Transcriptional fusions of promoters with a lux reporter, together with in vitro transcription, EMSA and footprinting assays revealed that: (1) the different sequence composition of the −35 promoter element is responsible for substantial divergence in strengths of the promoters; (2) variations in repression result from the TA repressor complex acting at different steps in the transcription initiation process; (3) transcription from an additional promoter upstream of atp also contributes to the observed inefficient repression of axe-txe module. This study provides evidence that even closely related TA cassettes with high sequence similarity in the promoter/operator region may employ diverse mechanisms for transcriptional regulation of their genes.Transcriptional repression is a mechanism which enables effective gene expression switch off. The activity of most of type II toxin-antitoxin (TA) cassettes is controlled in this way. These cassettes undergo negative autoregulation by the TA protein complex which binds to the promoter/operator sequence and blocks transcription initiation of the TA operon. Precise and tight control of this process is vital to avoid uncontrolled expression of the toxin component. Here, we employed a series of in vivo and in vitro experiments to establish the molecular basis for previously observed differences in transcriptional activity and repression levels of the  Escherichia coli σ70 subunit, responsible for transcription of most genes required for cell growth and maintenance (housekeeping genes), is composed of four separately folded domains connected by flexible linkers. Each of these domains interacts with the core enzyme and also makes specific contacts with corresponding promoter elements [Gene transcription is a multistep process carried out by DNA-dependent RNA polymerase (RNAP). Bacterial RNAP is composed of β, β′, ω and two α subunits which form a core enzyme capable of nonspecific DNA binding. However, specific promoter DNA sequence recognition enabling efficient transcription is performed by the RNAP holoenzyme which is assembled from the core enzyme and one of several σ factors. The elements ,2,3,4.70 contains a number of characteristic sequence elements. The −10 and −35 boxes, which are named to indicate their approximate distance from the transcription start site (+1) are known to make direct contacts with surface exposed regions of the σ subunit. The −10 and −35 hexamers have 5′-TATAAT-3′ and 5′-TTGACA-3′ consensus sequences, respectively [A typical bacterial promoter recognized by sigmaectively .In prokaryotes, transcription initiation is a multistep process ,10,11,12Critical contacts mediated between the promoter DNA elements and the RNAP holoenzyme modulate the frequency and efficiency of transcription initiation, and thereby regulate gene expression ,8,11. ItInhibition of transcription initiation is an important and common mechanism of gene expression regulation in bacteria. Proteins acting as repressors bind to specific sites, called operators, often positioned within or in close proximity of the promoter region. The level of gene expression is modulated as a result of steric hindrance or interactions between the transcription factor bound to these sites and RNA polymerase ,19,20. POne of examples where negative transcription regulation is a crucial strategy for precise gene regulation is illustrated by the toxin-antitoxin (TA) systems ,25,26. TyefM-yoeB cassette has been identified on E. coli chromosome and also in the genomes of many diverse bacterial species. Moreover, its homolog named axe-txe has been discovered on different Enterococcus faecium plasmids. Similarly to a typical type II TA system, the E. coli yefM-yoeB module undergoes transcriptional autoregulation where the YefM antitoxin serves as a weak repressor while YoeB acts as a co-repressor enhancing efficiency of promoter inhibition [yefM-yoeB systems were assessed in terms of their gene regulation and these investigations showed that in other organisms this process can be more complex than in the E. coli example. Expression of this locus in Streptococcus pneumoniae was shown to be driven from two different promoters located upstream of the antitoxin gene, one of them being regulated by the YefM-YoeB proteins while the other, weaker promoter is constitutive [yefM-yoeB system from Lactobacillus rhamnosus and for axe-txe from pRUM plasmid of E. faecium, where additional promoters were identified within the antitoxin gene and an antisense transcript was found to overlap with the toxin gene [The hibition ,38. To dtitutive . Even moxin gene ,41.Escherichia coli K-12 chromosomal yefM-yoeB locus and the axe-txe cassette encoded by the pRUM plasmid of Enterococcus faecium [yefM-yoeB and axe-txe operons is mediated by binding of the N-terminal domains of the antitoxin molecules to pairs of palindromes that overlap their cognate promoter  which differs from the consensus sequence (5′-TAtAAT-3′) at position −10 where there is a C instead of T, however, that position is the least conserved site in this region [TTGACA-3′) [yy,p the second T is replaced by A (5′-TaGttA-3′), while for atp all three nucleotides are the same as in the consensus sequence (5′-TTGctt-3′). In both promoters, the linker between −10 and −35 boxes has a length of 17 bp which is typical for most promoters. Regarding the transcription start site, there is a purine base in both promoters, A in atp and G in yyp.Analysis of the elements . Both pryefM-yoeB in genomes of many diverse bacterial species, suggesting that it is a key feature of yefM-yoeB transcriptional control [The operator region located within both promoters consists of long (L) and short (S) repeats which provide a binding site for the antitoxins, YefM or Axe. Both palindromes have a common 5′-TGTACA-3′ core motif separated by the same distance of 12 bp from center to center . This di control .atp and yyp promoters demonstrated that they may have quite different transcriptional activities [yyp experiments a lacZ reporter fusion was used, while for assessing atp promoter activity a lux operon fusion was applied. This makes a direct comparison of the activity of both promoters impossible. To unify experimental conditions, here we employed yyp and atp transcriptional fusions with a promoterless lux operon as a reporter, using the pBBRlux-amp vector. pBBRlux-pyy and pBBRlux-pat constructs were introduced into E. coli SC301467 strain which is a MG1655 derivative where 5 out of 13 known type II chromosomal toxin-antitoxin cassettes have been deleted, including yefM-yoeB, to avoid any possible cross-interactions with homologous systems [600~0.5 and the results were divided by optical density of the cultures. The at-luxp fusion produced over 5 × 106 RLU, while yyp-lux fusion gave an activity below 3 × 105, which is over 20 times less than the other fusion  crude extracts were prepared where YefM, YefM-YoeB, Axe and Axe-Txe were overproduced from appropriate pET22(b) derivatives . BL21 ceexcluded . Howeveryyp and atp were incubated with increasing concentrations of cell lysates. As a control, crude extract of cells with pET22(b) vector plasmid was used and in this case no binding was observed  into the SC301467 strain, and co-transformed them with pBAD33 derivatives overexpressing YefM and YefM-YoeB under L-arabinose inducible promoters. The activity of lux fusions was measured when bacterial cell cultures reached OD600~0.5. In this set of experiments, we also included the yyp promoter variant with −35 box mutated to that of atp. The reason for this was to have a stronger version of the yyp promoter as a control, i.e., to assess if the potential diversity between yyp and atp is not solely due to the difference in the RNA polymerase affinity to a weaker or stronger promoter sequence. The results obtained are presented in yyp and yyp−35mut, than on atp: YefM lowered the yyp promoter activity to 4% and that of yyp−35mut to 11%, while atp transcriptional activity was repressed to only 63% relative to control conditions. Moreover, the YefM-YoeB complex abolished activity of both yyp and yyp−35mut to almost background levels, in both cases to below 1%, while the activity of atp was still detected at a relatively high level of 32%  were incubated together for 10 min, and then reactions were treated with the DNaseI endonuclease. Fully developed footprints overlapping appropriate L and S palindromic repeats could be observed for all three tested templates at 0.5 µM TA complex concentration  was allowed to bind to the DNA and then different concentrations of RNAP were added. To stabilize the RNA polymerase interactions with promoter fragments, the first two ribonucleotides were included in the reaction mixtures. It has been previously shown that binding of the initiating NTPs helps to shift the closed RNAP-DNA complex to the more stable open complex. RNAP alone covered the promoter region extending from around −55 do +25 base pairs  and at2patp in which −10 box of the at2p promoter was disrupted by changes TAGAAT to CGGAAT (pTEpat2mut−10pat) into pTE103 vector. Our results undoubtedly show that the upper band is not present when the DNA template contains mutated or no at2p sequence  and that the more similar −10 and −35 boxes are to the consensus the stronger the promoter [atp all three nucleotides are the same as in the consensus sequence (5′-TTGctt-3′), while for yyp the second T is replaced by A (5′-TaGttA-3′) which explains a weaker activity of the latter. We have also tested the effect of transcriptional start site nucleotide, but changing A to G and vice versa did not exert any substantial effects on the promoter activity. This also confirms previously published data showing that both purine bases are more or less equally effective in transcription initiation [In the report presented here we first established experimentally that the nucleotide sequence of the −35 box is responsible for the differences in the basal strength of yefM-yoeB and axe-txe modules appear to share high structure similarity, their cognate partners do not cross-interact [yyp promoter and by Axe-Txe at the atp promoter. While yyp inhibition was reaching almost the background level, atp repression was not efficient and still a substantial promoter activity could be detected. After careful analysis of both promoter/operator regions, we noticed that although both repressor binding palindromes were mostly identical in sequence, they were located differently in relation to the main promoter elements. This prompted us to suspect that maybe different steps in the transcription initiation process are perturbated in each case.Previously, we have also shown that despite the fact the proteins of interact . Here, waxep) located within the axe antitoxin gene, which drives additional expression of the Txe toxin, makes it impossible to clone the axe-txe operon under a heterologous inducible promoter, like BADp used in these studies [axe-txe genes were cloned together with the atp promoter downstream of the T7 promoter. It was the only way we found to overexpress Axe-Txe, however, the stoichiometry of both proteins does not have to be optimal and can differ from naturally occurring in the wild type system. Apart from that, this fusion does not contain a His-tag which would enable to purify the Axe-Txe complex as for an unknown reason a His-tagged version of Txe did not allow for overexpression of the complex. Nevertheless, EMSA assays with lysates obtained by overexpression of proteins of both systems clearly indicate that proteins of the axe-txe and yefM-yoeB systems are able to bind to their cognate and non-cognate operator regions with similar efficiency. The atp activity in the presence of Axe and Axe-Txe was also previously measured in in cis lux-transcriptional fusions [in trans fusions and YefM-YoeB proteins. In our opinion, these data allow us to assume that experiments with proteins of only one of these systems are sufficient to produce relevant answers about mechanism of transcriptional repression of both tested toxin-antitoxin cassettes.We decided to perform most of experiments only with YefM-YoeB proteins for several reasons. The presence of additional promoter , including the well-studied Fur repressor of promoter ,50. Wherpromoter ,52,53. Oormation .yyp promoter, the L repeat, overlaps the −10 promoter box, while the S repeat encompasses the transcription start site. That kind of placement of the operator sequence suggests creation of a steric hindrance for RNA polymerase binding. Such mechanism of transcription repression has been confirmed by our EMSA and DNaseI footprinting experiments where the bound repressor either prevented RNAP from binding at the same time or did not allow RNAP to effectively displace it. On the other hand, in our studies, transcription inhibition mediated by the YefM-YoeB or Axe-Txe complexes at atp was not as efficient as at the yyp promoter, despite identical binding core sequences in both operators. We propose that the reason for that is different location of both operator sites relative to the promoter recognition elements resulting in different repression mechanisms. At atp the major repressor binding site, the L repeat, lies within the linker between −10 and −35 promoter boxes, while the S repeat partly overlaps the −10 hexamer. Such operator sequence location potentially enables simultaneous RNAP and repressor binding and transcription inhibition occurs at a later step. Binding site/s located within promoter spacer have been already identified for few repressors. For example, Arc protein of Salmonella typhimurium phage 22, MerR repressor and nucleoid-associated Fis protein have been demonstrated to slow the rate at which RNAP forms open complex or to inhibit promoter opening by trapping RNAP at a promoter [The primary binding site for YefM-YoeB complex at the promoter ,56,57.atp promoter enables simultaneous binding of the RNA polymerase and probably affects the open complex formation step. Repression at this step of transcription initiation seems to be less efficient than complete blocking of RNAP binding, like in the yyp example. Transcriptional autorepression is a characteristic feature of a vast majority of type II toxin-antitoxin systems, however to date a precise stage in the multistep transcription initiation process which is affected by repressor binding was not investigated in any of the known examples.To our knowledge, in all examples described to date where the repressor protein binds to the promoter spacer sequence, it influences the open complex formation step in the transcription initiation process. This is not surprising, since it has been shown that for effective promoter opening physical deformations causing conformational changes within the linker DNA occur in parallel with melting of the promoter sequence extending from the −10 box . Thus, ayefM-yoeB and axe-txe regulation is the presence of an additional active promoter located 40 bp upstream of the major atp promoter, which we called at2p. Its presence was discovered by in vitro transcription experiments where we noticed a band above the main transcript. To confirm this promoter’s activity in vivo, we have used transcriptional fusions with the lux reporter gene, which were devoid of the sequence fragment upstream of the atp promoter, including this promoter region. Transcription activity measured just for the main atp promoter appeared to be lower by around 28% in comparison with the sample where the two promoters were present. Its presence and activity was also confirmed in in vitro transcription assays. These experiments also indicate that at2p activity masks the repression level mediated by the TA complex at the atp promoter, since it does not seem to be regulated by this protein complex. Results presented in atp promoter is repressed by the YefM-YoeB complex, the higher transcript level initiating from the at2p promoter. We assume that this results from more RNAP molecules becoming available for binding on this DNA region and is not due to a direct activation by the TA complex. Computational analysis of E. coli and other bacterial species regulatory regions revealed that they contain clusters of promoter-like signals, in contrast to the coding regions. In other words, functional promoters occur mostly within regions with high density of overlapping putative promoters [Another element which we found to be different in the romoters . With grromoters . One canE. coli mazEFG module is preceded by two promoters located 13 bp apart, P2 and P3, whose relative strengths differ by approximately ten-fold [yefM-yoeB operon of Streptococcus pneumoniae provides another example of transcriptional control mediated by a pair of promoters located upstream of these TA genes. Both promoters are located 30 nt apart from each other. In this case, the expression from one of them, yefM1p is constitutive while the other, stronger promoter, situated more downstream, yefM2p, is negatively autoregulated by YefM-YoeB complex. yefM1P was found to be 15-fold weaker than yefM2p, and its presence was shown to decrease the overall promoter activity in yefM-yoeB operon [hicAB locus of E. coli was also shown to be transcribed from two promoters [P1, generates a hicAB transcript that produces both HicA and HicB. On the other hand, the downstream P2 promoter is autorepressed solely by HicB antitoxin and produces only HicB but not HicA toxin. These results indicate that the P1 and P2 generated transcripts are translated at highly different rates [There are only single examples of known class II TA cassettes whose transcription is directed by two adjacently positioned promoters. The ten-fold ,60. TranB operon . The hicromoters . It has nt rates . Transcrat2p promoter we cannot speculate about its regulation. However, it appears that the regulation of axe-txe expression in terms of the regulatory elements located upstream of the axe gene resembles the situation found in streptococcal yefM-yoeBSpn, where the strong main at2p promoter is precedent by a weaker, constitutive promoter at1p [at2p.As we have not found any obvious regulatory elements upstream of the ter pat1 . This maatp and at2p promoters is 40 bp it suggests that transcription from both promoters cannot occur simultaneously and is rather subjected to some kind of competition. Moreover, it is likely that both promoters are positioned on the same face of the DNA double helix since the distance between them is close to four full helical turns. RNA polymerase molecules may bind simultaneously to both promoter sites however at at2p only closed complexes could be formed, while at atp one αCTD of RNAP would be possibly displaced. Thus, it seems possible that each promoter may influence the other’s binding by RNAP and thus modulate the overall promoter activity of the axe-txe transcription unit. In the future it would be worth elucidating the interplay between them by mutational analyses where each of these two adjacent promoters would be inactivated and tested under repressive and non-repressive conditions.During formation of a loose, unstable initial closed complex (RPc) RNAP protects the promoter DNA from position −55 to −5 (around 50 nt). When RNA polymerase forms transcriptionally active complex (open complex) it covers the promoter sequence extending from about −50 to +20 (around 70 nt) in respect to the transcription start site. Since the distance between the 70 subunit of RNA polymerase. At yyp, steric hindrance occurs, repressor binding is strong, and RNAP cannot reach the crucial promoter elements and thus repression is efficient. In contrast, at atp, the repressor binding site only partially overlaps with the RNAP binding elements, so both protein complexes can bind DNA at the same time, and thus repression occurs at further stages of transcription initiation and this repression mechanism is not as effective. Moreover, a second promoter located upstream of atp, called at2p, contributes to gene expression of the axe-txe module and its transcription does not seem to be directly regulated by the TA complex bound to the main promoter  was used for crude extract preparation with Axe, Axe-Txe, YefM and YefM-YoeB overproduction from pET22axe and pET22at_axe-txe, pET22yefM and pET22yefM-yoeB, respectively. BL21(DE3) was used for YefM-YoeB6His overproduction and purification. Strain SC301467, a derivative of MG1655 devoid of mazF, chpB, relBE, dinJ-yafQ and yefM-yoeB [E. coli cells were grown in Luria-Bertani (LB) medium at 37 °C with shaking. Ampicillin and chloramphenicol were added to final concentrations of 100 µg/mL and 34 µg/mL, respectively, when required. Enterococcus faecalis strain OG1RF was used for β-galactosidase assays with derivatives of the pTCVlac vector [efM-yoeB . E. colic vector . For the600~0.5. Expression of axe (pET22axe), axe-txe (pET22at_axe-txe), yefM (pET22yefM) or yefM-yoeB (pET22yefM-yoeB) was induced with 1 mM IPTG and incubation continued for 3 h. Cells were harvested at 1600× g for 10 min. The pellet was resuspended in 1 mL of buffer containing 20 mM Tris-HCl pH 7.5 and 50 mM NaCl. The cells were sonicated and then centrifuged for 30 min at 15,500 × g at 4 °C. Supernatant was dialyzed against the same buffer containing 10% glycerol. Glycerol was added to the samples up to 50%, which were then aliquoted and stored at −20 °C.Bacteria were grown at 37 °C in 10 mL of LB medium with appropriate antibiotic until ODE. coli BL21(DE3) and purified by Ni2+ affinity chromatography essentially according to the Novagen technical manual. 150 mL of culture harboring the expression plasmid pETyefM-yoeB was grown at 37 °C until OD600 ≈ 0.8, expression was induced with 1 mM IPTG and growth continued for 3 h. Cells were harvested at 4000 rpm at 4 °C for 10 min. The pellet was resuspended in 4 mL of binding buffer  with lysozyme (0.1 mg/mL). Next, the cells were sonicated and then centrifuged for 1 h at 15,000 rpm at 4 °C. The supernatant was applied to a column containing 2 mL of His-tag resin (Roth) and equilibrated with the binding buffer. Binding of the fusion protein to the resin was allowed to continue for 1 h at 4 °C after which the column was washed with 15 mL of wash buffer . Elution was performed with 5 mL of elution buffer  and 1 mL fractions were collected. Two fractions containing the YefM-YoeB complex were combined and dialyzed against 1 L of storage buffer  overnight, and then for 2 h against fresh storage buffer. Glycerol was added to the samples up to 50%, which were then aliquoted and stored at −20 °C.YefM-YoeB complex was overproduced in E. coli was purified as described [Native RNA polymerase from escribed  with modescribed .lux operon under transcriptional control of the wild-type and mutated yyp or atp promoter fragments were used. PCR fragments were cloned into pBBRlux-amp between SpeI-BamHI restriction sites upstream of the promoterless luxCDABE to yield proper transcriptional fusions. Where indicated, the strains with pBBRlux fusions were additionally co-transformed with pBAD33 plasmids [yefM or yefM-yoeB genes under control of an arabinose-inducible promoter (pBADyefM and pBADyefMyoeB). Overnight cultures carrying recombinant plasmids were diluted (1:100) into fresh LB medium and grown until OD600~0.5. Where necessary, synthesis of antitoxin or toxin-antitoxin was induced by addition of 0.2% L-arabinose at time of culture dilution. Then, luminescence of 200 µL of cell cultures was measured in a luminometer . Results, obtained in relative light units (RLU), were divided by the optical density (OD600) of the cultures.Strain SC301467 harboring derivatives of pBBRlux-amp with the plasmids  encodingatp (primers 156 and 157) and yyp (primers 154 and 155) promoter regions were used in EMSA studies. Reactions containing 10 nM of cy5-labeled DNA and bacterial crude extract or purified YefM-YoeB6His complex were assembled in binding buffer , 2.5% glycerol) in final volumes of 20 µL and incubated for 20 min at RT. Then samples were electrophoresed on 6% native polyacrylamide gels in 0.5× TBE buffer for 120 min at 100 V at RT. Detection of the cy5–labeled DNA was performed using Typhoon scanner. Competitive EMSA with YefM-YeoB6His complex and E. coli RNA polymerase holoenzyme were performed on cy-5 one strand labelled DNA fragments  in the reaction mixture and conditions described above, however samples were run on 4% native polyacrylamide gels in 0.5× TBE buffer for 70 min at 100 V at RT.Cy5-labeled double-stranded PCR fragments that included the atp and yyp promoters was analyzed in multiround in vitro transcription assays performed with circular plasmid DNAs  (25 nM) and YefM-YoeB6His complex were added in the indicated order and samples were incubated for 10 min. The reactions were started by addition of ribonucleotides, final concentrations: 0.15 mM of GTP, ATP and CTP, 0.015 mM of UTP and 0.8 µCi α32P-UTP, and were run for 15 min. Then, 20 µL of stop solution  was added and samples were denatured for 5 min at 95 °C prior to loading 20 µL on a denaturing 7 M urea, 6% polyacrylamide gel, and quantified by phosphor-imaging with a Typhoon 9200 imaging system .Transcription activity of 3 vector ) as indi2, 100 mM NaCl, 1 mM β-mercaptoethanol. PCR fragments with 5′ cy5 label and containing the yyp (primers 154 and 181) or atp (primers 156 and 181) promoters were used as templates . To stabilize the RNAP-DNA open complexes, ATP and UTP (for atp) and GTP and UTP (for yyp) were added to 125 µM. RNAP was used at 0, 50, 100, 150, 200 nM, and YefM-YoeB6His was used at 1 µM. When RNAP or YefM-YoeB6His were assayed alone, the samples were incubated at 37 °C for 10 min, followed by an addition of DNase I  and further incubation at 37 °C for 2.5 min. In reactions where RNAP and YefM-YoeB6His were assayed together, one or the other was pre-incubated at 37 °C with DNA in the reaction buffer for 10 min, followed by the addition of the other protein and further incubation for another 10 min. Reactions were terminated by addition of EDTA to 25 mM and concentrated by vacuum evaporation. Next, samples were resuspended in 14 µL of loading solution , denatured at 100 °C for 2 min, and 7 µL were loaded on 7 M urea, 8% acrylamide gels along with sequencing reactions . Typhoon image system  was used for scanning and documentation.For these assays, protein-DNA binding reactions were performed in 20 µL in the following buffer: 50 mM Tris-Cl (pH 8.0), 10 mM MgCllacZ gene under transcriptional control of the atp or at2patp promoter fragments. PCR fragments were cloned into pTCVlac [EcoRI and BamHI restriction sites upstream of promoterless lacZ gene. Overnight cultures carrying recombinant plasmids were diluted (1:50) into fresh BBL Trypticase Soy medium and grown until OD600~0.5. Then β-galactosidase activity assay was performed with cells permeabilized with chloroform and SDS as described by Miller [Electrocompetent cells of OG1RF strain were electroporated with derivatives of pTCVlac vector bearing the  pTCVlac  between y Miller . We founy Miller .http://molbiol-tools.ca/promscan/).Promoter searches were performed using PromScan bioinformatic program (In most cases genes are not simply turned on or off, but instead their expression is precisely fine-tuned to suit the demands of the cell in response to changing environmental conditions, as well as to keep intracellular balance. How genes are modulated so precisely is still not well understood, but it appears that mechanisms driving this regulation are more complex and multilayered than previously expected. This is because many of these regulatory mechanisms/elements produce a subtle effect/change that easily can be overlooked or unnoticed under standard experimental conditions/procedures. It is obvious that in many cases only the major—i.e., giving the strongest effect—elements are being studied and described. Due to the engagement of type II TAs in virulence, antibiotic tolerance and biofilm formation, it is crucial to understand how these genes are regulated at a molecular level. Our work substantially adds to these studies.


Digestive cancers are the leading cause of cancer-related death worldwide and have high risks of morbidity and mortality. Histone methylation, which is mediated mainly by lysine methyltransferases, lysine demethylases, and protein arginine methyltransferases, has emerged as an essential mechanism regulating pathological processes in digestive cancers. Under certain conditions, aberrant expression of these modifiers leads to abnormal histone methylation or demethylation in the corresponding cancer-related genes, which contributes to different processes and phenotypes, such as carcinogenesis, proliferation, metabolic reprogramming, epithelial–mesenchymal transition, invasion, and migration, during digestive cancer development. In this review, we focus on the association between histone methylation regulation and the development of digestive cancers, including gastric cancer, liver cancer, pancreatic cancer, and colorectal cancer, as well as on its clinical application prospects, aiming to provide a new perspective on the management of digestive cancers. The latest epidemiological data indicate that an estimated 333,680 people will be diagnosed with digestive cancers in the United States in 2020 and that ~167,790 will die of these diseases; both of these estimates rank first among all cancers.2 For example, the transcription factor FOXA1 drives large-scale enhancer reprogramming through H3K27ac (acetylation of lysine 27 of histone 3) modulation, thereby promoting PC progression.3 An increase in H3K9me2 in the promoter region of RARRES3 gene represses its transcription, thereby contributing to cancer cell migration in hepatocellular carcinoma (HCC).4Therefore, further study of the molecular mechanism underlying digestive cancers is urgently needed. The interest in epigenetics has increased. Histone modifications, including acetylation, methylation, phosphorylation, and ubiquitylation, are a pivotal form of epigenetic information. Each of these modifications is associated with gene activity, gene silencing, or insulation between active and inactive gene regions.Since extensive studies have recently revealed the key role of histone methylation regulation in the development of digestive cancers, this review focuses on histone methylation. We highlight a cluster of histone methylation modifiers that play a vital role in digestive cancers—KMTs (lysine methyltransferases), KDMs (lysine demethylases), and PRMTs (protein arginine methyltransferases) Fig. . These ml-methionine to the ε-amino group of a histone lysine residue, thus regulating gene expression. For example, H3K27me3 (trimethylation of lysine 27 of histone 3) is often associated with transcriptionally repressed chromatin, and H3K4me3 is often linked to transcriptionally active chromatin. According to their defined protein domain or homologous sequence, KMTs are classified into eight distinct subfamilies: KMT1–8.5KMTs are a group of enzymes that catalyze monomethylation, dimethylation, or trimethylation by adding one, two, or three methyl groups, respectively, from S-adenosyl-6 the H3K9/56 methyltransferase G9a ,7 the H3K27 methyltransferase enhancer of zeste homolog 2 ,9 and the H3K4 methyltransferases KMT3E,11 KMT2A,13 KMT2D,14 etc., has been found to be ectopic expressed in digestive cancers. Among these KMTs, EZH2, a catalytic subunit of polycomb repressive complex 2 (PRC2),15 is one of the most commonly reported methyltransferases that represses gene expression in digestive cancers via H3K27me.16 EZH2 expression is elevated through the regulatory effects of various microRNAs and lncRNAs (long noncoding RNAs), thereby promoting the development of digestive cancers. For example, EZH2 is recruited by the lncRNA UCA1 and promotes direct interaction with the cyclin D1 promoter to activate the translation of cyclin D1, thereby fueling tumor growth in GC.17 Loss of miR-355 upregulates EZH2 expression by activating Sox4 in PC.18 In addition, loss of miR-26, miR-101, and miR-214 contributes to overexpression of EZH2, leading to the development of digestive cancers.23 Among the abovementioned microRNAs, the effect of miR-26 on EZH2 has been widely noted and has been identified in HCC, CRC, and GC.22 Another study identified that EZH2 can also bind to the lncRNA HOX-AS2 together with KDM1A to form a complex implicated in PC cell proliferation,24 and a similar function can also be performed by the lncRNA HOXA11-AS in GC.25 In addition to being modulated by microRNAs and lncRNAs, the expression of EZH2 is also promoted by cyclin-dependent kinase 4 (CDK4),26 Snail,27 Yin Yang-1 (YY-1),28 proviral integration site 1 (PIM-1) kinases,29 damage specific DNA-binding protein 2 (DDB2),30 nonacetylated homeobox B9 (HOXB9),31 and signal transducer and activator of transcription 3 (STAT3) signaling,32 thus contributing to the malignancy of digestive cancers. However, the activation of EZH2 may not be that straightforward; Battistelli et al. reported that the EMT-inducing transcription factor (EMT-TF) Snail recruits EZH2 to its target gene promoters in a HOTAIR-dependent manner,27 indicating that EZH2 activation is actually quite a complex process needing further exploration. Furthermore, the catalytic activity of EZH2 is not only determined by its own expression but also affected by its cofactors in PRC2. For example, activation of another core protein component of PRC2, embryonic ectoderm development (EED), promotes the catalytic function of EZH2, resulting in increased levels of H3K27me3 in PC cells.33 In addition, other studies have demonstrated that H3K27me3 levels in cancer cells are decreased concomitant with the reduction in the expression of suppressor of zeste 12 , inhibiting the development of HCC and GC.35A cluster of KMTs, including the H3K36 methyltransferase KMT3A,36 which possesses H3K4 methyltransferase activity and results in the installation of activating marks on chromatin at actively transcribed regions. Analysis of genomic and epigenomic features of HCC showed that p53 mutation led to independent transcriptional activation that could directly regulate a distinct set of chromatin regulators, including KMT2A.37 In addition, KMT2A expression is reduced by a decrease in the level of hPaf1/PD2 level, which decreases H3K4me2/3 and induces a corresponding decrease in the level of chromohelicase DNA-binding protein 1 (CHD1), an ATP-dependent chromatin remodeling enzyme that specifically binds to H3K4me2/3 marks.38 In addition to the influence of KMT2A targeting on MWRAD complex activity, the regulation of other components of the complex also affects its catalytic function.39 Studies have demonstrated that knockdown of WDR5 impacts H3K4 methylation in CRC cells and PC cells.41 Activation of WDR5 by the lncRNAs GCAWKR and GClnc1 facilitates H3K4me3 modification of the target genes protein tyrosine phosphatase type IVA  and superoxide dismutase 2  in GC.43 Furthermore, the lncRNA HOTTIP promotes HOXA9 expression by binding WDR5 to enhance Wnt/β-catenin pathway signaling, thus augmenting cancer stem cell (CSC) properties in human PC.44 Meanwhile, a study conducted by Mishra et al. has shown that KMT2A promotes leukemogenesis mainly through recruitment of MOF and forming H4K16ac (acetylation of lysine 16 of histone 4), not the intrinsic histone methyltransferase activity of KMT2A. However, the role of this mechanism in digestion cancers still needs further exploration.45KMT2A  is another histone methyltransferase attracting increasing attention in digestive cancers. KMT2A functions by forming the MWRAD complex with WD repeat domain 5 (WDR5), RBBP5, ASH2L, and DPY30,49 KDM6A, also called ubiquitously transcribed X (UTX chromosome tetratricopeptide repeat protein), is a Jumonji C (JmjC) domain-containing demethylase that targets H3K27 in complex proteins associated with Set1 (COMPASS), contributing to transcriptional activation.51 In solid pseudopapillary neoplasms (SPNs), which are borderline pancreatic tumors, recruitment of KDM6A to the enhancer of the SCL2A1 gene is inhibited due to deletion of BRCA1 associated protein-1 (BAP1),53 thus activating SCL2A1 transcription and driving the malignancy of SPNs. In addition to performing its histone methylation function alone, KDM6A can also interact with other histone modification enzymes. For example, KDM6A interacts with the histone acetyltransferase protein CBP and recruits it to the CDH1 (E-cadherin) promoter in HCT-116 CRC cells, resulting in increased H3K27ac, a transcription-activating histone mark.54 Similarly, during PC, KDM6A cooperates with the histone acetyltransferase p300 to regulate gene expression, while loss of KDM6A results in decreased H3K27ac levels in the promoter regions of tumor suppressor genes such as CDKN1A, LOXL1, SASH1, TXNIP, and IGFBP2.55 In addition, another study showed that loss of KDM6A rewires the enhancers and/or activated super-enhancers (SEs) of several tumor-related genes,56 which are formed by clusters of H3K27ac-marked enhancers.58KDMs are a class of enzymes that play a role in the demethylation of histone lysines. Similar to KMTs, KDMs are diverse, but recent whole-genome sequencing and copy number analysis identified KDM6A (lysine demethylase 6A) as one of the most frequently altered histone methylation modifiers in digestive cancers.59 KDM3A can also be activated by HIF1α via co-activators that bind to hypoxia response elements (HREs) in the doublecortin calmodulin-like kinase 1 (DCLK1) gene under hypoxic conditions in PC;60 DCLK1 is a marker of CSCs, and this process is promoted by protein inhibitor of activated STAT protein 4 (PIAS4).61 RUNX family transcription factor 3 (RUNX3)-mediated upregulation of miR-29b increases its targeting of the KDM2A 3′-UTR and decreases KDM2A expression, which suppresses the proliferation and migration of GC cells.62 Furthermore, KDM4A is activated by loss of miR-137,63 and KDM4C is activated by CircZMYM2 via its sponging of miR-335-5p in PC.64 Both KDM4A and KDM4C target H3K9me2/3, H3K36me2/3, and H3K56me2/3. Sun et al. also found that the lncRNA HOXA11-AS can recruit KDM1A (an H3K4me1/2 and H3K9me1/2 demethylase) and functions as a scaffold for KDM1A, EZH2, and DNA-methyltransferase 1 (DNMT1) in GC, resulting in the progression of cancer development.25 Moreover, KDMs are closely related both to each other and to other histone modification enzymes. For example, Tzatsos et al. found that KDM2B contributes to overexpression of KDM5A and EZH2, while KDM2B interacts with KDM5A and MYC to activate metabolic and ribosomal gene expression and binds EZH2 to silence the expression of lineage specification genes.65In addition to KDM6A, other KDMs are activated or inhibited under certain conditions, thus playing a corresponding role in digestive cancers. PHD finger protein 5A (PHF5A) K29 acetylation enhances KDM3A expression by stabilizing its mRNA. PHF5A acetylation increases the level of the H3K9me1/2 demethylase KDM3A by reducing its aberrant alternative splicing of its mRNA in CRC, thereby modulating stress responses and carcinogenesis in CRC.66Histone arginine methylation, which is catalyzed mainly by PRMTs, is also important in digestive cancers, but reports on histone arginine methylation are more scarce than those on histone lysine methylation to date. PRMTs can be divided into three categories according to their catalytic activity: type I , type II (PRMT5 and PRMT9) and type III (PRMT7). All PRMTs use S-adenosylmethionine (SAM) as the methyl donor to catalyze three major forms of arginine methylation on histones: monomethylarginine (MMA), asymmetric dimethylarginine (aDMA), and symmetric dimethylarginine (sDMA).68 and histone H3 arginine 2 (H3R2) methyltransferase PRMT6,70 are highly expressed in digestive cancer cells. During the development of colon cancer, intestine-specific peroxisome proliferator-activated receptor alpha (PPARa) deficiency promotes PRMT6 expression via the RB1/E2F pathway and increases the enrichment of H3R2 asymmetric dimethylation (H3R2me2a) in the promoter of p27.70 Another study demonstrated that PRMT9 promotes HCC invasion and metastasis by activating PI3K/Akt/GSK‐3β/Snail signaling.71 However, the study did not explain whether this activation was caused by histone methylation of related genes, and this possibility needs further investigation. In addition, PRMTs not only play a role through histone arginine methylation but can also be associated with other histone methyltransferases. For example, recent studies have demonstrated that upregulated PRMT5 can epigenetically silence the expression of the E3 ubiquitin ligase F-box and WD repeat domain-containing 7 (FBW7) by increasing H4R3me2s and H3K9me3 and decreasing H3K9ac in PC,72 indicating that PRMT5 can recruit histone KMTs and histone deacetylases to promote tumor development. This process results in c-Myc stabilization,72 and fibroblast growth factor-binding protein 1 (FGFBP1) is a downstream target of the FBW7/c-Myc axis; thus, the FGFBP1-mediated FGF signaling pathway may be involved in the process described above.73 However, in addition to the histone methylation function of PRMTs, their non-histone methylation function has also received increasing attention. For instance, PRMT3-mediated methylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and PRMT4-mediated methylation of malate dehydrogenase 1 (MDH1) are involved in the metabolic reprogramming and proliferation of PC cells.75Multiple studies have demonstrated that several PRMTs, such as the histone H3 arginine 8 (H3R8) and H4R3 methyltransferase PRMT5,Under stimulation by some external or internal factors, the regulation of histone methylation of cancer-related genes in normal digestive system cells may lead to abnormal cell growth and differentiation and thus to the occurrence of digestive cancer.76 Moreover, chromatin immunoprecipitation (ChIP) assays showed that during hepatitis B virus-induced hepatocarcinogenesis, HBV X protein (HBx) expression decreases the level of the silencing modification H3K27me3, while the level of the activating histone modification H3K4me1 is increased in the promoter of EpCAM, a host gene involved in HBV-mediated hepatocarcinogenesis.34 Similar conclusions were reached in a subsequent study conducted by Zhang et al.77Hepatitis caused by viruses, alcohol, or fat intake is a major cause of HCC, and histone methylation plays an important role in its progression. Fan et al. demonstrated that in mice, KMT1B-mediated H3K9me3 contributes to nonalcoholic steatohepatitis, which accelerates hepatitis-induced hepatocarcinogenesis. In hepatocytes, KMT1B-mediated H3K9me3 represses Sirt1 transcription, while in macrophages, KMT1B-mediated repression of PPARγ transcription favors the proinflammatory M1 phenotype over the anti-inflammatory M2 phenotype, thereby elevating hepatic inflammation.Helicobacter pylori significantly increases the occupancy of KDM4B and c-Jun, resulting in a significantly diminished H3K9me3 signal.78 Furthermore, another study identified three H3K27me modifier genes  that are individually associated with GC susceptibility via a synergistic triad interaction.46 During human colorectal carcinogenesis, mutations in Wnt/β-catenin signaling mediators may be one of the earliest events that initiates and drives tumor progression.79 Upregulation of disheveled segment polarity protein 22 (DVL), resulting from a significant decrease in H3K36me3 in the absence of KMT3A, leads to enhancement of Wnt/β-catenin signaling pathway activity and thereby drives colorectal carcinogenesis. Moreover, another study revealed the decrease in H3K27me3 and the increase in H3K4me3 in the WNT3 promoter region, indicating that histone methylation directly activates the Wnt/β-catenin signaling pathway to promote the initiation of CRC.80Regarding gastrointestinal carcinogenesis, researchers have found that KDM4B physically interacts with c-Jun on the promoters of IL-8, MMP1, and ITGAV via its demethylation activity, while infection with 82 At the early stage of definitive endoderm differentiation, KDM6A/B promote mesoderm differentiation by demethylating H3K27me3, which upregulates WNT3 expression and activates the WNT signaling pathway. At the late stage, KDM6A/B demethylates H3K27me3 to activate the WNT antagonist DKK1 in order to suppress the WNT signaling pathway and promote endoderm differentiation over mesoderm differentiation.82 However, under some conditions, such as injury to the pancreas or chronic inflammation, loss of EZH2 results in impaired pancreatic regeneration and accelerates KrasG12D-driven neoplasia.83 Further studies showed that EZH2 is upregulated in injured pancreatic cells, while the presence of KRAS reverses the function of EZH2 during late-stage regeneration, thus sustaining the expression of nuclear factor of activated T cells 1 (NFATc1), a crucial driver of pancreatic carcinogenesis and progression, through a decrease in H3K27me3 and increase in H3K4me3.84 This event integrates signals from inflammatory networks to control cellular plasticity and counteracts TGFβ-mediated apoptosis induction.87In the pancreas, histone methylation plays a crucial role in the growth and differentiation of pancreatic cells even under physiological conditions. Studies have shown that KDM6A/B are crucial in endoderm differentiation in the pancreas.89 This process, in conjunction with the expression of oncogenes such as K-ras, may trigger the development of PC.90 ChIP-seq data showed a significant gain of H3K27me3 and H2AK119ub (ubiquitination of lysine 119 of histone 2) at the regulatory acinar cell fate genes RBPJL, PTF1A, NR5A2, and BHLHA15 in pancreatic cells that had undergone ADM, and additional significant loss of H3K4me3 on these genes was found in PC cells.91 In addition, H3K4me3 on the Sox9 promoter increases as a result of NFATc1 activation during the ADM process, resulting in elevated expression of Sox9, a downstream effector of oncogenic Kras, and accelerates the formation of premalignant lesions in the pancreas.93Acinar-to-ductal metaplasia (ADM), which is essential for PC carcinogenesis, is a reprogramming event in which acinar cells display marked cellular plasticity and transiently transform into progenitor-like cells exhibiting a ductal morphology in pancreatitis.94 Histone methylation plays a key role in the uncontrolled proliferation of digestive cancer cells. For example, repressive H3K27me3 marks installed by EZH2 are enriched in the Kruppel-like factor 2 (KLF2) promoter, leading to a growth advantage in the tumor cell population in GC and CRC,96 via the HIF-1α/Notch-1 signaling pathway and the Hedgehog pathway.98 In addition, the proliferation of CRC cells is significantly accelerated by the EZH2-mediated H3K27me3 modification of the dual specificity phosphatase 5 (DUSP5) gene,99 a negative regulator of the mitogen-activated protein kinase (MAPK)-signaling pathway.100Uncontrolled proliferation is an important characteristic of tumor cells. Under appropriate conditions, cancer cells can multiply indefinitely and become immortalized.101 suggesting that histone methylation is indeed involved in cell cycle regulation. Further studies have focused mainly on the regulation of CKIs.Abnormalities in cell cycle network are closely related to the proliferation of tumor cells. During the development of digestive cancers, histone methylation can regulate cell cycle by regulating cyclins, CDKs cyclin-dependent kinase inhibitors (CKIs), thus causing cell cycle dysregulation and uncontrolled cell proliferation. Previous experiments with human pancreatic cell lines showed that KMT2D silencing leads to a decrease in the number and proportion of cells in G0/G1, accompanied by a global reduction in H3K4me1/2/3,104 CRC,106 HCC,108 and PC.109 This process, triggered by upstream lncRNAs such as BLACAT1, SNHG17 and CASC15, can inhibit the expression of P15 and P21 and lead to G0/G1 checkpoint deficiency.112 In addition, Zhang et al. found that EZH2-mediated H3K27me3 downregulates the expression of the p57 gene in addition to p15 gene, contributing to the regulation of the GC cell cycle and GC cell proliferation.113 In addition, TWIST recruits EZH2 to induce H3K27me3, which represses the transcription of the p16 gene by binding to the E-box in its promoter in PC cells under hypoxic conditions,114 and the same modification can be initiated by the lncRNA PVT1 in GC.115 P27kip1 is similarly inhibited by EZH2-mediated H3K27me3; the only difference is that EZH2 binds and modifies exon 1 rather than the promoter region.116 Another study suggested that EZH2 induced by KRAS mutation installs H3K27me3 in the promoter of miR-34a, thus epigenetically inactivating miR-34a expression to suppress the effects of p53 transactivation.117The genes encoding P15 and P21, two commonly mentioned CKIs, exhibit increased levels of H3K27me3 and H3K9me3 and decreased levels of H3K4me2/3 in digestive cancers, including GC,118 In addition, the transcription of CDK2 is promoted by KMT3E-mediated increases in H3K4me3 enrichment at the corresponding promoter sites.119 Regarding cyclins, the level of cyclin D1 is significantly increased due to decreases in repressive H3K9me2 marks and increases in activating H3K4me3 marks installed by KDMA/B and KMT2A, respectively.120 In addition, upregulation of Forkhead box transcription factor M1 (FOXM1) by KMT4-induced H3K79me2 in PC significantly increases the expression of its classical target genes cyclin A2 and cyclin B1 through the Wnt5a signaling pathway.121Similarly, histone methylation also regulates cyclin and CDK in digestive cancer cells. According to recent studies, Jumonji domain-containing protein Jumonji domain-containing protein 6 (6JMJD6), a histone arginine demethylase, activates CDK4 expression by decreasing the H4R3me2s level in the CDK4 promoter in HCC.122 All six of these apoptosis-regulating genes are involved in PC growth and progression.126 In addition, a genome-wide ChIP-sequencing analysis identified that H3K9me3 is enriched in the FAS promoter in metastatic human colon carcinoma cells; this enrichment inhibits the expression of Fas and results in resistance to Fas-mediated apoptosis.127 Furthermore, EZH2-mediated H3K27me3 by the lncRNA HOTAIR in the death receptor 5 (DR5) gene promoter results in resistance to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis;128 the same modification is found in the proapoptotic genes growth differentiation factor 15 (GDF15) and KLF2.130In addition to the uncontrolled proliferation caused by cell cycle dysregulation, abnormal negative regulatory mechanisms of cell proliferation, especially immune escape, can also cause abnormal proliferation during the development of digestive cancers. For example, researchers have demonstrated that during PC, H3K4me3 in the BCL2L1, CFLAR, and MCL-1 gene promoters upregulates the expression levels of the anti-apoptotic proteins Bcl-x, FLIP, and Mcl-1 and that H3K9me3 in the BAK1, BAX, and BCL2L11 gene promoters downregulates the expression levels of the pro-apoptotic proteins Bak, Bax, and Bim.131 However, the expression of CXC chemokine receptor 4 (CXCR4) is promoted by EZH2-mediated loss of miR-622,132 and CXCR4 can facilitate the evasion of immune surveillance by binding to CXCL12.133 Another experiment conducted by Bugide et al. showed that overexpressed EZH2 associates directly with the promoters of natural killer (NK) cell ligands, such as ULBP1 and MICA in HCC cells and then catalyzes the installation of repressive H3K27me3 marks on these promoters.134 In addition, Zhou et al. found that in PC, upregulation of FOXM1 by KMT4-induced H3K79me2 significantly attenuates antitumor responses, including bone marrow-derived dendritic cell (BMDC) maturation, cytokine secretion, and T cell activation, through the Wnt5a signaling pathway.121 Moreover, in PC cells, KMT2A overexpression increases the H3K4me3 level in the CD274 promoter region and upregulates the expression of PD-L1,135 a T cell inhibitory receptor ligand that results in potent immunosuppression.136In addition to apoptosis resistance, the production of the Th1-type chemokines CXC chemokine ligand 9 (CXCL9) and CXCL10, mediators of effector T cell trafficking, is suppressed by H3K27me3 in their gene promoters in colon cancer.137 Similarly, the Warburg effect can be induced in digestive cancer cells through histone methylation or demethylation of related genes. Glucose transporter 1 (GLUT1), one of the major components regulating glucose homeostasis,138 can be activated by p-ERK/KDM4B-mediated removal of the repressive H3K9me3 mark, thus contributing to glucose uptake in colon cancer cells under glucose deprivation conditions.139 KMT2D inhibition mediates the loss of H3K4me3 in metabolic pathway genes, which results in alterations in aerobic glycolysis and lipid levels via GLUT3-mediated processes.140 As GLUT3 exhibits a high affinity for glucose, thus ensuring efficient glucose uptake into cancer cells,141 this process induces aerobic glycolysis and increases the levels of lipids, such as docosadienoic acid, docosatrienoic acid, and docosatetraenoic acid, which harbors oncogenic properties in PC cells.140 In addition, Sakamoto et al. demonstrated that H3K4me2 demethylation mediated by KDM1A overexpression suppresses the expression of two metabolism-related genes, PGC-1α and LCAD, which are involved in the transcriptional control of genes related to mitochondrial oxidative metabolism and fatty acid oxidation, respectively.142 Similar modifications can occur on the glyceronephosphate O-acyltransferase (GNPAT) gene to activate its transcription; the only difference is that this process is triggered by c-Myc. GNPAT recruits the enzyme USP30, which deubiquitylates and stabilizes dynamin-related protein 1 (DRP1), thus promoting dysregulation of mitochondrial morphology and lipid metabolism in HCC.143 Moreover, KDM5A-mediated H3K4me3 demethylation of the mitochondrial pyruvate carrier 1 (MPC-1) gene transcriptionally inhibits its expression, leading to elevated mitochondrial pyruvate metabolism and inhibition of glycolysis. As a result, a series of metabolic disorders occur, such as hyperpyruvatemia and lactic acidosis.144 In addition, another study confirmed that cooperative binding of KDM2B, KDM5A, and/or MYC activates the expression of genes involved in metabolic homeostasis and protein synthesis, such as ATP5G2 and RPL3/7/24/30, via enrichment of H3K4me3 on these genes.65To support the ability for uncontrolled proliferation, tumor cells have adapted many aspects of their metabolic patterns. One of the most well-known examples is the Warburg effect, through which the rate of aerobic glycolysis is increased in cancer cells, thus leading to decreased glucose oxidation and enhanced flux through the anabolic side branches of glycolysis.145 In addition, Oliver et al. demonstrated that 6-aminonicotinamide (6AN), a 6-phosphogluconate dehydrogenase (PGD) inhibitor, induces broad enrichment of H3K9me2 across the CDH2 (N-cadherin) and topoisomerase 2β (TOP2B) genes, causing distant metastasis of PC.146Regulation of histone methylation can affect metabolism in digestive cancer cells, and the metabolites in digestive cancer cells can reciprocally affect histone methylation. For example, FH (fumarase), which catalyzes the reversible hydration and dehydration of fumarate, can bind to the c-Jun gene promoter, inhibiting KDM2A activity through local fumarate production and promoting H3K36me2.147 while progression of PC induces a hypoxic microenvironment through the Warburg effect and results in the production of a large amount of HIF1.149 A recent study conducted by Jiang et al. in CRC also supported this conclusion. O-linked N-acetylglucosamine transferase (OGT), which is negatively regulated by miR-101, enhances EZH2 protein stability and function and promotes the enrichment of repressive H3K27me3 marks on the miR-101 promoter region, resulting in the upregulation of OGT.150 This process creates a vicious cycle and further exacerbates tumor progression.Interestingly, collectively, these studies suggest that histone methylation and metabolic reprogramming may form a positive feedback loop to promote digestive cancer progression. For instance, Yi et al. reported that HIF1a stabilization under hypoxic conditions leads to KDM1A upregulation at the mRNA and protein levels in PC,151 Accumulating data have indicated that EMT leads to enhanced cell invasion and migration in digestive cancers.153 Moreover, the results of a tag and track experiment in pancreatic epithelial cells conducted by Rhim et al. indicated that EMT can occur even before the formation of cancer, showing that pancreatic epithelial cells can invade and enter the bloodstream to become circulating epithelial cells (CECs), maintaining the mesenchymal phenotype in pancreatic intraepithelial neoplasia (PanIN), a common precancerous lesion of PC.154 Several histone methylation modifications are involved in EMT in digestive cancers, especially EZH2-mediated H3K27me3 on the E-cadherin gene promoter. For example, Zhou et al. found that loss of ten-eleven translocation 1 (TET1) activates EZH2, which represses E-cadherin gene transcription by catalyzing H3K27me3 modification in the E-cadherin promoter in CRC cells.155 This modification leads to a decline in the expression of E-cadherin, a well-known hallmark of EMT.156 The same CDH1 gene promoter modification in CRC was also found in two other studies; one study showed that this modification was triggered by the lncRNA SNHG622 and the other showed that it was induced by the EMT-TF Snail2.157 During PC, EZH2 can also be recruited by TWIST and loss of microRNA-101, which binds to one or more E-boxes in the E-cadherin gene promoter and increases repressive H3K27me3 marks.158 In addition, EZH2-mediated H3K27me3 can regulate EMT indirectly through microRNAs and lncRNAs. EZH2 epigenetically silences miR-34a through H3K27me3 enrichment in the miR-34a promoter region. Downregulation of miR-34a in turn activates c-Met and thus results in transcriptional activation of Snail, thus contributing to the EMT process in GC cells and accelerating tumor metastasis.159 Another study demonstrated that miR-34a, along with miR-203, is repressed by EZH2-mediated H3K27me3, thereby activating the Snail1 and Snail2 EMT-TFs to downregulate E-cadherin.160 MiR-139-5p transcription is inhibited by EZH2 through an increase in H3K27me3; therefore, the levels of the EMT-TFs ZEB1 and ZEB2 increase, while the E-cadherin level decreases.161EMT is an important pathophysiological event that results in the loss of cell–cell adhesion, abnormal apical–basal polarity, and cytoskeletal reorganization, thereby enabling polarized, immotile epithelial cells to acquire mesenchymal abilities such as invasiveness and motility.54 In addition, G9a-mediated upregulation of H3K9me2, a heterochromatic histone methylation marker, has been observed at the same site in HCC cells and results in further inhibition of E-cadherin expression.163 Moreover, recent studies showed that ChIP assays can detect decreases in H3K9me3 and increases in H3K4me3, which are catalyzed by KDM4B and KMT7, respectively, in the ZEB1 promoter region.165 Meanwhile, KMT5C silences expression of the mesenchymal-to-epithelial transition (MET)-promoting transcription factors FOXA1, OVOL1, and OVOL2 via its repressive mark H4K20me3 in PC.166 Furthermore, PRMT1 can bind to the promoter region of CTNNB1 and increase the β-catenin protein level in PC cells,167 but whether this effect is related to histone methylation needs further exploration.In addition to EZH2-mediated H3K27me3, other histone methylation modifications are involved in regulating EMT in digestive cancers. Loss of KDM6A negatively regulates E-cadherin expression by coordinating the regulation of H3K27 methylation in the E-cadherin promoter in HCT-116 colon cancer cells, switching the transcriptionally active state to a transcriptionally repressive state.Invasion and migration of cancer cells into the surrounding tissue and vasculature is an essential initial step in cancer metastasis, which contributes to the terminal stages of cancer. During this process, digestive cancer cells invade deep tissue and enter lymphatic and blood vessels for dissemination into the circulation via regulation of histone methylation. This dissemination enables the cancer cells to colonize distant organs.168 as MMP7 facilitates tumor cell invasion and HMGA2 promotes invasion and migration through EMT.170 Moreover, two other MMP family proteins, MMP9 and MMP14, are also activated during the development of digestive cancers. The level of H3K9me3 in the MMP9 promoter is markedly decreased,171 while MMP14 exhibits robust accumulation of H3K4me3 and loss of H3K27me3.91For example, decreases in H3K27me3 in the MMP7 and HMGA2 promoter regions promote the progression of PC in Brg1-depleted PC cells,173 The opposite modification, demethylation of H3K9me3, occurs in the promoter of MALAT1, thereby upregulating the expression of MALAT1 and enhancing the activity of the β-catenin signaling pathway.174 In addition to H3K9 modification, decreases in H3K4me3 in BMP7, WIF1, and TIMP2/3 also significantly enhance the invasion and migration abilities of cancer cells in HCC and CRC,176 thus accelerating tumor progression. H3K27me3 also participates in this process. Tang et al. proved that aberrant KDM6B expression decreases H3K27me3 in the SLUG gene promoter and activates the transcription of SLUG, which promotes migration, invasion, and stem cell-like behaviors in HCC.177 Similar modifications can be detected in the Wnt10b promoter region in HCC; these modifications activate the Wnt/β-catenin signaling pathway and reinforce cell proliferation, migration, and invasion.178 Moreover, PRMT6-overexpressing GC cells also acquire invasiveness through direct transcriptional inhibition of PCDH7 by increasing H3R2me2as level.179 Another study conducted by Li et al. demonstrated that H3K27me3 and H3K9me2, mediated directly and indirectly by EZH2, are enriched in the miR-218-2 promoter, thus leading to downregulation of gene expression in PC. Loss of miR-218 increases invasion and migration through the effect of the UDP-glycosyltransferase 8 (UGT8) protein.180 In addition to EZH2, KDM6B is also involved in the processes of invasion and migration. Loss of this histone demethylase enhances the invasion and migration of PC cells through downregulation of CCAAT-enhancer-binding protein alpha (CEBPA); mechanistically, loss of KDM6B induces H3K27me3 in the region upstream of the CEBPA transcription start site.181In addition to MMP family proteins, researchers have also found that ectopic expression of a cluster of genes that participate in tumor invasion and metastasis in the digestive system is associated with abnormal regulation of histone methylation. For instance, the invasion and migration abilities of cancer cells are promoted by H3K9me3-mediated epigenetic silencing of numerous related genes, such as IGFBP3, CXCL3, NOS3, and SLIT1.182 was found to be downregulated in PC cells via a lncRNA HOTAIR-induced increase in the H3K27me3 level in its promoter.129 Moreover, researchers have proven that EZH2-mediated H3K27me3 suppresses RUNX3 gene expression in PC and GC.184 Notably, RUNX3 has traditionally been considered a tumor suppressor, but an article published in Cell in 2015 reported that it had a reinforcing effect on metastatic seeding and colonization via extracellular matrix (ECM) remodeling in PC.185 Therefore, in this case, inhibition of this suppressor reduces the malignant behavior of digestive cancer cells rather than promoting tumor development, indicating that EZH2 can function as both an oncogene and a tumor suppressor.However, conflicting results have been obtained. For example, inconsistent with the above results, GDF15, a migration-promoting factor,186 Moreover, the lack of effective drugs for chemotherapy and targeted therapy and resistance to existing drugs are other important reasons for the dismal prognosis. For these reasons, discovering effective drug therapies for affected patients is of paramount importance. As discussed above, regulation of histone methylation plays a pivotal role in digestive cancer development. Thus, strategies that regulate histone methylation modifiers and thus control histone methylation may constitute potential treatments for digestive cancers.As mentioned above, the regulation of histone methylation has substantial clinical potential, especially in treatment strategies for digestive cancers. Currently, radical surgical resection is the most effective treatment. However, the prognosis of digestive cancers is still not very optimistic. In PC, for example, fewer than 20% of patients have the opportunity for surgery, and of those who undergo resection followed by adjuvant therapies, more than 80% relapse and ultimately die of their disease.187 GC,188 CRC, and PC,191 but their therapeutic effect differs widely across patients. Studies have revealed that regulation of histone methylation plays an important role in determining the efficacy of and resistance to chemotherapy and targeted therapy;193 EZH2-mediated H3K27 methylation and KDM1A-mediated H3K4/9 demethylation194 are especially important. Lima-Fernandes et al. demonstrated that inhibition of the H3K27 methyltransferase EZH2 by UNC1999 resulted in increased sensitivity to 5-fluorouracil in CRC cells, accompanied by downregulation of H3K27me3 in the promoter of Indian Hedgehog and decreased self-renewal of CRC-initiating cells.195 In addition, metformin decreases the gene expression of EZH2 in pancreatospheres derived from gemcitabine-resistant PC cells, thus increasing the sensitivity of PC cells to gemcitabine via targeted killing of CSCs.196 Two other types of agents, lactate dehydrogenase A (LDHA) inhibitors and curcumin, have also recently been shown to display synergistic cytotoxic activity with gemcitabine in PC; the underlying mechanism is at least partially mediated by inhibition of EZH2.198Chemotherapy and targeted therapy are still the first-line treatment modalities for digestive cancers, including HCC,199 Further research proved that KDM1A inhibitors, such as pargyline and GSK2879552, dramatically suppress the stem-like properties of sorafenib-resistant HCC cells, including resensitization to sorafenib. Mechanistically, these KDM1A inhibitors negatively regulate the Wnt/β-catenin pathway through enrichment of activating H3K4me1/2 marks in the promoter of the Wnt antagonists Prickle1, APC and Sfrp5.200 This finding is consistent with the results reported by Lei et al., which indicated that knockdown of KDM1A sensitizes HCC cancer cells to cisplatin and sorafenib by increasing the expression of several suppressors of β-catenin signaling, especially Prickle1 and APC.201Suppression of KDM1A might be an attractive target for regorafenib sensitization and clinical HCC therapy, but an in-depth mechanistic investigation is lacking.202 Moreover, the G9a inhibitor UNC0638 notably enhances the cytotoxicity of topoisomerase-based treatment in CRC; mechanistically, H3K9me2 in the PP2A promoter is decreased, thus activating the PP2A–RPA axis.203 Verticillin A is a selective HMTase inhibitor that inhibits SUV39H1, SUV39H2, G9a, GLP, NSD2, and KMT2A, which decreases the H3K9me3 level in the FAS promoter and restores Fas expression, thus alleviating 5-fluorouracil resistance in CRC. Verticillin A is less toxic but exhibits greater efficacy than decitabine and vorinostat.127 Lu et al. proved that a sublethal dose of verticillin A effectively overcomes the resistance of human PC cells to gemcitabine and suppresses tumor growth.122 Furthermore, verticillin A was recently found to have a synergistic effect with anti-PD-L1 therapy in PC.135In addition to studies on targeting EZH2 and KDM1A, recent studies have demonstrated that small molecule inhibitors of KMT-4 (EPZ5676) combined with 5-fluorouracil or PARP inhibitors, which are used to treat CRCs, show additive effects accompanied by decreased H3K79me3 levels.204 DZNep (3-deazaneplanocin A), a potent chemical inhibitor of S-adenosylhomocysteine hydrolase that modulates chromatin accessibility through inhibition of histone methyltransferases, including EZH2,205 can lead to a significant reduction in H3K27me3 with a marked reduction in cell proliferation and migration in CRC. Similar effects can also occur in PC, decreasing the global H3K27me3 level and subsequently causing reexpression of miR-218, thus inhibiting cell proliferation, promoting apoptosis, and inducing cell cycle arrest in PC cells.180 A later study found that DZNep significantly alters miR-663a and miR-4787-5p expression and suppresses TGFb1-induced EMT signaling in PC.206 GSK126 is another EZH2 inhibitor that leads to epigenetic reprogramming in cancer cells, thus successfully promoting the infiltration of functional CD8þ T cells and substantially suppressing HCC growth.207 In addition, Huang et al. reported that suppressing EZH2-mediated H3K27me3 with GSK126 results in increased numbers of myeloid-derived suppressor cells (MDSCs) and decreased numbers of CD4+ and IFN-γ+ CD8+ T cells, which are closely related to antitumor immunity in CRC.208 UNC1999, an EZH2-specific inhibitor, not only reduced the aberrant H3K27 methylation that characterizes PC cells but also slowed the proliferation of cancer cells in three model systems.209 In addition, chaetospirolactone has been demonstrated to suppress the activity of the epigenetic regulator EZH2 and consistently decrease H3K27me3 to allow the transcription of DR4,210 which binds to TRAIL and leads to activation of the initiator caspase-8 and assembly of the death-inducing signaling complex (DISC).211 Subsequently, diosgenin, garcinol, FBW7, and the curcumin analog CDF were also identified as potential agents targeting EZH2 and thus hindering the development of PC.215In addition to reversing the effects of chemotherapeutic drug resistance, histone methylation can also constitute a direct therapeutic target in digestive cancers. In this regard, inhibition of EZH2-mediated H3K27me and G9a-mediated H3K9me/H3K56me are the most widely reported strategies. In fact, EZH2 inhibitors have even been identified as cancer prevention drugs. A recent study showed that suppressing EZH2 activity with GSK343 ameliorated experimental intestinal inflammation and delayed the onset of colitis-associated cancer accompanied by a gradual loss of H3K27me3 expression.7 Another study conducted by Kim et al. showed similar results in GC cells, while G9a inhibition in GC was mediated by kaempferol.216 A novel G9a inhibitor, BRD4770, was discovered to reduce cellular levels of H3K9me2 and H3K9me3 without inducing apoptosis, to induce senescence through ATM pathway activation, and to inhibit proliferation in the PC cell line PANC-1.218 Moreover, recent research has led to innovations in drug administration. Nanodiamond-mediated delivery of the G9a inhibitor UNC0646 maintained the biological functionality of UNC0646, with improved efficacy in reducing H3K9 methylation as well as enhancing the suppression of invasion in HCC cells,219 laying the foundation for future administration of small molecule histone methylation-regulating drugs. Similarly, dual targeting of G9a and DNMT1 by compounds such as CM-272 has also been proposed, thus providing a new perspective on HCC treatment.220Inhibition of not only EZH2 but also G9a counteracts the development of digestive cancers. For example, pharmacological knockdown of G9a activity triggers an increase in autophagy by decreasing histone H3K9me2 and increasing histone H3K9ac, which leads to elevated expression of autophagy genes, such as LC3B, WIPI1, DOR, and p62, in PC cells, ultimately decreasing cell viability.221 thus forming a positive feedback loop. Moreover, combination treatment with the pan-H3K9me HMT inhibitor chaetocin and an aurora kinase A (AURKA) inhibitor diminishes H3K9 methylation at centromeres, induces mitotic aberrations, triggers an abnormal mitotic checkpoint response, and ultimately leads to mitotic catastrophe in PC.222 At the same time, the prospect of arginine methylation as a therapeutic target has been revealed gradually. For example, a recent study found that targeting PRMT5 activity by DW47800 inhibits the malignancy of HCC by downregulating the binding of H4R3me2s to the HNF4α promoter.223 Meanwhile, AMI-1, a small molecule inhibitor of PRMTs, was demonstrated to strongly inhibit proliferation and migratory activity in HCC and GC, along with a decreased expression levels of H4R3me2s and H3R8me2s.225In addition to EZH2 and G9a, the novel curcumin analog L48H37 downregulates the histone methyltransferase KMT2D, as shown in a recent study, and KMT2D deficiency augments L48H37-induced apoptosis and blocks migration,226 Furthermore, the mechanism by which histone methylation regulates gene expression is still controversial. In contrast to the traditional understanding, some researchers believe that H3K4me3 may be not the “cause” but rather the “effect” of upregulated gene expression. Demethylation of CpG islands may shape the distribution of H3K4me3, and, in turn, H3K4me3 may influence the chromatin landscape at CpG islands.228 Therefore, the detailed mechanism linking H3K4me3 to upregulation of gene expression remains to be explored.As stated above, increasing attention has been focused on the role of histone methylation in the development of digestive cancers and its potential in clinical application. However, current studies still have limitations. For example, EZH2 has been found to act as both an oncogene and a tumor suppressor, since it maintains, rather than specifies, the transcriptional repression state of thousands of target genes. EZH2 acts most often as a mediator in tumor development; thus, it may play different roles under different conditions.229 Another study proved upregulation of PRMT5 induces increasing H4R3me2s and H3K9me3 in FBW7 gene in PC, indicating that PRMT5 can recruit histone KMTs and histone deacetylases to promote tumor development. These patterns suggest that interaction indeed occurs between histone arginine methylation and lysine methylation. Such interaction and internal mechanisms should be further investigated, such as the event upstream of this action and explanation of how histone modifiers are recruited. To broaden the research scope, the interactions among histone methylation, histone acetylation, DNA methylation, and other epigenetic modifications should also be given full attention.In addition, most current studies have focused on histone lysine methylation, and the role of histone arginine methylation has been relatively neglected. The crosstalk between histone arginine methylation and lysine methylation is also important, and may play important roles in the development of digestive cancers. Since histone methylation can regulate gene expression, histone lysine methylation may occur on histone arginine methyltransferase genes to regulate histone arginine methylation, and vice versa. Demetriadou et al. reported that activating H3K4me3 marks are reduced but repressive H3K27me3 marks are significantly enriched in the PRMT5 gene promoter in HCT116 cells with depletion of N-alpha-acetyltransferase 40 (NAA40).Moreover, although new therapeutic targets and molecular mechanisms are being revealed, only a modest number of clinical trials have been conducted Table . FurtherSupplementary Information