their high copy number masks the effects of any recessive changes . However , recent high throughput sequencing analyses identified a high incidence of somatic mutations of critical histone lysines into methionine ( K-to-M ) in multiple cancers ( Schwartzentruber et al . , 2012; Sturm et al . , 2012; Wu et al . , 2012; Behjati et al . , 2013; Shah et al . , 2014 ) . The most prominent examples include the association of a K27M mutation in H3 . 3 ( a variant form of H3 ) with pediatric high-grade glioblastoma , a highly aggressive brain tumor , and the association of a K36M mutation in H3 . 3 with chondroblastoma , a tumor of the bone . The high frequency of such mutations indicates that they are driver mutations of tumorigenesis . The role of histone K-to-M mutations in cancer is under intense investigation , but the mechanism is not well understood and controversial . For example , cells containing H3 . 3K27M show reduced H3K27 trimethylation ( H3K27me3 ) levels on wild type histones , and an H3K27M peptide inhibits polycomb repressive complex 2 ( PRC2 ) -mediated H3K27 methylation in vitro , suggesting that these mutations function as inhibitors of H3K27 methyltransferases ( Bender et al . , 2013; Chan et al . , 2013a; Lewis et al . , 2013; Venneti et al . , 2013 ) . However , ChIP-seq analyses of cancer cells containing H3 . 3K27M show that in addition to a global reduction of H3K27me3 , a large number of ectopic H3K27me3 peaks are also detected , arguing against a simple inhibitory model ( Bender et al . , 2013; Chan et al . , 2013a ) . Other histone K-to-M mutations also dominantly block the methylation of their corresponding lysine residues ( Chan et al . , 2013b; Lewis et al . , 2013; Herz et al . , 2014; Lu et al . , 2016; Fang et al . , 2016 ) , suggesting a similar mechanism of function . Interestingly , mass spectrometry analyses of H3K9M-containing nucleosomes purified from human cells show increased association of an H3K9 demethylase KDM3B , suggesting that recruitment of a demethylase contributes to the effects of H3K9M on H3K9 methylation ( Herz et al . , 2014 ) . One of the difficulties in reconciling these results is the complication of the mammalian system . First , there are multiple copies of genes encoding histone H3 and H3 . 3 ( Maze et al . , 2014 ) , but it is not clear whether each gene is equivalent in its expression profile . Second , there are multiple methyltransferases that modify each lysine ( Greer and Shi , 2012 ) , and they are differentially inhibited by K-to-M mutations ( Fang et al . , 2016 ) . Another difficulty in studying the mechanism of histone K-to-M mutations is the paucity of structural data that explains how the methionine substitution functions to inhibit histone methyltransferases . There are two recent structures of an H3K27M peptide in association with the H3K27 methyltransferase PRC2 , which give conflicting interaction details . The structure of Chaetomium thermophilum PRC2 reveals that an arginine residue ( R26 ) adjacent to H3K27M occupies the expected lysine-binding channel ( Jiao and Liu , 2015 ) , whereas the structure of human PRC2 indicates that the methionine side chain occupies this channel ( Justin et al . , 2016 ) . There is currently no structure that examines the interaction between other K-to-M mutations and their corresponding histone methyltransferases . The fission yeast Schizosaccharomyces pombe shares highly conserved chromatin modification pathways with mammals , but has key advantages such as relatively facile genetics and single representatives of most key families of mammalian chromatin-modifying factors ( Wood et al . , 2002 ) . For example , a single histone H3K9 methyltransferase Clr4 is critical for H3K9me across the entire genome and regulates the assembly of heterochromatin ( Rea et al . , 2000; Nakayama et al . , 2001; Cam et al . , 2005 ) . Moreover , there are only three copies of histone H3 genes with identical protein sequences ( Mellone et al . , 2003 ) . The simplicity of the fission yeast system allows us to demonstrate that H3K9M blocks H3K9 methylation through trapping of Clr4 at heterochromatin nucleation centers to prevent its spreading . In addition , H3K9M interacts directly with Clr4 and the interaction is dramatically enhanced by S-adenosylmethionine ( SAM ) , the methyl donor for histone methyltransferases . Moreover , we determined a high-resolution crystal structure of H3K9M in complex with an H3K9 methyltransferase G9a and SAM , which provides a detailed mechanism for the role of methionine in trapping histone methyltransferases . These results provide clear mechanistic insights of histone H3K9M in regulating H3K9me levels .
Fission yeast contains three histone H3 genes: hht1+ , hht2+ , and hht3+ , which produce identical proteins ( Mellone et al . , 2003 ) . We generated Flag-tagged versions of each of these genes at their endogenous chromosomal loci . Western blot analyses showed that the three proteins were expressed at similar levels ( Figure 1—figure supplement 1 ) . We then introduced the K9M mutation into each histone H3 gene at its endogenous chromosome locus . The three mutant histone H3s were also expressed at similar levels ( Figure 1—figure supplement 1 ) . In fission yeast , heterochromatin is mainly present at pericentric region , subtelomeres , and the silent mating-type region , which all contain a similar repetitive DNA element ( Grewal and Jia , 2007 ) . These regions contain high levels of histone H3K9 methylation and the transcription of the underlying repeats is repressed . Reporter genes inserted within these repeats , such as otr::ura4+ inserted at the dh repeat of pericentric region and Kint2::ura4+ inserted at the cenH repeat of the mating-type region ( Figure 1A ) ( Allshire et al . , 1995; Grewal and Klar , 1997 ) , are silenced , resulting in cells that only grow weakly on medium without uracil . These cells grow well on medium containing 5-fluoroorotic acid ( FOA ) , which is converted to a toxic form ( 5-flurouracil ) in cells expressing ura4+ ( Figure 1B , and Figure 1—figure supplement 2 ) . Loss of heterochromatin , such as in clr4∆ , results in the expression of these reporters and robust cell growth on medium without uracil and decreased growth on FOA-containing medium . Introducing a K9M mutation into any H3 gene resulted in silencing defects similar to that of clr4∆ , even though there were two other wild type histone H3 genes present ( Figure 1B , and Figure 1—figure supplement 2 ) . Therefore , we used only hht3-K9M for all subsequent analyses . In contrast , an hht3-K9R mutation ( which is expected to abolish methylation of lysine 9 only on Hht3 ) had little effect on gene silencing ( Figure 1—figure supplement 2 ) , suggesting that the effect of H3K9M is specific . 10 . 7554/eLife . 17903 . 003Figure 1 . H3K9M affects H3K9me and heterochromatin assembly . ( A ) Schematic diagrams of reporter genes used . Red bars indicate primers used for ChIP-qPCR and qRT-PCR analyses . ( B ) Left , ten-fold serial dilution analyses of indicated yeast strains grown on indicated media to measure the expression of otr::ura4+ . Right , ChIP analyses of H3K9me3 and H3K9me2 levels at heterochromatin-associated repeats , normalized to act1+ , and qRT-PCR analysis of repeat transcript levels , normalized to act1+ . Data presented is mean ± s . d . of four technical repeats . ( C ) ChIP-seq analysis of H3K9me3 levels across the genome . Data presented is average of two sequencing runs . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 00310 . 7554/eLife . 17903 . 004Figure 1—figure supplement 1 . Western blot analyses of histone H3 and Tubulin levels . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 00410 . 7554/eLife . 17903 . 005Figure 1—figure supplement 2 . Ten-fold serial dilution analyses of indicated yeast strains grown on indicated media to measure the expression of otr::ura4+ . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 00510 . 7554/eLife . 17903 . 006Figure 1—figure supplement 3 . ChIP-seq analyses of H3K9me3 levels at centromeres and telomeres . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 006 ChIP analyses showed that H3K9me3 levels were abolished and H3K9me2 levels were significantly reduced at the heterochromatic dh and cenH repeats ( Figure 1B ) . Moreover , transcripts of these repeats were dramatically increased ( Figure 1B ) . Further ChIP-seq analyses showed that H3K9me3 was reduced to background levels across the entire genome , similar to clr4∆ ( Figure 1C and Figure 1—figure supplement 3 ) . Unlike the presence of additional H3K27me3 peaks in H3 . 3K27M containing mammalian cells ( Bender et al . , 2013; Chan et al . , 2013a ) , we did not detect any additional peaks of H3K9me3 in hht3-K9M cells . Therefore , H3K9M functions dominant-negatively to regulate H3K9 methylation across the genome . It has been shown that an H3K27M peptide inhibits the enzymatic activity of the H3K27 methyltransferase PRC2 in vitro ( Lewis et al . , 2013 ) . To examine whether H3K9M also similarly inhibits the enzymatic activity of Clr4 , we performed in vitro histone methyltransferase assays using a recombinant Clr4 SET domain ( amino acids 190–490 ) , recombinant mono-nucleosomes , and 3H labelled S-adenosylmethionine ( SAM ) . The incorporation of radioactively labelled methyl group into histone H3 was significantly reduced in the presence of an H3K9M peptide , suggesting that H3K9M directly inhibits the enzymatic activity of Clr4 in vitro ( Figure 2A ) . 10 . 7554/eLife . 17903 . 007Figure 2 . H3K9M traps Clr4 at nucleation centers to prevent heterochromatin spreading . ( A ) In vitro histone methyltransferase assay with recombinant Clr4 SET domain and recombinant nucleosomes . ( B ) Top , a schematic diagram of the 3xgbs-ade6+ reporter . Left , ten-fold serial dilution analyses of indicated yeast strains grown on low adenine media ( YE ) to measure the expression of 3xgbs-ade6+ . Right , ChIP analyses of H3K9me3 levels at 3xgbs , normalized to act1+ . Data presented is mean ± s . d . of four technical replicates . ( C ) ChIP analysis of Clr4 levels at heterochromatin associated repeats , normalized to act1+ . Data presented is mean ± s . d . of four technical replicates . ( D ) Western blot analysis of Flag-Clr4 protein levels . ( E ) Live cell imaging of cells expressing GFP-Clr4 . Scale bar is 1 μm . ( F ) Top , a schematic diagram of the mating type region , with shaded area representing heterochromatin . The PCR fragments used for qPCR analyses are labeled . Bottom , ChIP analysis of Flag-Clr4 , H3K9me3 , and H3K9me2 levels across the mating type region , normalized to act1+ . Data presented is mean ± s . d . of four technical replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 00710 . 7554/eLife . 17903 . 008Figure 2—figure supplement 1 . Yeast strains were grown on low adenine media ( YE ) to measure the expression of 3xgbs-ade6+ . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 00810 . 7554/eLife . 17903 . 009Figure 2—figure supplement 2 . H3K9M affects mating-type switching . ( A ) Schematic diagram of the mating-type region . mat2P and mat3M embedded with heterochromatin are used as donors to replace sequence at mat1 , which determine the mating-type of the cell . ( B ) A competitive PCR analysis to show the relative amount of cells of each mating type . ( C ) Iodine staining of indicated colonies . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 009 To test the inhibitory effects of H3K9M in vivo , we used an ade6+ reporter that is adjacent to 3 copies of Gal4-binding sites ( 3xgbs-ade6+ ) . In wild type cells , the reporter gene is expressed , resulting in the formation of white colonies on low adenine medium ( YE ) . In contrast , artificially targeting Clr4 through a Gal4 DNA binding domain silences the reporter , resulting in red colonies ( Kagansky et al . , 2009 ) . In hht3-K9M cells , the reporter gene was not silenced and no H3K9me3 was detected ( Figure 2B and Figure 2—figure supplement 1 ) , suggesting that H3K9M inhibits the enzymatic activity of Clr4 in vivo . Surprisingly , ChIP analyses showed that Clr4 protein levels were much higher at dh and cenH repeats in hht3-K9M cells ( Figure 2C ) , even though overall Clr4 protein levels were unchanged ( Figure 2D ) . Consistently , live-cell imaging analyses showed stronger GFP-Clr4 foci in hht3-K9M cells ( Figure 2E ) . These results suggest that hht3-K9M does not affect the targeting of Clr4 to chromatin , but instead traps Clr4 at heterochromatic locations . Heterochromatin formation is divided into two distinct steps: initiation and spreading ( Wang et al . , 2014 ) . First , Clr4 is recruited to nucleation centers to initiate H3K9me . H3K9me recruits additional Clr4 , through the chromo domain of Clr4 and Swi6 proteins , leading to methylation of adjacent nucleosomes ( Hall et al . , 2002; Zhang et al . , 2008a; Al-Sady et al . , 2013 ) . The repetition of such cycles promotes heterochromatin spreading across large chromosome domains . Both dh and cenH , which showed higher levels of Clr4 , are heterochromatin nucleation centers ( Hall et al . , 2002 ) . To investigate the effects of H3K9M on heterochromatin spreading , we measured Clr4 , H3K9me3 , and H3K9me2 levels at the silent mating-type region , which has been extensively used to examine heterochromatin initiation and spreading ( Hall et al . , 2002 ) . At this region , cenH recruits Clr4 , which then spreads into a 20 kb domain that are marked by two inverted repeats ( IRL and IRR ) ( Figure 2F ) . We found that in hht3-K9M cells , Clr4 was highly enriched at cenH , but not at the surrounding regions ( Figure 2F ) . Furthermore , H3K9me3 was completely abolished across the entire locus , whereas low levels of H3K9me2 were restricted to cenH ( Figure 2F ) . Therefore , H3K9M traps Clr4 at nucleation centers and prevents its spreading into neighboring regions . Heterochromatin at the silent mating-type locus is essential for the correct choice of donors during mating-type switching , when heterochromatin-embedded mat2P or mat3M are used as a donor to replace the DNA sequence at mat1 ( Jia et al . , 2004 ) ( Figure 2—figure supplement 2A ) . Loss of heterochromatin leads to the predominant use of mat3M as a donor , the accumulation of the M mating type within a switching competent population , and reduced mating efficiency ( indicated by lightly iodine-stained colonies on medium that induces mating and meiosis ) ( Jia et al . , 2004 ) ( Figure 2—figure supplement 2B and C ) . As expected , hht3-K9M cells were predominantly of the M mating type and formed lightly iodine-stained colonies similar to clr4∆ cells ( Figure 2—figure supplement 2B and C ) . One possible reason for the increased levels of Clr4 at heterochromatin nucleation centers is that Clr4 has a higher affinity for H3K9M . However , the fact that H3K9M-containing nucleosomes outside of heterochromatin failed to trap Clr4 suggests that the interaction between Clr4 and H3K9M is regulated . Clr4 contains a catalytic SET domain as well as a chromo domain that interacts with H3K9me . We found that the chromo domain of Clr4 ( 1–190 ) interacted with an H3K9me3 peptide and to a lesser extent with an H3K9me2 peptide , but showed no interaction with the H3K9M peptide ( Figure 3A ) . In addition , none of the other H3K9me-interacting chromo domains in fission yeast , including Swi6 , Chp2 , and Chp1 ( Sadaie et al . , 2008; Schalch et al . , 2009 ) , interacted with the H3K9M peptide ( Figure 3—figure supplement 1 ) . Interestingly , the SET domain of Clr4 ( 190–490 ) showed stronger interaction with an H3K9M peptide than a wild type histone H3 tail peptide in a histone methyltransferase buffer ( 50 mM Tris , pH 8 . 0 , 1 mM EDTA , 50 mM NaCl , 1 mM DTT ) supplemented with 100 μM SAM ( Figure 3B ) . In addition , such interaction was significantly reduced in the absence of SAM ( Figure 3B ) . The binding is not covalent as Clr4 was removed under stringent washing conditions ( data not shown ) . Moreover , the binding is significantly reduced when S-adenosyl-homocysteine ( SAH ) was used in place of SAM ( Figure 3C ) . Furthermore , the SET domain of human H3K9 methyltransferase G9a , which is inhibited by H3K9M in vitro ( Lewis et al . , 2013 ) , also showed stronger binding to the H3K9M peptide than the wild type H3 tail peptide in the presence of SAM ( Figure 3D ) , suggesting a similar binding mechanism . 10 . 7554/eLife . 17903 . 010Figure 3 . H3K9M interacts directly with H3K9 methyltransferases . ( A ) Binding assays with recombinant Clr4 chromo domain and histone peptides in PBS buffer . ( B , C ) Binding assays with recombinant Clr4 SET domain and histone peptides in histone methyltransferase buffer supplemented with SAM or SAH . ( D ) Binding assays with recombinant G9a SET domain and histone peptides in histone methyltransferase buffer supplemented with SAM . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 01010 . 7554/eLife . 17903 . 011Figure 3—figure supplement 1 . Binding assays with recombinant GST-tagged chromo domains and histone peptides in PBS buffer . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 01110 . 7554/eLife . 17903 . 012Figure 3—figure supplement 2 . H3K9M functions through the SET domain of Clr4 . ( A ) ChIP analyses of H3K9me2 and H3K9me3 levels at cenH , normalized to act1 . Data presented is mean ± s . d . of four technical repeats . ( B , C ) ChIP analysis of Clr4 levels at cenH , normalized to act1 . Data presented is mean ± s . d . of four technical repeats . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 012 The binding data suggests that H3K9M exerts its effects through the SET domain of Clr4 . To rule out the contribution of the chromo domain of Clr4 to the trapping of Clr4 by H3K9M in vivo , we generated a W31G mutation within the chromo domain , which abolishes its interaction with H3K9me3 , but does not affect the enzymatic activity of Clr4 in vitro ( Nakayama et al . , 2001; Zhang et al . , 2008a ) . Similar to previous observations , introducing the W31G mutation at the endogenous clr4+ locus resulted in a severe reduction of H3K9me3 , but had little effects on H3K9me2 at cenH , the heterochromatin nucleation center of the silent mating-type region ( Figure 3—figure supplement 2A ) ( Al-Sady et al . , 2013 ) . The localization of Clr4 to cenH was severely reduced in clr4-W31G cells , due to the dependence of H3K9me3 for the stable association of Clr4 with heterochromatin ( Figure 3—figure supplement 2B ) ( Zhang et al . , 2008a ) . Consistent with the idea that H3K9M exerts its effects through the Clr4 SET domain , Clr4-W31G showed enhanced localization at cenH in hht3-K9M cells ( Figure 3—figure supplement 2C ) . To understand the molecular details for the interactions between H3K9M and its methyltransferases , we determined a 1 . 7 Å resolution crystal structure of G9a SET domain in complex with an H3K9M peptide ( ARTKQTARMSTGGKA ) and SAM ( Supplementary file 1 ) . Clear electron density was observed for residues 3–12 of the peptide based on the crystallographic analysis ( Figure 4A ) . We did not include additional SAM during purification and crystallization , but a SAM molecule was observed in the electron density ( Figure 4B ) , which was likely copurified with the G9a SET domain from E . coli lysates . 10 . 7554/eLife . 17903 . 013Figure 4 . Structure of the H3K9M-G9a-SAM complex . ( A ) Omit Fo–Fc electron density at 1 . 7 Å resolution for the H3K9M peptide , contoured at 3σ . ( B ) Omit Fo–Fc electron density at 1 . 7 Å resolution for SAM , contoured at 3σ . ( C ) Overall structure of G9a ( cyan ) in complex with the H3K9M peptide ( green , stick models ) and SAM ( magenta ) . Several zinc ions bound to the protein are shown as pink spheres . The bound position of dimethylated H3K9 peptide to GLP1 is also shown ( gray ) . ( D ) Detailed interactions between G9a ( cyan ) and the H3K9M peptide ( green ) near Met9 . The structure of GLP1 in complex with the dimethylated H3K9 ( Mly9 ) peptide and SAH is also shown ( gray ) . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 01310 . 7554/eLife . 17903 . 014Figure 4—figure supplement 1 . Structure of the H3K9Nle-G9a-SAM complex . ( A ) Omit Fo–Fc electron density at 1 . 9 Å resolution for the H3K9Nle peptide , contoured at 3σ . ( B ) Detailed interactions between G9a ( cyan ) and the H3K9Nle peptide ( green ) near Nle9 . The structure of H3K9M in complex with G9a is also shown for comparison . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 01410 . 7554/eLife . 17903 . 015Figure 4—figure supplement 2 . Sequence alignment of SUV39 family H3K9 histone methyltransferases . Arrows indicate residues of G9a that interact with the methionine side chain . Red arrow indicates Y451 of Clr4 . s . p . stands for Schizosaccharomyces pombe , d . m . stands for Drosophila melanogaster , h . s . stands for homo sapiens . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 01510 . 7554/eLife . 17903 . 016Figure 4—figure supplement 3 . Characterization of the Clr4-Y451N mutant . ( A ) In vitro binding assays with recombinant SET domain of Clr4 and histone peptides in the presence of SAM . ( B ) Western blot analysis of Flag-Clr4 levels . ( C ) Mass spectrometry analyses of purified Clr4 complexes . The number of peptides identified and the percentage of protein these peptides cover are indicated . ( D ) Left , ten-fold serial dilution analyses of indicated yeast strains grown on indicated media to measure the expression of otr::ura4+ . Right , ChIP analyses of H3K9me3 and H3K9me2 levels at pericentric dh repeats , normalized to act1+ . Data presented is mean ± s . d . of four technical repeats . ( E ) In vitro histone methyltransferase assay with recombinant Clr4 SET domain and histone octamers . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 016 Compared with the structure of another H3K9 methyltransferase G9a-like protein 1 ( GLP1 ) in complex with a dimethylated H3K9 peptide and SAH ( Wu et al . , 2010 ) , the binding mode of the H3K9M peptide to G9a is essentially identical ( Figure 4C and D ) . The methionine residue of H3K9M occupies the same binding pocket as the dimethylated lysine residue ( Figure 4D ) . Five aromatic residues , Tyr1067 , Tyr1087 , Phe1152 , Tyr1154 and Phe1158 surround the methionine side chain . While the dimethylated Lys side chain assumes a fully extended conformation , there is a sharp bend at the sulfur atom of methionine , such that its end methyl group is in closer contact with Tyr1067 and the sulfur is in closer contact with Phe1152 and Tyr1154 . The methionine side chain does not extend as deeply into the pocket , and the SAM compound at the bottom of the pocket moves closer to the side chain , by ~0 . 8 Å compared to the position of SAH in GLP1 . SAM does not make direct contributions to the interactions with the methionine side chain , suggesting that its role is to stabilize the structure of the protein . Interestingly , the methyl group of SAM is 3 . 4 Å away from the side chain of Tyr1154 , which is part of the conformational change to stabilize the peptide-binding pocket . In contrast , the G9a-SAH structure does not have this interaction ( Wu et al . , 2010 ) . It has been demonstrated that the hydrophobicity of the side chain is critical for H3K27M to inhibit PRC2 , and changing methionine to norleucine produces a more potent inhibitor ( Lewis et al . , 2013; Brown et al . , 2014 ) . To further understand the contribution of the aliphatic side chain to the interaction between H3K9M and G9a , we performed binding assays with an H3K9-norleucine ( H3K9Nle ) peptide . We found that H3K9Nle also enhanced binding of the H3 tail peptide to G9a similar to H3K9M ( data not shown ) . We also solved a 1 . 9Å structure of G9a in complex with an H3K9Nle peptide and SAM ( Figure 4—figure supplement 1 ) . The structure showed that the norleucine side chain adopts a similar orientation as the methionine side chain , with a bend of the end methyl group ( Figure 4—figure supplement 1 ) . To characterize the importance of residues in the binding site for the interactions with the H3K9M peptide , we mutated Tyr451 of Clr4 , which is equivalent to Tyr1154 of G9a , to asparagine ( Figure 4—figure supplement 2 ) . Consistent with our expectations , the binding of Clr4-SET-Y451N to the H3K9M peptide was significantly reduced ( Figure 4—figure supplement 3A ) . We then introduced the Y451N mutation into the endogenous clr4+ locus . The mutant protein was expressed at similar levels as that of wild-type Clr4 and mass spectrometry analysis showed efficient association with other components of the Clr4 complex ( Hong et al . , 2005; Horn et al . , 2005; Jia et al . , 2005 ) ( Figure 4—figure supplement 3B and C ) . The localization of Clr4-Y451N to pericentric dh repeats was completely abolished , and no trapping of Clr4 was observed in hht3-K9M cells ( data not shown ) . The complete loss of localization of Clr4-Y451N to dh repeats is due to the positive feedback mechanism between H3K9 methylation and Clr4 localization ( Zhang et al . , 2008a ) , as the Y451N mutation cannot methylate histones in vitro and in vivo ( Figure 4—figure supplement 3D and E ) . Therefore , although the Y451N mutant demonstrates the critical role of this residue in mediating interaction with H3K9M in vitro , it cannot distinguish in vivo the contribution of its being catalytically inactive or inability to bind H3K9M . We have also mutated two other residues of Clr4 that are expected to interact with H3K9M , Y357N and F383N . However , both of them behaved similarly as Y451N , with complete loss of enzymatic activity in vitro and in vivo , but associated efficiently with other components of the Clr4 complex ( data not shown ) . Given that the aromatic residues interacting with the methionine side chain are also required for interaction with the normal lysine substrate , it is not surprising that mutating these residues affected Clr4 enzymatic activity . We therefore explored other ways to relieve trapping of Clr4 by H3K9M . The enzymatic activity of Clr4 in vitro is influenced by modifications of residues adjacent to H3K9 such as phosphorylation of serine 10 ( Nakayama et al . , 2001 ) . We suspected that phosphorylated S10 might interfere with the binding of Clr4 with H3K9M peptide . Indeed , in vitro binding of Clr4 SET domain was significantly reduced when an H3K9M peptide containing phosphorylated S10 was used ( Figure 5A ) . In addition , the in vitro binding of Clr4 to an H3K9MS10D peptide ( to mimic the phosphorylated state of S10 ) was also dramatically reduced ( Figure 5A ) . 10 . 7554/eLife . 17903 . 017Figure 5 . Reduced binding between H3K9M and Clr4 alleviates its inhibitory effects . ( A ) Binding assays with recombinant Clr4 SET domain and histone peptides in histone methyltrasnferase buffer containing SAM . ( B ) Western blot analyses of histone H3 and Clr4 levels . ( C ) ChIP analysis of Clr4 levels at heterochromatin associated repeats , normalized to act1+ . Data presented is mean ± s . d . of four technical repeats . ( D ) Left , ten-fold serial dilution analyses of indicated yeast strains grown on indicated media to measure the expression of otr::ura4+ . Right , ChIP analyses of H3K9me3 and H3K9me2 levels at heterochromatin-associated repeats , normalized to act1+ , and qRT-PCR analysis of repeat transcript levels , normalized to act1+ . Data presented is mean ± s . d . of four technical repeats . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 01710 . 7554/eLife . 17903 . 018Figure 5—figure supplement 1 . S10D has to be present in cis with K9M to detoxify H3K9M . Ten-fold serial dilution analyses of indicated yeast strains grown on indicated media to measure the expression of otr::ura4+ . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 018 To examine whether alleviating the trapping of Clr4 by H3K9M can detoxify H3K9M in vivo , we constructed an hht3-K9MS10D transgene to mimic the constitutively phosphorylated S10 in vivo . The mutant histone was expressed at similar levels to hht3-K9M , and the mutation had no effects on Clr4 protein levels ( Figure 5B ) . Consistent with our hypothesis , Clr4 was localized to pericentric dh repeat in hht3-K9MS10D cells at levels very similar to those in wild-type cells , demonstrating that trapping by H3K9M was alleviated by the adjacent S10 modification ( Figure 5C ) . Moreover , hht3-K9MS10D cells showed normal heterochromatin as indicated by silencing of otr::ura4+ , wild type levels of H3K9me3 and H3K9me2 at pericentric repeats , and wild type levels of dh RNA transcripts ( Figure 5D ) . The S10D mutation only detoxified H3K9M when it was present in cis with K9M , as introducing an hht1-S10D mutation in trans could not rescue silencing defects associated with hht3-K9M ( Figure 5—figure supplement 1 ) . To further examine the mechanism by which H3K9M regulates H3K9 methylation , we introduced a genomic DNA library into cells containing hht3-K9M and otr::ura4+ and looked for clones that conferred resistance to FOA ( Figure 6A ) . We identified two clones , and sequencing of these two plasmids showed that one contains the clr4+ gene and the other contains histone H3 gene hht2+ ( Figure 6B and Figure 6—figure supplement 1A ) . A similar screen with a cDNA library also identified three suppressors that all encode histone H3 genes ( Figure 6B and Figure 6—figure supplement 1A ) . These results raise the possibility that the relative dosage of Clr4 and histone H3 is essential for the phenotypes associated with the H3K9M mutation . 10 . 7554/eLife . 17903 . 019Figure 6 . The effects of H3K9M on heterochromatin depend on the relative concentrations of H3K9M and Clr4 . ( A ) A schematic diagram of the screen procedure for overexpression suppressors of hht3-K9M . ( B ) List of genomic and cDNA clones identified in the screen . ( C , D , E ) Left , ten-fold serial dilution analyses of indicated yeast strains grown on indicated media to measure the expression of otr::ura4+ . Right , ChIP analyses of H3K9me3 and H3K9me2 levels at pericentric dh repeat , normalized to act1+ , and qRT-PCR analysis of dh repeat transcript levels , normalized to act1+ . Data presented is mean ± s . d . of four technical replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 01910 . 7554/eLife . 17903 . 020Figure 6—figure supplement 1 . Ten-fold serial dilution analyses of indicated yeast strains grown on indicated media to measure the expression of otr::ura4+ . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 020 To further test the effects of Clr4 dosage on hht3-K9M , we replaced the clr4+ gene promoter with an inducible nmt41 promoter . Induction of clr4+ partially rescued the silencing defects associated with hht3-K9M ( Figure 6C ) . The effect is Clr4 dosage dependent , as the stronger nmt1 promoter provided better rescue and the weaker nmt81 promoter conferred less rescue ( Figure 6—figure supplement 1B ) . In contrast , overexpression of Swi6 , an HP1 family protein that binds to H3K9me ( Bannister et al . , 2001; Lachner et al . , 2001; Nakayama et al . , 2001 ) , could not rescue silencing defects associated with hht3-K9M ( Figure 6—figure supplement 1B ) . To investigate the effects of histone dosage on hht3-K9M , we constructed diploid cells containing one or two copies of hht3-K9M . Diploid cells containing two copies of hht3-K9M are similar in mutant histone dosage as haploid cells containing one copy of hht3-K9M , and behaved similarly ( Figure 6D ) . However , diploid cells containing one copy of hht3-K9M resulted in only partial desilencing of otr::ura4+ , with normal H3K9me2 and about a 50% reduction of H3K9me3 ( Figure 6D ) . Therefore , H3K9M acts in a dosage-dependent manner to affect H3K9 methylation and heterochromatin assembly . We also tested the effects of histone demethylation in hht3-K9M cells . In fission yeast , genetic studies suggest that the JmjC domain protein Epe1 is an H3K9 demethylase , although it should be noted that no demethylase activity of Epe1 is detected in vitro ( Tsukada et al . , 2006; Zofall and Grewal 2006; Trewick et al . , 2007; Audergon et al . , 2015; Ragunathan et al . , 2015 ) . We found that epe1∆ hht3-K9M cells partially rescued silencing defects of otr::ura4+ , accompanied by the restoration of H3K9me2 levels and a reduction of pericentric dh repeat transcript levels , although H3K9me3 levels were only marginally rescued ( Figure 6E ) . Therefore , the balance of heterochromatin promoting and destabilizing forces also contribute to the effects of hht3-K9M .
Histone K-to-M mutations are associated with distinct types of cancers and they function in a dominant fashion to block the methylation of lysine residues . However , the molecular mechanism by which these mutations block histone methylation is not well understood . Mammalian cells contain large number of histone genes ( and their variants ) and histone methyltransferases , which complicate mechanistic analysis . Here , we established an H3K9M model in a much simpler organism , fission yeast , which contains only three histone H3 genes and a single H3K9 methyltransferase Clr4 . We found that cells containing one copy of mutant H3K9M completely abolished H3K9me3 on the two other wild copies of histone H3 across the entire genome . The formation of heterochromatin is divided into two distinct steps: initiation and spreading . The histone methyltransferase Clr4 is first targeted to heterochromatin nucleation centers to initiate H3K9 methylation . The spreading of heterochromatin requires Clr4 mediated H3K9 methylation , which creates binding sites for the chromo domain proteins such as Swi6 and Clr4 , thus enhancing the binding of Clr4 to methylate adjacent histones ( Figure 7 ) . We found that Clr4 is trapped at heterochromatin nucleation centers in cells containing H3K9M . The fact that H3K9M enhances the interaction between the SET domain of Clr4 and H3 tail , and that compromising such interaction alleviates the trapping of Clr4 by H3K9M to allow heterochromatin assembly suggest that the trapping of Clr4 is mediated by a direct interaction between H3K9M and the Clr4 SET domain . Since Clr4 enzymatic activity is required for heterochromatin spreading and H3K9M inhibits Clr4 activity , it is also possible that the lack of H3K9me also contributes to the failure of Clr4 spreading in addition to the interaction between Clr4 and H3K9M . The fact that trapping only occurs at heterochromatin nucleation centers suggests that recruitment of Clr4 to H3K9M-containing nucleosomes is essential for trapping of Clr4 . Alternatively , other factors might also contribute to the trapping of Clr4 in vivo . 10 . 7554/eLife . 17903 . 021Figure 7 . A model of H3K9M dominantly blocking histone H3K9 methylation . In wild type cells , Clr4 is first recruited to heterochromatin nucleation centers to initiate H3K9 methylation . Methylated nucleosomes recruit additional Clr4 to methylate neighboring nucleosomes , leading to heterochromatin spreading . In the end , low levels of Clr4 will cover the entire heterochromatin domain . In H3K9M expressing cells , Clr4 is still recruited to heterochromatin nucleation centers , but will be trapped there due to its interaction with H3K9M containing nucleosomes ( yellow ) . This result in the failure of Clr4 spreading to form heterochromatin domains . DOI: http://dx . doi . org/10 . 7554/eLife . 17903 . 021 It should be noted that in hht3-K9M cells , although H3K9me3 is completely abolished from the entire genome , there are still significant amounts of H3K9me2 present at heterochromatin nucleation centers . Given that the interaction between H3K9M and Clr4 is not covalent , Clr4 is expected to be in an equilibrium between H3K9M bound and free states . Therefore , although Clr4 is trapped at heterochromatin nucleation centers for a majority of the time , there will be short intervals when Clr4 is in the free form and thus available to methylate nearby nucleosomes . Since the kcat of Clr4 for H3K9me3 is 10 times slower than that of H3K9me2 ( Al-Sady et al . , 2013 ) , it is much easier for Clr4 to catalyze H3K9me2 than H3K9me3 , therefore resulting in a much stronger effects on H3K9me3 than H3K9me2 at heterochromatin nucleation centers , where Clr4 levels are high . Our structure of G9a in complex with H3K9M and SAM provides the molecular details of how methionine interacts with histone methyltransferases . In contrast to Clr4 , which can perform H3K9me3 and regulates heterochromatin assembly , G9a only performs H3K9me2 and mainly acts at euchromatin . Nonetheless G9a is inhibited by H3K9M in vitro ( Lewis et al . , 2013 ) and its SET domain is highly conserved with that of Clr4 . Therefore , we used the G9a-H3K9M-SAM structure as a guide for further analyses . The crystal structure of G9a-H3K9M-SAM complex clearly shows the methionine side chain of H3K9M . It occupies a similar position as that of a normal lysine and interacts with several aromatic residues of G9a through van der Waals interactions . The most obvious difference between the methionine and lysine side chains is the bending of the end methyl group of methionine . When the methionine was substituted with Norleucine , a similar bend of the methyl group was also observed . Such bending increases the diameter of the side chain to enhance van der Waals interactions , therefore explaining the increased binding of H3K9M and H3K9Nle to H3K9 methyltransferases . Although SAM enhanced the binding between H3K9M and H3K9 methyltransferases , SAM does not make strong direct contributions to the interactions with the methionine side chain , suggesting that its role is to stabilize the structure of the protein to properly form the binding pocket . In addition , the position of SAM is incompatible with dimethylated lysine within G9a , suggesting that the binding of SAM helps release the wild type histone H3 tail after methylation reactions . Therefore , the structure explains why SAM is critical for stabilizing the interaction between H3K9M and H3K9 methyltransferases . The binding mode of H3K9M-G9a-SAM differs from the structure of H3K27M in complex with Chaetomium thermophilum PRC2 , in which an adjacent arginine occupies the lysine channel ( Jiao and Liu 2015 ) , even though H3K9M also has an adjacent arginine . It is consistent with the structure of the H3K27M-human PRC2 complex , with methionine occupying the expected lysine-binding channel ( Justin et al . , 2016 ) . Such a binding state can explain the fact that all known K-to-M mutations , even those without adjacent arginine ( such as H3K36M and H3K4M ) , inhibit the enzymatic activity of their corresponding histone methyltransferases ( Chan et al . , 2013b; Lewis et al . , 2013; Herz et al . , 2014 ) . The high conservation of aromatic residues mediating interactions with the methionine side chain suggests that other K-to-M mutations might function in a similar fashion . Indeed , mutant histones show increased association with histone methyltransferases in vivo and in vitro ( Bender et al . , 2013; Chan et al . , 2013a; Justin et al . , 2016; Lu et al . , 2016; Fang et al . , 2016 ) . The strengthened interaction might similarly prevent the turnover of these enzymes to block the methylation of other wild type histones . Finally , our results also suggest several ways to detoxify the effects of H3K9M . First , the effect of H3K9M can be modulated by a modification on the K9M containing histone tail that affects its interaction with Clr4 . It is interesting to note that H3K27 is also adjacent to a serine ( S28 ) . S28 phosphorylation reduced the inhibitory effects of H3K27M on PRC2 activity in vitro , and introduction of a H3K27MS28E mutant reduced the effects of H3K27M on H3K27me3 in vivo ( Brown et al . , 2014 ) . Another way to modulate the effects of H3K9M is through changing Clr4 levels or mutant-to-wild type histone ratios , suggesting that the effects of H3K9M are exerted through a direct competition between H3K9M and normal H3 for a limited pool of Clr4 protein . Finally , tipping the balance between heterochromatin assembly and disassembly , such as the deletion of putative histone demethylase Epe1 , can also partially detoxify H3K9M . This is consistent with an earlier observation that inhibiting H3K27 demethylase JMJD3 alleviates the effects of H3K27M mutation in glioblastoma ( Hashizume et al . , 2014 ) . Given the similarities between different histone K-to-M mutations , it is highly likely that these approaches can be used to detoxify other histone K-to-M mutations as well .
Yeast strains containing epitope tagged histones and their mutants were generated by a PCR-based module method . All other strains were constructed through genetic crosses . A list of yeast strains used is provided in Supplementary file 2 . For serial dilution plating assays , ten-fold dilutions of a mid log-phase culture were plated on the indicated medium and grown for 3 days at 30°C . Log-phase yeast cells were incubated at 18°C for 2 hours and then fixed for 30 minutes in 3% freshly made formaldehyde . The crosslinking reaction was stopped by the addition of 2 . 5 M Glycine to make a final concentration of 0 . 125 M . The cells were pelleted and washed with PBS ( phosphate buffered saline ) before resuspended in ChIP lysis buffer ( 50 mM HEPES-KOH , pH 7 . 5 , 140 mM NaCl , 1% Triton X-100 , 0 . 1% Deoxycholate supplemented with cOmplete protease inhibitors cocktail ( Roche ) ) . Ice cold glass beads were added and the mixtures were vigorously disrupted in a beadbeater . The lysates were collected and subjected to sonication to reduce chromatin size to 500–1000 base pairs . The cleared cell lysates were incubated with antibodies: H3K9me3 ( Abcam ) , H3K9me2 ( Abcam ) , and Flag ( Sigma ) over night at 4°C . Protein G beads were then added to isolate the antibodies and associated chromatin fragments . The beads were then washed with ChIP lysis buffer twice , ChIP lysis buffer containing 0 . 5 M NaCl , Wash buffer ( 10 mM Tris , pH 8 . 0 , 250 mM LiCl , 0 . 5% NP-40 , 0 . 5% Deoxycholate , 1 mM EDTA ) , and TE ( 50 mM Tris pH 8 . 0 , 1 mM EDTA ) . The bound chromatin fragments were eluted with TES ( 50 mM Tris pH 8 . 0 , 1 mM EDTA , 1% SDS ) and the crosslinking was reversed by incubating at 65°C overnight . The protein DNA mixture were then subjected to proteinase K treatment and phenol:chloroform extraction before the DNA was precipitated by ethanol . Quantitative real-time PCR ( qPCR ) was performed with Maxima SYBR Green qPCR Master Mix ( Fermentas ) in a StepOne Plus Real Time PCR System ( Applied Biosystems ) . DNA serial dilutions were used as templates to generate a standard curve of amplification for each pair of primers , and the relative concentration of target sequence was calculated accordingly . An act1 fragment was used as a reference to calculate the enrichment of ChIP over WCE for each target sequence . A list of DNA oligos used is provided in Supplementary file 3 . For ChIP-seq , DNA samples were prepared according to TruSeq ChIP sample preparation guide ( Illumina ) and sequenced on the Illumina HiSeq 2500 system by 100 bp paired-end sequencing . The raw reads were trimmed by Trimmomatic ( v0 . 35 ) ( Bolger et al . , 2014 ) to remove potential adapter contamination and regions with bad sequencing qualities . The trimmed reads were aligned to the S . pombe reference genome ( Ensembl version: ASM294v2 . 29 ) by bwa ( 0 . 7 . 12-r1039 ) ( http://bio-bwa . sourceforge . net/ ) . The resulting sam files were further processed by Samtools ( v1 . 2 ) ( Li et al . , 2009 ) , picard-tools ( v2 . 0 . 1 ) ( http://broadinstitute . github . io/picard/ ) and GATK ( v3 . 5 ) ( McKenna et al . , 2010 ) for indexing , sorting , removing PCR duplicates , and local-realignment . The per-based mapping depth was calculated by bedtools ( v2 . 25 . 0 ) ( Quinlan and Hall , 2010 ) and the sliding window plots ( window size = 100 bp , step size = 50 bp ) were created by in-house Perl and R scripts . We also employed MACS ( v1 . 4 . 2 ) ( Zhang et al . , 2008b ) to contrast each ChIP sample versus WCE sample for peak calling . Total cellular RNA was isolated from log-phase cells using MasterPure yeast RNA purification kit ( Epicentre ) according to the manufacturer’s protocol . Quantification with real time RT-PCR was performed with Power SYBR Green RNA-to-CT one-step Kit ( Applied Biosystems ) . RNA serial dilutions were used as templates to generate a standard curve of amplification for each pair of primers , and the relative concentration of the target sequence was calculated accordingly . An act1 fragment served as a reference to normalize the concentration of samples . The concentration of each target gene in wild type was arbitrarily set to 1 and served as reference for other samples . Histone peptides ( H3 , 1–21 ) with a C-terminal biotinylated lysine were synthesized by Anaspec at 90% purity and confirmed by mass spectrometry analyses . Clr4 chromo domain ( 1–190 ) and SET domain ( 190–490 ) were cloned into a pGEX vector . Expression plasmid for GST tagged Swi6 , Chp2 , and Chp1 chromo domains are gifts from Dr . Jun-ichi Nakayama . The GST fusion proteins were purified with Glutathione beads ( GE Healthcare ) according to the manufacturer’s protocol . To examine the enzymatic activity of Clr4 mutants , histone methyltransferase assays were performed with 0 . 5 µg of recombinant Clr4 SET domain and 2 . 6 µg of Hela histones in a histone methyltransferase buffer ( 50 mM Tris , pH 8 . 0 , 1 mM EDTA , 0 . 5 mM DTT ) supplemented with 3H-SAM for 30 minutes at 30°C . The samples were resolved by SDS-PAGE and subjected to Coomassie staining to visualize the proteins and then treated with EN3HANCE ( Perkin Elmer ) to visualize labeled substrates . To examine the inhibitory effects of H3K9M peptide , 0 . 1 µg recombinant SET domain of Clr4 was incubated with 1 µg recombinant human nucleosomes in a methyltransferase buffer ( 100 mM Tris , pH 8 . 8 , 100 mM KCl , 5% glycerol , 1 mM MgCl2 , 20 µM ZnSO4 , 10 mM β-mercaptoethanol ) supplemented with 10 µM non-radioactive SAM and 2 µM [3H]-labeled SAM for 30 minutes at 30°C . The H3K9M peptide was incubated with Clr4 SET domain for 15 minutes at 30°C before the addition of mono-nucleosomes . Protein binding assays with recombinant chromo domains were performed by incubating recombinant proteins with biotinylated histone peptides in PBS buffer for 30 minutes at 30°C . Streptavidin beads were added to isolate biotinylated peptide and associated proteins . The beads were washed three times with PBS buffer . The proteins bound to the beads were resolved by SDS-PAGE and stained with Coomassie blue . Protein binding assays with recombinant SET domains were performed by incubating recombinant proteins in histone methyltransferase buffer ( 50 mM Tris , pH 8 . 0 , 50 mM NaCl , 1 mM EDTA , 0 . 5 mM DTT ) supplemented with 50 µM SAM or SAH for one hour at 30°C . Streptavidin beads were added to isolate biotinylated peptide and associated proteins . The beads were washed three times in histone methyltransferase buffer before resolved by SDS-PAGE and stained with Coomassie blue . To examine whether the binding was covalent , ChIP lysis buffer ( 50 mM HEPES-KOH , pH7 . 5 , 500 mM NaCl , 1% Triton X-100 , 0 . 1% sodium deoxycholate ) was used to perform the washes . The expression and purification of the SET domain of human G9a ( residues 913–1193 ) followed a published procedure ( Wu et al . , 2010 ) . The expression plasmid ( gift of Prof . Matthieu Schapira ) was transformed into competent cells and induced using 0 . 25 mM isopropyl-1-thio-D-galactopyranoside when A600 is 0 . 7 . After incubation overnight at 16°C , the cells were harvested and resuspended in lysis buffer ( 20 mM Tris ( pH 8 . 0 ) , 250 mM NaCl , and 5% ( v/v ) glycerol ) supplemented with 2 mM β-mercaptoethanol , 0 . 1% Igepal and 1 mM phenylmethylsulfonyl fluoride and lysed with ultrasonication . The lysate was incubated with Ni-NTA resin ( QIAGEN ) and washed with 10 column volume lysis buffer containing 50 mM imidazole . Bound protein was eluted with lysis buffer containing 250 mM imidazole , and was digested overnight with thrombin to remove the 6xHis-tag . After concentration , the protein was loaded onto a Superose 6 column ( GE Healthcare ) equilibrated with gel filtration buffer ( 20 mM Tris ( pH 8 . 0 ) , 150 mM NaCl ) . The fractions of the protein peaks were collected and concentrated . The protein was incubated with the H3K9M peptide at a molar ration of 1:10 on ice for 30 min , and the sample was then set up for crystallization with the hanging-drop vapor diffusion method at 20°C . The reservoir solution contained 0 . 1 M Bis-Tris propane ( pH 7 . 5 ) , 18% ( w/v ) PEG3350 , 0 . 2 M NaF , and 5% ( v/v ) ethylene glycol . Using mother liquor supplemented with 15% ( v/v ) glycerol as cryo-protectant , the crystals were frozen in liquid nitrogen before data collection . X-ray diffraction data were collected at 100K at NE-CAT beamline 24-ID-C of Advanced Photon Source ( APS ) at Argonne National Laboratory and processed using program HKL2000 ( Otwinowski and Minor , 1997 ) . The structure was solved through molecular replacement using Phaser-MR in program PHENIX ( Adams et al . , 2002 ) . The structure refinement was carried out using PHENIX , and manual model building with Coot ( Emsley and Cowtan , 2004 ) . ChIP-seq data is available at Arrayexpress under accession number E-MTAB-4776 . The atomic coordinates have been deposited at the Protein Data Bank , with accession codes 5T0K ( Clr4-H3K9M-SAM ) and 5T0M ( Clr4-H3K9Nle-SAM ) .
Histone lysine-to-methionine ( K-to-M ) mutations are associated with multiple cancers , and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones . However , their mechanisms of function are controversial . Here we show that in fission yeast , introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome . In addition , H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM ( S-adenosyl-methionine ) -dependent manner , and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of large heterochromatin domains . We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM , which reveales that the methionine side chain had enhanced van der Waals interactions with G9a . Therefore , our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation .
Cells switch their genes on or off in order to respond to changes in their environment . A group of proteins called histones are partly responsible for regulating gene activity . Like all proteins , histones are made from smaller building blocks called amino acids . Enzymes can chemically modify specific amino acids in histone proteins , which allows the histones to switch nearby genes on or off . One of these modifications is called methylation , and the methylation of specific “lysine” amino acids in histone proteins regulates gene activity in different ways . Previous research has shown that , in certain types of cancer cells , lysines that can be methylated are often replaced with another amino acid , a methionine . These substitutions stop the histones from correctly controlling the activity of nearby genes because methionine cannot be methylated like lysine . Additionally , even if only a small number of histones have methionine in place of lysine , this change can have a widespread effect because the few histones with the methionine can block other histones from being methylated too . However , previous studies did not provide a clear mechanism for why this is the case . In the fission yeast Schizosaccharomyces pombe an enzyme called Clr4 methylates a histone protein at a lysine named H3K9 . Now , Shan , Wang , Xu et al . show that substituting this lysine with a methionine ( referred to as H3K9M ) stops the widespread methylation of histones by trapping the Clr4 enzyme . Specifically , Clr4 becomes stuck to the H3K9M histones , and is therefore unable to modify any other histones . Shan et al . went on to carry out a more detailed study of the structure of H3K9M attached to another enzyme called G9a . This enzyme is found in human cells and is similarly inhibited by H3K9M . This investigation identified additional chemical interactions that explain why Clr4 and G9a become trapped by the H3K9M histone but not by normal histones . Future studies are needed to explore whether other altered histones are able to trap enzymes in the way that H3K9M traps Clr4 and G9a . In addition , this work could eventually lead to new cancer therapies .