1、ReviewControlling the Controllers:Regulation ofHistone Methylation by PhosphosignallingRyan J.Separovich,1Chi Nam Ignatius Pang,1and Marc R.Wilkins1,*Histone methylation is central to the regulation of eukaryotic transcription.Here,we review how the histone methylation system itself is regulated.The
2、re is sub-stantial evidence that mammalian histone methyltransferases and demethylasesare phosphorylated and regulated by upstream signalling pathways.Functionalstudiesofspecificphosphosites are revealing which kinases andpathwayssignaltothehistonemethylationsystemandarediscoveringthediverseeffectso
3、fphos-phorylation on enzyme function.Nevertheless,the majority of phosphosites haveno known kinase or function and our understanding of how histone methylation isregulated is fragmentary.Improved approaches are needed to establish andstudy the key regulatory phosphorylation sites on histone methyltr
4、ansferasesand demethylases,to avoid focus on constitutive sites which may have littleregulatory purpose.Histone Methylation Is a Central Regulatory Process in the Eukaryotic CellEukaryotic cells respond to internal and external stimuli via signalling systems.Associatedchanges in gene expression are
5、mediated by epigenetic modifications(see Glossary)ofhistone proteins,which affect chromatin compaction and the recruitment of transcriptionalcoregulators 1.Histone methylation is a key epigenetic modification that regulates many nuclearprocesses,including transcription 2,replication 3,and DNA repair
6、 4.Specific histone methylmarkscan haveeitheractivatingor repressiveeffects on transcription depending on theirpositionand methylation state 2.All eukaryotic histone methylation sites demarcate functionalsequences in the genome,both throughout a genes body and within noncoding and regulatoryelements
7、,such as promoters and enhancers 5,6.Importantly,the genomic distribution ofhistone methylation changes dramatically during growth 7,differentiation 8,and in responseto exogenous perturbation 9,to bring about widespread transcriptional reprogramming.Despite its major regulatory role,little is known
8、about how histone methylation as a systemreceives information from upstream signalling pathways.As such,it remains unclear how thisepigenetic modification is controlled by intracellular and extracellular signals.This review high-lights recent efforts to delineate the connection between cellular sign
9、alling and histone methyla-tion and discusses the challenges associated with integrating two central regulatory systems ofthe cell.Human Histone Methylation:The Methyltransferases and DemethylasesThe mammalian genome encodes 35 histone methyltransferases and 23 demethylases 1013.Histone residues mod
10、ified by these enzymes in the human cell are summarised in Figure 1.Despite much progress in mapping enzymes to their corresponding methylation sites,there isstill contention surrounding the specificity of many human methyltransferases and demethylases1417.For example,SMYD3 was initially reported to
11、 have H3K4 methyltransferase activity,butwassubsequentlyshowntoinsteadmethylateH4K5andnon-histonesubstrates16.Althoughitwas originally classified as a H3K4 methyltransferase,MLL5 does not have methyltransferaseHighlightsHistone methylation is an importantepigenetic modification that controlseukaryot
12、ic transcription,and its dysregu-lation contributes to the aetiology ofhuman disease.Recent phosphoproteomic studieshave revealed that mammalian histonemethyltransferase and demethylaseenzymesareextensivelyphosphorylated.Kinases that phosphorylate histonemethyltransferaseanddemethylaseenzymes are fr
13、om many different signaltransduction pathways,however,mostphosphosites have no known kinase.Phosphorylation has diverse effectson histone methyltransferase anddemethylase biology,affecting theircatalytic activity,chromatin binding,and degradation.Targeted studies have investigated a tinyproportion o
14、f phosphorylation sites,meaning that our understanding of howthe intracellular signalling network con-nects with histone-based gene regula-tory systems is rudimentary.1School of Biotechnology andBiomolecular Sciences,University ofNew South Wales,New South Wales,2052,Australia*Correspondence:m.wilkin
15、sunsw.edu.au(M.R.Wilkins).Trends in Biochemical Sciences,Month 2020,Vol.xx,No.xxhttps:/doi.org/10.1016/j.tibs.2020.08.0041 2020 Elsevier Ltd.All rights reserved.Trends in Biochemical SciencesAn official publication of the INTERNATIONAL UNION OF BIOCHEMISTRY ANDMOLECULAR BIOLOGYTIBS 1716 No.of Pages
16、14activity 15.The overlapping site specificity of histone methyltransferases and demethylases(Figure 1)makes it likely that additional enzymes will be found to methylate or demethylatehuman histones.Indeed,of the 55 SET domain-containing methyltransferases in the humanproteome,approximately half hav
17、e no known substrates 18.Given its central role in cellular processes,there is a need to establish how histone methylation isregulated.Underscoring its functional importance,aberrant histone methylation is linked to theaetiology and progression of many diseases 13,19,and histone methyltransferases a
18、nddemethylasesareemergingasanticancertherapeutictargets11,20.Consequently,thetranscrip-tionalandpost-transcriptionalregulationofanumberofkeymethylation-relatedenzymeshasbeeninvestigated.For example,expression of the oncogenic H3K27-specific methyltransferase,enhancer of zeste homolog 2(EZH2),is dire
19、ctly controlled by the c-Myc transcription factor 21,and abundance of the resultant transcript is negatively regulated by miRNA-101 22.This typeof regulation,however,does not fully explain the complexity of the histone methylation system asit only affects the amount of enzyme present.The post-transl
20、ational modification of the enzymesthemselves,being a poorly understood aspect of histone methyltransferase and demethylasebiology,is emerging as an important regulatory process;one which connects the intracellularsignalling network with histone-based gene regulatory systems,controlling when and whe
21、re themethylating and demethylating enzymes act.Mammalian Histone Methylation Enzymes Are Extensively PhosphorylatedHistone methyltransferase and demethylase enzymes are post-translationally regulated by anumber of different modifications,including phosphorylation 23,methylation 24,acetylation25,ubi
22、quitination 26,and glycosylation 27.Phosphorylation is of particular interest andhigh-throughput phosphoproteomic studies are revealing that histone methyltransferase anddemethylase enzymes are extensively phosphorylated.Our collation of phosphorylation sitesfrom two phosphoproteomic datasets 23,28
23、identified 2511 phosphosites on 58 enzymes,ofwhich 1744 are on 35 histone methyltransferases and 767 are on 23 histone demethylases(Figure 2).Phosphorylation of Histone Methyltransferase and Demethylase Families Provides Insights intoTheir RegulationThe phosphorylation profiles of histone methyltran
24、sferase and demethylase enzymes in the samefamily show strong similarity(Figure 2).By contrast,different histone methyltransferase anddemethylase families are phosphorylated to markedly different extents,and have differentpatterns of phosphosites in sequence and structural features.These observation
25、s are importantand suggestthatthe cell uses distinct regulatory mechanisms to controlspecific enzymefamilies.A number of histone methyltransferase and demethylase families are extensively phosphorylatedand likely to be subject to complex post-translational regulation.For example,all six members ofth
26、eMLLmethyltransferasefamilyharbouratleast60phosphositesperprotein(Figure2A).Similartrends areobserved for the ASH1L methyltransferasefamilymembers,whichare also extensivelyphosphorylated.Enzymes with very large numbers of phosphosites are likely to be importantintegrators of information 29,receiving
27、 signals from multiple independent signalling path-ways to fine-tune their function.Several histone methyltransferase and demethylase families receive little information from up-stream signalling pathways.For example,arginine methyltransferases(PRMTs)have a smallernumber of phosphosites compared wit
28、h their lysine methyltransferase counterparts(Figure 2A).All five PRMT members have less than 20 phosphosites and show similar phosphorylationGlossaryConstitutive phosphorylation site:aphosphorylation site that is alwayspresent and is likely essential for proteinfunction and/or structure.Crosstalk:w
29、hen multiple post-translationalmodifications,adjacentonaprotein,affect one another or thecognate proteins interactions.Disordered region:a part of a proteinwhich lacks a defined 3D structure;frequently involved in proteinproteininteractions.Epigenetic modification:heritablealterations in gene expres
30、sion that arenot due to changes in the underlyingDNA sequence.Integrators of information:proteinswhich receive information from manydifferent inputs to determinedownstream functional outcomes.Thistype of regulation is a hallmark ofimportant regulatory proteins(cf.,p53)Phosphodegron:a short linear mo
31、tifthat,when phosphorylated,is thenubiquitinated by a corresponding E3ligase,targeting the protein forproteasomal degradation.Phosphoproteomics:proteome-wideanalysis of phosphorylation sites,typically involving affinity-basedenrichment and mass spectrometricidentification of phosphopeptides andthus
32、phosphoproteins.Stoichiometry:the proportion of aprotein that is modified at a particularsite.Calculating stoichiometry requireshigh-resolution quantitative techniquesto determine the amount of modifiedprotein present,compared with the totalprotein abundance.Trends in Biochemical SciencesAn official
33、 publication of the INTERNATIONAL UNION OF BIOCHEMISTRY ANDMOLECULAR BIOLOGY2Trends in Biochemical Sciences,Month 2020,Vol.xx,No.xxprofiles.Enzymes with few phosphosites,suchasthe PRMTs,mightbe activated/deactivated onphosphorylation of a specific residue or may predominantly be subject to transcrip
34、tional control.Consistent with this is the observation that a single phosphorylation event on tyrosine 291 ofPRMT1 alters its substrate specificity and proteinprotein interactions 30.Phosphorylation Sites in Kinase Motifs Reveal Connections to Known Signalling PathwaysOf the 2511 phosphosites on hum
35、an histone methyltransferase and demethylase enzymes,62%arelocatedwithinaknownhumankinaserecognitionmotif(Figure2A,C).Thesemotifshelprevealthe signalling pathways that connect to and communicate with the histone methylation system.The most common motifs are for constituents of the MAPK/ERK,mTOR/PI3/
36、Akt,Wnt/-catenin,cyclin-dependent,and cAMP-dependent signal transduction pathways.This highlightsthe potential for diverse signalling pathways to transmit cellular information to histone methyl-transferase and demethylase enzymes.It also shows that methyltransferase and demethylaseenzymes are critic
37、al connectors between the intracellular signalling network and the histonemethylation system;two major regulatory processes in the cell.Phosphorylation Sites in Domains and in Regions of DisorderPhosphorylation sites within protein domains can regulate catalytic activity and interaction part-ners31.
38、Theproportion of phosphorylationsiteswithindomains of histone methylationenzymesis appreciably lower than expected by chance,highlighting a negative enrichment for phospho-sites within such domains(Figure 2B,D).Regulation of histone methyltransferase anddemethylase function by phosphorylation of dom
39、ains may thus be less widespread or of lowerimportance.Of the small number of sites residing in domains,several are located within methyl-transferase(SET,DOT1,and PRMT)and demethylase(JmjN and JmjC)domains.Suchsitesmayact as molecular switches,whereby catalytic activity is turned on or off in respon
40、se to a specificsignal,or could target histone methylation enzymes to chromatin features including specificlocations in the genome.Consistent with this,several phosphosites are located within chromatininteraction domains(chromodomains,bromodomains,and PHD).Functions of phosphosites areexplored in gr
41、eater detail,as described below.TrendsTrends inin BiochemicalBiochemical Sciences SciencesFigure 1.Mammalian Histone Methylation and Its Phosphoregulation.Histone proteins H2A,H2B,H3,and H4 are methylated at lysine(K)and/or arginine(R)residues along their N-terminal tails by methyltransferase enzyme
42、s(green).These methyl marks can be subsequently removed by demethylase enzymes(pink).Allhuman histone methyltransferases and demethylases have been shown to be phosphorylated,suggesting regulation from upstream signalling pathways.The kinasesresponsible for phosphorylating enzymes are shown in mauve
43、,where known.Kinases that are unknown are shown as question marks.Trends in Biochemical SciencesAn official publication of the INTERNATIONAL UNION OF BIOCHEMISTRY ANDMOLECULAR BIOLOGYTrends in Biochemical Sciences,Month 2020,Vol.xx,No.xx3TrendsTrends inin BiochemicalBiochemical Sciences Sciences(See
44、 figure legend at the bottom of the next page.)Trends in Biochemical SciencesAn official publication of the INTERNATIONAL UNION OF BIOCHEMISTRY ANDMOLECULAR BIOLOGY4Trends in Biochemical Sciences,Month 2020,Vol.xx,No.xxDisordered regions in proteins play a key role in signalling as different post-tr
45、anslationalmodifications alter their conformational fold,binding affinity to specific interaction partners 32,and liquidliquidphasetransitions33.Incontrast to thesmallproportionofsites within domains,histone methyltransferase and demethylase enzymes are extensively phosphorylated withinregions of di
46、sorder.Correspondingly,74%of mammalian histone methylation enzymes werepositively enriched for phosphosites within disordered sequences(Figure 2B,D).Whilehigh,this is consistent with the proportion of phosphosites in disorder across the entirephosphoproteome 34.Notably,however,some enzyme families(e
47、.g.,MLL methyltransferasesand JMJD3 demethylases)are extensively phosphorylated within disordered regions whereasothers are not(e.g.,SMYD methyltransferases and JARID demethylases).Sites in regions ofdisorder can mediate scaffolding and proteinprotein interactions of histone methyltransferaseand dem
48、ethylase enzymes,and will be of importance for targeted analysis.Phosphorylation Has Diverse Effects on Histone Methyltransferase andDemethylase FunctionPhosphorylation of histone methyltransferases and demethylases can have different effects ondownstream cellular processes.A review by Trevio et al.
49、in 2015 concisely summarised targetedstudieswhichdefinedtheeffectofphosphoregulationforspecifichumanhistonemethyltransferasesand demethylases 35.Here,we comprehensively review recent studies of methyltransferase anddemethylase regulation by phosphorylation.Collectively,these have highlighted the eff
50、ects thatphosphorylation of histone methyltransferases and demethylases can have on enzymatic activity,proteinprotein interactions,chromatin binding,and stability(Figure 3).Effects on Methyltransferase and Demethylase ActivityA single phosphorylation event can affect the activity of histone methyltr
