Determining HMTase specificity

The most parsimonious scenario would predict that each H3K36 methyltransferase enzyme catalyses a transition between two distinct methylation states (for example, between unmethylated H3K36 and monomethylated H3K36 (H3K36me1)). However, this is not necessarily the case. There are many discrepancies as to the level of methylation (mono-, di- and tri-) imparted, as well as the residue (or residues) that is targeted, by the various H3K36-specific methyltransferases; these discrepancies may arise from multiple factors, including the nature of the substrate tested (for example, peptides and histones versus nucleosomes), the source of enzyme (for example, full length versus SET domain only) and the assay conditions themselves (for example, antibody specificity versus mass spectrometry). Physiologically relevant substrates, characterized using nucleosomes and/or loss-of-function experiments, have been determined for some H3K36 methyltransferases (TABLE 1); but for enzymes such as SETD3, the enzymatic activities reported so far are based on analyses of peptides and core histones^11,12.

NSD1 as a mono- and dimethylase for H3K36

Although it was originally shown to bind steroid nuclear receptors¹³, nuclear receptor SET domain-containing 1 (NSD1; also known as KMT3B) was subsequently reported to use its SET domain to methylate H3K36 as well as H4K20 (REF. 14). However, NSD1 also methylates non-histone substrates, such as the p65 subunit of nuclear factor-κB (NF-κB)¹⁵. Indeed, several HMTases have multiple substrates, including non-histones, and it is important to consider this when interpreting phenotypes derived from loss-of-function experiments.

NSD1 has specific mono- and dimethylase activity for H3K36 (REFS ^16–18), generating H3K36me1 and dimethylated H3K36 (H3K36me2), and there are also mixed reports as to whether it has specificity for H4K20 (REFS ^14,19,20). Enzymatic assays using recombinant nucleosomes containing unmethylated H3K36 or a mimic of H3K36me1 (REF. 21) were shown to serve as specific substrates for NSD1 (REF. 16). However, when histone octamers were used as substrates, NSD1 was found to additionally methylate histone H4 as well as histones H2A and H2B, suggesting that nucleosomes contribute to H3K36 specificity. Structural data have also confirmed the specificity of NSD1 as a mono- and dimethylase that targets H3K36 (REF. 18). Intriguingly, this study showed that the substrate-binding channel of NSD1 is blocked by an autoinhibitory loop that resides between the SET and post-SET domains. As Lys36 resides near the core of the nucleosome, interactions between the enzyme and DNA may allosterically relieve inhibition by this loop, a result that is consistent with the preference of NSD enzymes for nucleosome substrates, as opposed to octamers. This furtherunderscores the necessity for using physiologically relevant substrates in order to properly characterize enzyme specificity towards H3K36.

NSD1 controls the levels of methylated H3K36 within and surrounding the body of the bone morphogenic protein 4 (BMP4) gene in human HCT116 colorectal cancer cells¹⁷. In the absence of NSD1, BMP4 expression is significantly impaired and the levels of all three forms of methylated H3K36 are reduced within the body of the gene. These data could support a model in which NSD1 catalyses H3K36 trimethylation (H3K36me3), but a more likely scenario is that the loss of mono- and dimethylation at H3K36 also inhibits trimethylation by failing to provide another HMTase, SETD2 (also known as KMT3A and HYPB), with its substrate. This is an important distinction to make when considering which HMTases regulate the formation of a trimethylated residue.

The role of NSD1 as a mono- and dimethylase also seems to be conserved in other metazoans. Worms and flies each have one NSD1 orthologue called maternal-effect sterile 4 (MES-4)^22,23. In Caenorhabditis elegans, MES-4 has been reported to be a dimethylase that is specific for H3K36 (REF. 22). Similarly to NSD1, Drosophila melanogaster MES-4 catalyses global mono- and dimethylation of H3K36 in vivo, but SET2, the fly orthologue of human SETD2, regulates all trimethylation at this site; again, MES-4 is probably required to provide the proper dimethylated H3K36 substrate for the SET2 enzyme.

In worms, mutation of the SETD2 orthologue histone methyltransferase-like 1 (met-1) significantly reduced global H3K36me3 levels²⁴. This supports the notion that the worm MES-4 and MET-1 proteins coordinate dimethylation and trimethylation, respectively²⁴. These experiments are generally consistent with studies of mammalian NSD proteins and SETD2 (REFS ^16,25). By contrast, separate studies showed by immunostaining that worm embryos individually deficient in mes-4 or met-1 retain significant levels of H3K36me3, but embryos doubly deficient in mes-4 and met-1 are completely devoid of H3K36me3 (REFS ^26,27). This suggests that MES-4 collaborates with MET-1 to perform trimethylation. As each of these studies has used different antibodies, it will be important to validate these experiments using additional, antibody-independent techniques, such as mass spectrometry. Collectively, the combination of in vitro and in vivo data suggests that NSD1 and its orthologues probably catalyse the addition of either the monomethyl or dimethyl groups onto H3K36 and so indirectly regulate the levels of trimethylation by altering the availability of monomethyl and dimethyl substrates for the trimethylating enzymes.

NSD2 as a mono- and dimethylase for H3K36

Similarly to NSD1, NSD2, the product of Wolf–Hirschhorn syndrome candidate gene 1 (WHSC1; also known as MMSET), has been reported to target H3K36, as well as H4K20, H3K4 and H3K27 (REFS ^16,28–32). Here again, these discrepancies may lie, at least in part, with the nature of the substrates used in the assays. For example, NSD2 acts as a dimethylase towards H3K36 when presented with nucleosomes, but it preferentially dimethylates H4K44 when presented with octamers¹⁶. Interestingly, when short single-stranded and double-stranded DNA molecules that are notably smaller than the length that is necessary to generate a nucleosome are added to octamers, NSD2 preferentially dimethylates H3K36, a result that has been attributed to the ability of DNA to act as an allosteric effector of NSD2 (REF. 16). This is consistent with another study, in which mass spectrometry and immunoblotting against native and recombinant nucleosomes showed that NSD2, as well as its major splicing variant, RE-IIBP, acts as a mono- and dimethylase specific for H3K36 (REF. 30). By contrast, there have been reports that the NSD2 isoform RE-IIBP is an H3K27-specific HMTase²⁸ and that H3K36 trimethylation is lost in embryonic stem (ES) cells derived from NSD2-defective mice³². But the idea that NSD2 might act as a trimethylase would not be consistent with a previous study that identified SETD2 as the sole trimethylase in mammalian cells²⁵. Furthermore, H3K36me3 levels are reduced when SETD2 is depleted, despite normal levels of H3K36me2 (REFS ^33,34). Regardless, these data are all consistent with a role for NSD2 in H3K36 methylation. This is also supported by the observation that, in ~15% of patients with multiple myeloma (a haematological malignancy that accounts for 1% of all cancers³⁵), NSD2 is overexpressed as a result of a translocation with the immunoglobulin locus (t(4;14)⁺) and the global levels of H3K36me2 are increased³⁶.

Possible specificity of NSD2 for H4K20

Despite strong evidence linking NSD2 to H3K36me, it may also act as an H4K20-specific methyltransferase during the cellular response to DNA double-strand breaks (DSBs)^37,38. NSD2 was isolated in an RNA interference screen for genes involved in resistance to hydroxyurea, a DNA-replication inhibitor³⁸. Consistently, NSD2-defective cells are sensitive to DNA damage and NSD2 localizes to sites of DNA damage. Moreover, NSD2 is phosphorylated at Ser102 by the kinase ataxia telangiectasia mutated (ATM) upon damage³⁷, and this mediates the recruitment of NSD1 to sites of DNA damage, where it has been suggested to dimethylate H4K20, which is an important event in recruiting the DNA damage response regulator p53-binding protein 1 (53BP1)³⁹. Although one could envision that NSD2 phosphorylation and damaged DNA may both alter the catalytic specificity of NSD2 through allosterism, it should be noted that recombinant NSD2 methylates H4 in in vitro assays with H4 peptides³⁷. Thus, it is possible that NSD2 does have some specificity for H4 in addition to H3 even in the absence of DNA damage.

The Set2 trimethylase

In yeast, Set2 is non-essential, performs all three methylation reactions at H3K36, and couples H3K36me3 with transcriptional elongation through an interaction with Rpb1, the large subunit of RNAPII¹⁰. The human orthologue of Set2, SETD2, also interacts with RNAPII during elongation, but it is an essential protein and has been identified as a huntingtin-interacting protein. The role of SETD2 in Huntington’s disease is obscure. SETD2 is a trimethylase^25,33,34, and some reports have indicated that, in vitro, recombinant human SETD2 can add all three methyl groups to H3K36 (REFS ^32,34). By contrast, in vivo knockdown experiments targeting SETD2 revealed reduced levels of H3K36me3 only³⁴. This underscores the importance of using multiple approaches to determine enzyme specificity and also raises an important point about enzyme processivity. Does a trimethylase obligatorily require a dimethyl substrate or can it processively place all three groups on a Lys substrate? SETD2 associates with heterogeneous nuclear ribonucleoprotein L (hnRNPL), and hnRNPL knockdown analyses show decreased levels of H3K36me3 but not H3K36me1 or H3K36me2 (REF. 34). Thus, the ability to act as a processive enzyme could depend on the presence and regulation of distinct cofactors, as well as on the presence of a previously methylated substrate.

Defining further HMTase specificities for H3K36

Like other NSD family members, the HMTase NSD3 (also known as WHSC1L1) appears to be specific for H3K36 (REFS ^16,40). So far, relatively little is known about the substrate specificities of the Trithorax protein ASH1-like (ASH1L) (an orthologue of the fly protein Absent, small and homeotic discs 1 (ASH1)), SET domain and mariner transposase fusion gene-containing (SETMAR; also known as METNASE), SETD3 and SET and MYND domain-containing 2 (SMYD2). Each has been reported to methylate H3K36, particularly with respect to dimethylation, but other substrate specificities have also been described for these enzymes^12,41–43. However, the assays for SETD3 specificity were performed with peptides¹¹, and it will be important to confirm this enzyme specificity using nucleosome-based assays^16,44.

In addition to targeting H3K36, SMYD2 methylates H3K4 and non-histone substrates, such as p53 and retinoblastoma (RB)^45,46. The in vitro methylase activity of SMYD2 towards H3K4, but not towards H3K36, depends on its ability to bind heat shock protein 90α (HSP90α)⁴². Although the expression of SMYD2 appears to be largely confined to the brain and heart, its conditional deletion from mouse cardiomyocytes showed that it is dispensable for heart development and, surprisingly, its deletion had no effect on the global levels of methylated H3K36 (REF. 47). This may suggest that the physiological target for SMYD2 is a non-histone substrate, providing an additional example of how the interpretation of knockout phenotypes for HMTases must be carefully considered.

Rigorous experiments using recombinant nucleosomes have demonstrated that ASH1L is a dimethylase that is specific for H3K36 (REFS ^41,48). However, ASH1L has also been reported to target H3K4 and to localize within the transcribed regions of active target genes⁴⁹; and in vivo levels of H3K4me3 were reduced in the absence of ASH1L. Additional studies have shown that ASH1L also targets H3K36 and H4K20 (REFS ^44,46). Thus, it remains to be seen what the full substrate repertoire of ASH1L is. Notably, similarly to NSD1, ASH1L has an autoinhibitory loop that resides between the SET and post-SET domains, which is a feature that has been proposed as a hallmark feature of H3K36-specific enzymes^41,48.

H3K36 methylation and transcriptional activation

Numerous studies in multiple systems support a role for H3K36 methylation in transcriptional activation. It has been observed that, in general, there is a progressive shift from monomethylation to trimethylation of H3K36 between the promoters and the 3′ ends of genes⁵⁸. However, in zebrafish, somatic cells also show a bias for H3K36me3 in the 3′ end of actively transcribed genes, but this mark is curiously present in the 5′ promoter regions of quiescent genes that are developmentally regulated during spermatogenesis⁵⁹. The functional significance of this is unknown, and it will be important to determine whether promoter-proximal H3K36me3-containing nucleosomes can recruit repressive histone deacetylases to promote gene silencing.

Multiple lines of evidence in budding yeast have showed that Set2, a non-essential enzyme that is responsible for all three forms of methylated H3K36, is coupled to transcriptional elongation. Deletions in yeast Set2 cause sensitivity to the elongation inhibitor 6-azauracil and phenocopy mutations in other known elongation factors¹⁰. However, it remains possible that this phenotype may arise from indirect effects on nucleotide metabolism. Nevertheless, Set2 associates with the hyperphosphorylated form of RNAPII and deposits the trimethyl group onto H3K36 during elongation^10,60–62 in various systems, including yeast and humans. This interaction is regulated by phosphorylated residues in the CTD of Rbp1, the large subunit of RNAPII: the Ser2-phosphorylated form of the CTD is indicative of elongation and the Ser5-phosphorylated form is characteristic of a paused polymerase at a promoter⁶³. Indeed, human SET2 proteins also appear to bind RNAPII and to target H3K36 (REFS ^34,64).

One well-established function of Set2 in yeast is the prevention of aberrant transcriptional initiation within coding sequences (FIG. 2). Set2 catalyses H3K36 methylation co-transcriptionally and recruits the reduced potassium dependency 3 small (Rpd3S) deacetylase complex here^65–67 through association of the chromodomain-containing Rpd3S subunit ESA1- associated factor 3 (Eaf3) with H3K36me3; this enforces a deacetylated chromatin state in the wake of transcribing RNAPII^65,68. Rpd3S preferentially associates with histones containing H3K36me2 and H3K36me3 but not H3K36me1 (REF. 57). The PHD finger of the Rpd3S subunit Rco1 can also cooperate with the Eaf3 chromo-domain to promote these interactions⁶⁹. This indicates that H3K36me2 and H3K36me3 might act redundantly in the Set2–Rpd3S pathway.

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Figure 2

H3K36me3-dependent prevention of aberrant transcription in yeast

Actively transcribing RNA polymerase II (RNAPII) displaces acetylated nucleosomes. These evicted histones are reincorporated into nucleosomes and chromatin behind the polymerase. As SET domain-containing 2 (Set2) binds RNAPII, this promotes the trimethylation of Lys36 on histone H3 (H3K36me3) in the newly incorporated nucleosomes. H3K36me3 serves as a ‘mark’ that the reduced potassium dependency 3 (Rpd3) deacetylase complex binds; this complex facilitates local nucleosome deacetylation, preventing aberrant, spurious transcription in the wake of RNAPII progression through a region. CTD, carboxy-terminal domain; HAT, histone acetylase; m⁷G, 7-methylguanosine.

The role of histone Lys methylation in the maintenance of a repressive chromatin environment during transcriptional elongation is also used in humans but may be independent of acetylation⁷⁰. In this case, Lys demethylase 2 (LSD2; also known as KDM1B) demethylates methylated H3K4 in the intragenic regions of active target genes. LSD2 resides in complexes with NSD3, which acts as a mono- and dimethylase that is specific for H3K36 (REF. 16). Additionally, LSD2 binds the H3K9-specific HMTase G9A (also known as EHMT2). Consistent with this, the LSD2 complex has robust HMTase activity towards H3K9 and, to a lesser extent, H3K36 (REF. 70). Moreover, in chromatin immuno-precipitation followed by microarray (ChIP–chip) experiments, depletion of G9A alters both H3K9 methylation levels and transcriptional regulation at LSD2 targets. In light of these observations and the fact that LSD2 resides in complexes with elongation factors such as cyclin T1 (CCNT1; also known as PTEFb) and the Ser2-phosphorylated form of RNAPII, it appears that the methylation-dependent enforcement of a repressive chromatin state through H3K9 and possibly H3K36 methylation at actively transcribed genes is evolutionarily conserved. Additionally, it seems that NSD3 can participate in transcriptional elongation⁷⁰. Interestingly, the LSD2 complex contains multiple proteins with PWWP domains, including NSD3. Originally identified in NSD2, PWWP domains also bind H3K36me3 to act as reader proteins^7,8,71. In particular, the PWWP domain of DNA methyltransferase 3A (DNMT3A) binds H3K36me3 and methylates nearby DNA, demonstrating a link between H3K36me3 and DNA methylation that could represent a new mechanism for H3K36 methylation-mediated repression of gene expression.

H3K36 methylation in dosage compensation

Coordination between methylation and acetylation also occurs during dosage compensation in D. melanogaster⁷². To compensate for having only one X chromosome, male flies use the Male-specific lethal (MSL) complex to upregulate the expression of X-linked genes by a factor of two in order to achieve the same level of transcription as females, which have two X chromosomes. This may occur through a two-step model requiring H3K36me3 and increased elongation^73–75. The MSL complex is initially recruited to X-linked genes through a GA-rich recognition site and subsequently facilitates transcriptional elongation through an enhancement of RNAPII activity. Next, the MSL3 subunit (an orthologue of yeast Eaf3) is thought to promote spreading of the upregulated state through interactions that involve the chromodomain of MSL3 and H3K36me3 (REF. 73). Additional active marks, such as H4K16 acetylation, also affect X-linked gene expression, requiring the Males absent on the first (MOF) subunit of MSL. Thus, a collection of activating marks together achieves chromosome-wide gene expression⁷⁴. Although interactions between chromodomain proteins and H3K36me3 marks regulate acetylation in both yeast and D. melanogaster, they have opposite effects: transcription is repressed in yeast but upregulated in flies. This clearly illustrates that the function of a given histone modification, such as H3K36me3, is context-dependent and is much more complicated than a simple code.

NSD enzymes and transcriptional initiation

NSD1 regulation of H3K36 methylation appears to affect transcriptional initiation¹⁷. For example, NSD1 binds upstream of the promoter of BMP4 and regulates the levels of H3K36me1, H3K36me2 and H3K36me3 within the body of the gene. This seems to be required for the recruitment of RNAPII to the BMP4 promoter, an observation that links NSD1 to initiation events through RNAPII. NSD3 can also bind promoters and then influence the levels of H3K36 methylation within the body of a gene⁴⁰. For example, the extraterminal domains of bromodomain-containing 4 (BRD4) specifically recruit NSD3 to the promoter region of the gene encoding CCND1 and the decapping enzyme DCPS, and loss of either BRD4 or NSD3 reduces the levels of H3K36me3, particularly within the body of the gene. This is consistent with the NSD1-dependent regulation of BMP4, during which promoter-proximal bound NSD1 regulates H3K36me3 levels within the body of the gene¹⁷. Although NSD3 localization is biased towards the promoter of CCND1, it also can be found, albeit at reduced levels, at the 3′ end of the gene⁴⁰, suggesting that NSD3 participates in initiation and elongation events. Moreover, NSD3 levels were found to be higher in the coding regions than in the promoter of the proto-oncogene Ser/Thr kinase PIM2 (REF. 40). Thus, NSD3 appears to reside in multiple complexes (including LSD2 and BRD4 complexes) and can localize to either promoter or internal regions to promote H3K36 methylation and thereby influence initiation and elongation processes. This is in apparent contrast to NSD1, which has been reported to localize primarily near the 5′ ends of its targets¹⁷; it will be important to confirm this using high-resolution ChIP followed by sequencing (ChIP–seq) analysis. However, previous ChIP–seq data show that NSD2 is enriched near transcription start sites³⁰.

H3K36 methylation in transcriptional repression

Fascinating clues have emerged regarding the relationship between methylation at H3K36, transcriptional repression and worm development^26,27. In worms, maternally provided MES-4 is essential for germ cell viability and is involved in X-chromosome silencing in the germline^26,27. Early primordial germ cells (PGCs) do not engage in transcription as they lack the active hyperphosphorylated CTD form of RNAPII. But, in the absence of mes-4, active RNAPII persists, indicating that MES-4 is important for the establishment and/or maintenance of transcriptional repression in late stage PGCs^26,27. Loss of MES-4 also reduces the levels of H3K36me3, independently of RNAPII function. Thus, MES-4 maintains the H3K36me3 state in germline precursors independently of transcription — an observation that has not been made in other organisms so far.

Beyond H3K36 methylation-mediated regulation of spurious initiation, H3K36 methylation-mediated repression has also been reported. Yeast Set2 represses transcription from a lacZ reporter and, in Set2-deletion mutants, the basal levels of GAL4 transcription are increased^76,77; however, in these studies, Set2 is artificially recruited to the promoter, so it will be important to confirm whether this reflects the true nature of Set2 activity. Initial studies with tethered reporters in mammalian cells have implicated NSD1 in both transcriptional activation and repression¹³, although its targets have not yet been determined. In addition, an RNA interference study has shown that NSD1 represses the expression of the homeobox regulator MEIS1 in neuronal cell lines¹⁹. This repressive event is likely to be direct, as NSD1 localizes to the promoter of MEIS1 (REF. 78). Furthermore, NSD2 collaborates with the cardiac-specific factor NKX2-5 to repress targets such as platelet-derived growth factor-α (PDGFRα), probably through modulating H3K36 methylation levels³². Thus, H3K36 methylation appears to act as both an activating and inhibitory signal, and the overall biological readout might depend on the context of additional surrounding marks and their corresponding reader proteins.

H3K36 methylation and exon definition

There has been increasing support for the idea that biased nucleosome positioning favours exonic over intronic sequences^79–81. Such a bias seems to be accompanied by distinct histone modifications, including H3K36me3. This should not be surprising, as the 147-base-pair length of a DNA fragment associated with a histone octamer correlates with the average length of an internal exon. This is likely to be more than just mathematical serendipity; it is probably evidence of interplay between chromatin and splicing. Several large-scale bioinformatics studies have analysed both the positions of nucleosomes and their modification status within the genomes of humans, C. elegans, D. melanogaster and mice^79–81. In each case, nucleosomes were enriched specifically at exonic sequences. Although the increased deposition of nucleosomes at exons guarantees a bias in histone modifications within exons relative to those within introns, it is also clear that a subset of modifications is specifically enriched here. This is particularly true for H3K36me3 but also includes methylation at H3K79, H4K20 and H2BK5 (REF. 80). Each analysis also found that the H3K36me3 bias is more pronounced within exons further downstream of the transcription start site. This preference may reflect the propensity of RNAPII to abort transcription early in the transcription cycle, thereby reducing the number of nucleosomes that are displaced further downstream. The known association between Set2 and the RNAPII CTD may also further explain the particular increase in H3K36me3 signatures seen at downstream exons^61,82. The implication of nucleosome enrichment and the increase in H3K36me3 modifications is twofold. First, nucleosomes probably act as intrinsic pause sites for elongating RNAPII which could alter splice site recognition and, hence, change exon inclusion. Others have found that the introduction of pause sites within minigenes can increase the inclusion of alternatively spliced exons^83,84. Furthermore, expression of a ‘slow’ mutant RNAPII in D. melanogaster results in different inclusion patterns of the exons within the Ultrabithorax mRNA⁸⁵. A second possibility, which is not mutually exclusive with effects on RNAPII pausing, is that the H3K36me3 modification relays a specific signal to the splicing machinery to alter how it defines exons, leading to the specific inclusion or exclusion of particular exons.

Although the global analyses of H3K36me3 positioning in various genomes provide compelling evidence that this modification affects splicing, functional evidence beyond this has been lacking. However, an interesting connection has been made between SETD2, the reader protein MORF-related gene 15 (MRG15; which contains a chromodomain) and polypyrimidine tract-binding protein (PTB), the last of which is a known antagonist of exon definition that affects splicing of fibroblast growth factor receptor 2 (FGFR2) pre-mRNA^86,87. FGFR2 contains two mutually exclusive exons (IIIb and IIIc) that encode a region within the extracellular immunoglobulin-like domain and are each responsible for receptor binding to a unique range of FGFs⁸⁸. Exon IIIb is included in epithelial cells through the action of epithelial splicing regulatory protein (ESRP), whereas exon IIIc is included in cells of mesenchymal origin⁸⁹ (FIG. 3). Furthermore, the splicing pattern switches from exon IIIb to exon IIIc inclusion as prostate epithelial cells become androgen-independent, an important factor in metastasis. Analysis of nucleosome modifications throughout FGFR2 show that H3K36me3 is specifically enriched within exon IIIb and is restricted to mesenchymal cells, which exclude this exon. This H3K36me3 modification is recognized by MRG15, which also interacts with PTB. Thus, by recruiting PTB to its target exon, these interactions position PTB to bind to its intronic splicing silencer sites, which flank the repressed exon as they emerge from the transcribing RNAPII complex (FIG. 3). PTB repression of this exon can be alleviated by downregulating either MRG15 or SETD2. This regulatory module also exists at other alternatively spliced exons, with a bias towards exons that contain weaker PTB-binding sites⁸⁶. What remains to be seen is how two distinct cell types achieve this differential methylation of H3K36 within nucleosomes at alternatively spliced exons in order to regulate splicing.

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Figure 3

H3K36me3 influences alternative splicing in a cell-type specific manner

The fibroblast growth factor receptor 2 (FGFR2) locus that undergoes alternative splicing consists of two mutually exclusive exons, IIIb and IIIc, which are located between the constitutive exons 7 and 10. Mesenchymal stem cells favour the inclusion of exon IIIc and achieve this by repressing splicing of exon IIIb. Nucleosomes present near exon IIIb contain the SET domain-containing 2 (SETD2)-dependent trimethylated Lys36 on histone H3 (H3K36me3) ‘mark’ and its reader protein MORF-related gene 15 (MRG15). MRG15 also interacts with polypyrimidine tract-binding protein (PTB), a known repressor of exon inclusion, and this may be the mechanism by which the methylated H3K36 mark can influence splicing at this locus. In epithelial cells, FGFR2 expresses exon IIIb but excludes exon IIIc. Epithelial splicing regulatory protein (ESRP) is expressed and stimulates the inclusion of exon IIIb; reduced levels of H3K36me3 present at this exon, possibly as a result of lower SETD2 levels, allow its derepression. The role of dimethylases, such as the proteins of the nuclear receptor SET domain-containing (NSD) family, in this process has yet to be determined but these enzymes could also influence H3K36 methylation here.

This example of FGFR2 control exemplifies how the methylation status of H3K36 can affect splicing. However, this crosstalk is bidirectional: mutations in splice sites that abrogate intron removal of β-globin reporter genes cause a shift in H3K36me3 signatures towards the 3′ region of genes⁹⁰. Moreover, others have observed that H3K36me3 signatures are markedly lower in genes without introns⁹¹. In both cases, inhibition of splicing with pharmacological agents causes a rapid and global redistribution of H3K36me3 throughout the genome and reduces SETD2 recruitment to these genes^90,91. Although it is unclear how the spliceosome regulates H3K36 methylation, these studies suggest that it is a general phenomenon.

We apologize to those researchers whose work could not be cited owing to space constraints. We thank B. Martinez and K. Eterovic for assistance with the figures, as well as S. Ercan, P. Masamha and A. Sataluri for critical comments regarding the manuscript. Work in the laboratory of P.B.C. has been supported in part by the Robert A. Welch Foundation (AU-1569). Work in the laboratory of E.J.W. is supported by the US National Institutes of Health (5R00GM080447).