Transcription factors as readers and effectors of DNA methylation.pdf

Transcription factors as readers and effectors of DNA methylationHeng Zhu1,2,Guohua Wang3,and Jiang Qian2,31Department of Pharmacology and Molecular Sciences,Johns Hopkins School of Medicine,Edward Miller Research Building,733 North Broadway,Baltimore,Maryland 21205,USA2The Sidney Kimmel Comprehensive Cancer Center,Johns Hopkins School of Medicine,Baltimore,Maryland 21287,USA3The Wilmer Eye Institute,Johns Hopkins School of Medicine,The Smith Building,400 North Broadway,Baltimore,Maryland 21287,USAAbstractRecent technological advances have made it possible to decode DNA methylomes at single-base-pair resolution under various physiological conditions.Many aberrant or differentially methylated sites have been discovered,but the mechanisms by which changes in DNA methylation lead to observed phenotypes,such as cancer,remain elusive.The classical view of methylation-mediated protein-DNA interactions is that only proteins with a methyl-CpG binding domain(MBD)can interact with methylated DNA.However,evidence is emerging to suggest that transcription factors lacking a MBD can also interact with methylated DNA.The identification of these proteins and the elucidation of their characteristics and the biological consequences of methylation-dependent transcription factor-DNA interactions are important stepping stones towards a mechanistic understanding of methylation-mediated biological processes,which have crucial implications for human development and disease.DNA methylation,one of the best-studied epigenetic marks in eukaryotes,is a biological process in which a methyl group is covalently added to a cytosine,yielding 5-methylcytosine(5mC)13(BOX 1).The methylation process is carried out by a set of enzymes called DNA methyltransferases(DNMTs)4,which are encoded in many genomes,from bacteria to plants and mammals5,6.The evolutionary conservation of these enzymes suggests that DNA methylation provides a selective advantage to the organism.However,the percentage of Correspondence to H.Z.and J.Q.hzhu4jhmi.edu;jiang.qianjhmi.edu.Competing interests statementThe authors declare no competing interests.DATABASESENCODE:http:/encodeproject.orgENCSR000EBQ|ENCSR000EBV|ENCSR000BSI|ENCSR000ECC|ENCSR000ECF|ENCSR000BJW|ENCSR000BHOFURTHER INFORMATIONIrreproducible Discovery Rate:https:/www.encodeproject.org/software/idrSUPPLEMENTARY INFORMATIONSee online article:S1(figure)ALL LINKS ARE ACTIVE IN THE ONLINE PDFHHS Public AccessAuthor manuscriptNat Rev Genet.Author manuscript;available in PMC 2017 August 17.Published in final edited form as:Nat Rev Genet.2016 August 01;17(9):551565.doi:10.1038/nrg.2016.83.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptmethylated cytosine varies substantially across species.For example,vertebrates and plants often have a high percentage of methylated CpG dinucleotides outside CpG islands,whereas invertebrates typically exhibit intermediate levels or no methylation7,8.With the development of more sensitive methodological approaches,such as methylated DNA immunoprecipitation followed by bisulfite sequencing(MeDIPBS-seq)which sequences bisulfite-converted DNA species after enrichment for methylated DNA fragments using an anti-5mC antibody some genomes previously considered not to have any DNA methylation(for example,that of Drosophila melanogaster)have now been found to be methylated at a limited number of cytosines911.In most animals,DNA is methylated predominantly at CpG dinucleotides,whereas in plants and fungi,a large fraction of DNA methylation also occurs at CHG or CHH(where H can be any nucleotide but G)1215.That said,it was recently discovered that a small fraction of non-CpG methylation also occurs in animals(BOX 1).DNA methylation has a critical role as a means to control gene expression;for example,during development to ensure X-chromosome inactivation or genomic imprinting16,17 through various mechanisms.Furthermore,aberrant DNA methylation is a hallmark of many diseases,including various types of cancers18.Indeed,abnormal gains in methylation in normally unmethylated CpG islands have been linked to the inactivation of tumour suppressor genes1921.Such abnormal promoter CpG island methylation is emerging as a potential biomarker for cancer detection,diagnosis and prognosis22,23.More recently,DNA methylation has also been implicated in non-cancerous diseases,such as schizophrenia24 and autism spectrum disorders25,26.Thanks to rapid technological advances,especially the range of techniques based on deep sequencing,it is now possible to monitor the dynamics of the DNA methylome at single-nucleotide resolution2729.These developments have provided new insights into how the epigenome is shaped and how it regulates different biological processes,such as cellular differentiation and cancer development.For instance,comparing methylation profiles under different physiological conditions revealed tissue-specific or disease-specific differentially methylated regions3032,suggesting that the role of DNA methylation in gene regulation is multifaceted and goes beyond simple repression of gene expression.Despite the fast accumulating profiles of DNA methylomes in various biological processes and species,the interpretation of these data sets often falls short of providing a mechanistic understanding of the dynamic changes in DNA methylation levels.It still remains a challenge to establish the causality between DNA methylation and physiological outcomes in the epigenetic field.In our view,the first step towards a mechanistic understanding of the DNA methylome is to determine the proteinDNA interactions associated with the dynamics of the DNA methylome.In other words,the identification of DNA methylation readers and effectors,which translate methylation signals into biological actions,will be crucial to decipher the epigenetic code of methylation-mediated biological processes.In this Analysis article,we review the discovery of a new class of methylated-DNA-binding proteins,namely transcription factors(TFs),and the approaches used to discover these Zhu et al.Page 2Nat Rev Genet.Author manuscript;available in PMC 2017 August 17.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptinteractions.We focus on the interaction partners with methylated CpG sites in mammals,with a brief discussion of other methylation derivatives(BOX 1).We then summarize the specific properties of methylation-dependent interactions between TFs and DNA,and discuss the causal relationship between TFDNA interactions and DNA methylation,before concluding with an overview of potential biological consequences of methylation-dependent proteinDNA interactions.Readers of methylated DNAThe classical view of methylation-mediated protein DNA interactions is that only proteins with a methyl-CpG(mCpG)-binding domain(MBD)can recognize and bind to methylated CpG dinucleotides3,6,3335(FIG.1).The MBD protein family has five known members in mammals,including MeCP2(methyl-CpG-binding protein 2),MBD1,MBD2,MBD3 and MBD4.Except for MBD3,which does not bind to methylated DNA,all MBD proteins bind to methylated DNA in a non-sequence-specific manner36,37.Comparison of MBD proteins from different species showed conservation and divergence in terms of the number of MBD genes and the composition of the MBD domains6,38.Interestingly,the extent of genomic methylation generally correlates with the number of MBD proteins in a species6.Dysfunction of MBD proteins is associated with human diseases.For example,mutations in the gene encoding MeCP2 cause the neurodevelopmental disorder Rett syndrome39,40.Over the past 15 years,evidence has emerged that suggests that some TFs lacking MBDs are able to interact with methylated DNA2327(FIG.1).Unlike MBD proteins,a handful of mammalian TFs were found to possess sequence-dependent mCpG-binding activity in a few studies.For example,the transcriptional regulator Kaiso,which contains POZ(pox virus and zinc-finger)and zinc-finger domains,was found to bind to a specific methylated sequence with its C2H2 zinc-finger domains41.In other studies,the basic leucine zipper(bZIP)CCAAT/enhancer-binding protein-(CEBP)42,the zinc-finger protein ZFP57 and its cofactor KRAB-associated protein 1(KAP1;also known as TIF1)43,44 were shown to interact with specific methylated sequences.In addition to mammalian proteins,a bZIP herpesvirus protein,Zta,was found to bind to methylated regulatory elements and control the epigenetic landscape during the latency-to-lytic phase transition in infected mammalian cells45.Two amino acids,one cysteine and one serine,were found to interact with 5mC45.In rice,the nuclear protein MVBP(methylated VBE-binding protein)was shown to bind to a rice tungro bacilliform virus promoter region only when the promoter was methylated46.As the discovery of these mCpG-binding proteins was often serendipitous,whether TFs represent a new class of DNA methylation readers and,potentially,effectors,and whether sequence-specific mCpG-dependent binding activity is a widespread phenomenon or merely an exception,remained questionable.In addition,recent large-scale analyses of gene expression profiles and DNA methylomes showed that a substantial portion of DNA methylation sites is positively correlated with gene expression31.This finding may result from the high levels of DNA methylation in the gene body of highly expressed genes;however,it also raises the possibility that some TFs bind to methylated regulatory elements and activate gene expression.It should be noted that DNA methylation of a promoter or an enhancer has been shown to be correlated with increased transcription of a target gene47,49,Zhu et al.Page 3Nat Rev Genet.Author manuscript;available in PMC 2017 August 17.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptalthough most of the evidence showing a positive correlation between methylation and expression seems to result from methylation downstream of the transcription start site.Intrigued by these observations,several research groups have conducted unbiased,high-throughput screens to search for such a correlation in higher eukaryotes(TABLE 1).High-throughput reader discoveryTandem mass spectrometryOne systematic approach for the discovery of mCpG-binding proteins is based on tandem mass spectrometry(MS/MS)50,51.A recent study used a generic DNA sequence harbouring an mCpG site to pull down interacting proteins from nuclear extracts of cultured cells51.Proteins bound to methylated DNA sequences were then identified by MS/MS.Based on this approach,19 proteins were identified that interact preferentially with the methylated DNA probe rather than the non-methylated counterpart in mouse embryonic stem(ES)cell nuclear extracts.Besides the known MBD proteins(such as MeCP2,MBD1 and MBD4),many TFs(such as MHC class II regulatory factor RFX1,zinc-finger homeobox 3(ZFHX3),lysine-specific histone demethylase 1A(LSD1),zinc-finger and BTB domain-containing protein 44(ZBT44)and thymocyte nuclear protein 1(THYN1;also known as THY28),and the Krppel-like factors(for example,KLF2,KLF4 and KLF5),were identified as new mCpG-binding proteins(TABLE 2).The authors applied the same approach to neuronal progenitor cells and found that a large and distinct set of proteins showed preferential binding to mCpG sites,suggesting that the interaction with mCpG is dynamic and thus varies under different physiological conditions.A similar approach was also used to identify nucleosome-interacting proteins that are affected by DNA methylation50.Although proteins from cell extracts are in a more native state in the MS/MS-based approach,the DNA probes used in this approach are typically generic and,therefore,sequence specificity of observed interactions remains elusive.Functional protein microarrayFunctional protein microarrays have been used as a powerful tool to profile proteinDNA interactions in the past52.A comprehensive examination of sequence-specific mCpG-binding activities was conducted by sequentially probing a human protein microarray containing 1,321 TFs and 210 cofactors with 154 DNA motifs that each carried at least one mCpG site53.To identify human TFs that preferentially bind to methylated DNA motifs,each methylated motif was mixed with its unlabelled and unmethylated counterpart in tenfold excess in the binding assays.This competition assay ensures that the identified interactions are indeed methylation-dependent,rather than due to CpG-flanking sequences.Of the 154 methylated motifs examined,150 showed strong binding signals to at least one protein on the microarray.In total,41 TFs and 6 cofactors were found to bind to at least one methylated sequence.Most of these factors were found to bind to only a few methylated sequences,suggesting that the interactions are not only methylation-dependent but also sequence-specific.Interestingly,the factors that showed binding activity to methylated sequences were widespread among various TF subfamilies,such as zf-C2H2,homeobox,bHLH(basic helixloophelix),forkhead,bZIP and HMG(high-mobility group)box.Many of these factors are known to be involved in tissue development or have been associated with Zhu et al.Page 4Nat Rev Genet.Author manuscript;available in PMC 2017 August 17.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptcancer.A subsequent validation assay showed that some of these TFs indeed bind to methylated DNA in vivo and regulate gene expression53.DNA microarrayDNA(or protein-binding)microarray technology has been used to determine the binding specificity of TFs54,55.A double-stranded DNA microarray,typically comprising 40,000 unique DNA sequences that cover all possible combinations of 810-nucleotide-long DNA sequences that could constitute a binding motif,is incubated with a purified TF so that its binding preference can be accurately determined.In a recent study,the bacterial DNMT SssI was used to methylate the CpG sites of the sequences on the array56,followed by individual probing with eight purified proteins containing bZIP domains.By comparing the binding profiles of each protein obtained on the methylated and unmethylated microarrays,proteins that preferentially bind to specific sequences were determined.Among the eight bZIP proteins,CEBP and CEBP were found to specifically bind to a methylated sequence56.This approach enables a large amount of DNA sequences to be surveyed for proteinDNA interactions;accurate sequence specificity can,therefore,be determined for a given protein.However,prior knowledge of a candidate TF is required because it can be cumbersome to survey an entire TF family.Therefore,this approach is ideally used for fine-mapping sequence specificity of a previously identified mCpG-binding protein.ChIPBS-seqTo determine methylation-dependent protein-DNA interactions in vivo,chromatin immunoprecipitation followed by bisulfite sequencing(ChIPBS-seq)is an ideal approach.ChIP is first performed to obtain the DNA sequences tha