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Published in final edited form as: Curr Opin Struct Biol. 2019 Jun 21;59:98–106. doi: 10.1016/j.sbi.2019.05.009

Activation and regulation of H2B-Ubiquitin-dependent histone methyltransferases

Evan J Worden 1, Cynthia Wolberger 1,*
PMCID: PMC6888998  NIHMSID: NIHMS1530939  PMID: 31229920

Abstract

Covalent modifications of histone proteins regulate a wide variety of cellular processes. Methylation of histone H3K79 and H3K4 is associated with active transcription and is catalyzed by Dot1L and Set1, respectively. Both Dot1L and Set1 are activated by prior ubiquitination of histone H2B on K120 in a process termed “histone-crosstalk”. Recent structures of Dot1L bound a ubiquitinated nucleosome reveled how Dot1L is activated by ubiquitin and how Dot1L distorts the nucleosome to access its substrate. Structures of Dot1L-interacting proteins have provided insight into how Dot1L is recruited to sites of active transcription. Cryo-EM and crystallographic studies of the complex of proteins associated with Set1 (COMPASS) uncovered the architecture of COMPASS and how Set1 is activated upon complex assembly.

Introduction

The packaging of eukaryotic DNA into chromatin governs all cellular processes requiring access to DNA, such as transcription [1], DNA replication [2], and DNA repair [3]. The minimal organizational unit of chromatin is the nucleosome, which comprises 147 base pairs of DNA wrapped around an octameric core of histone proteins H2A, H2B, H3 and H4 [4]. The cell regulates the higher-order organization and position of nucleosomes through post-translational modification of histone proteins. These modifications modulate the packaging of DNA into chromatin and recruit a variety of protein complexes and enzymes [5,6]. Most posttranslational histone marks are small chemical modifications such as phosphorylation, methylation, and acetylation, which alter the steric bulk or charge of the modified histone sidechain. Histones can also be modified with the 76-amino acid protein, ubiquitin [7], a comparatively large modification that regulates a wide array of cellular processes including transcription activation, silencing, DNA repair and chromatin compaction [8,9]. The structural motifs that recognize ubiquitin [10] and ubiquitinated nucleosomes [11] have been reviewed recently. The precise patterning of histone modifications – termed the “histone code” [6] – is therefore required to properly regulate processes that access DNA. Dysregulation of histone modifications can lead to a variety of human diseases.

Some histone marks trigger modification of other histone residues. This interdependence of histone modifications, where recognition or deposition of one mark depends on the presence of another, is called “histone crosstalk” [12]. A well-established example of histone crosstalk is the dependence of histone H3K4 and H3K79 methylation on prior monoubiquitination of histone H2B K120 (H2B-Ub) (in humans; H2B K123 in yeast) [13-16]. H3K79 and H3K4 methylation are catalyzed by two enzymes: disruptor of telomeric silencing-like protein (Dot1L) [17], which methylates H3K79 in humans, and the complex of proteins associated with Set1 (COMPASS), which methylates H3K4 [18]. Dot1L contains a catalytic domain that resembles arginine methyltransferases [19] and the catalytic subunit of COMPASS (Set1) contains a SET lysine methyltransferase domain [20-22], [23]**, [24]**. Dot1L and COMPASS are evolutionarily unrelated enzymes that are conserved from yeast to humans. Recent structures of Dot1L bound to a ubiquitinated nucleosome [25]**, [26]**, [27]**, [28]*, [29]*, Dot1L-binding partners, and COMPASS [23]**, [24]** have provided crucial insights into how Dot1L recognizes and is activated by ubiquitin and how the COMPASS subunits activate Set1

Activation of Dot1L by H2B-Ub nucleosomes

In contrast to most sites of histone modifications, which occur on flexible histone tails, H3K79 is located in the folded histone octamer core of the nucleosome. Methylation of H3K79 by Dot1L is broadly associated with transcription [30], telomeric silencing [31] and the DNA damage response [32-34], although the mechanism by which H3K79 methylation promotes or regulates these processes remains unknown. The dependence of H3K79 methylation by human Dot1L and yeast Dot1p on H2B-Ub has been well established in vivo [15,16] and, in the case of Dot1L, in vitro [35]. Muir and colleagues have shown that a surface on ubiquitin centered on L73 and L71 is critical for Dot1L activation [36,37]. Dot1L activity also depends on a patch of basic residues in the flexible N-terminal tail of histone H4 [38-40], but a structural explanation for these observations had been lacking. In addition, although structures of yeast Dot1p [41] and human Dot1L [19] have been available for quite some time, it had not been possible to construct a model for how the relatively inaccessible H3K79 side chain enters the enzyme active site.

Recent cryo-EM structures of Dot1L bound to an H2B-Ub nucleosome have revealed how Dot1L recognizes ubiquitin and the acidic patch of the nucleosome [25]**, [26]**,[27]**, [29]* Four recent studies captured Dot1L in a state in which the active site is oriented far from the H3K79 sidechain, a configuration that would presumably exist either directly before or after methyl transfer (Figure 1a). In this “poised” state, Dot1L is anchored to one side of the nucleosome though direct interactions with the H2B-linked ubiquitin (Figure 1 b, c). Dot1L binds ubiquitin using a C-terminal helix and loop, which bears no resemblance to any previously identified ubiquitin-binding motifs. This region of Dot1L binds to a hydrophobic patch on ubiquitin that includes I36, L71 and L73 (Figure 1c), the latter two of which had previously been shown to be important for activating Dot1L [36]. There is no connecting density between Ubiquitin and H2B in any of the structures, indicating that this connection is not ordered and thus explaining how ubiquitin modifications near H2BK120 can also stimulate Dot1L [37]. In addition, Dot1L contacts the conserved H2A/H2B acidic patch with a single arginine, R282, which is clearly visible in all poised state structures (Figure 1b, c) [25]**, [26]**, [27]**, [29]*. This provides yet another example of a so-called “arginine anchor” contacting the nucleosome acidic patch, which is a hot spot of interactions with nucleosome binding proteins [42]. A fifth structure reported at 6.8 A resolution shows a similar overall orientation of Dot1L on the nucleosome [28]*. In all poised state structures, weak density at the N-terminal end of Dot1L, which is the most distant from the ubiquitin anchor point, indicates that the enzyme can access multiple positions while tethered at its C-terminus by ubiquitin and the acidic patch. Interestingly, classification of the poised state structure by Anderson et. al. [26]** isolated several states of Dot1L that are rotated to varying degrees about the arginine anchor and ubiquitin contact, showing that Dot1L can sample a wide area of the nucleosome surface. Furthermore, structures of Dot1L bound to an unmodified nucleosome, [27]** and [29]*, showed that Dot1L binds to essentially the same nucleosome surface with or without ubiquitin. However, the Dot1L density in these structures is very weak, indicating that the enzyme is much more mobile without the H2B-Ub modification.

Figure 1. Dot1L recognition of an H2B-Ubiquitinated nucleosome.

Figure 1.

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(a) Dot1L (2-416) switching from the poised (yellow) to the active (green) state as shown from the side (top) and top (bottom). The nucleosome is shown as a semi-transparent gray surface. (b) Structure of the active state complex between Dot1L and the H2B-Ub nucleosome (PDB: 6NJ9). (c) Close up view of Dot1L interactions with ubiquitin and the acidic patch. Interface residues are shown as sticks. (d) Formation of the Dot1L active site enclosure. Dot1L, H3 and H4 are shown as green, blue and red cartoons respectively. The SAM cofactor is shown as magenta sticks. (e) Dot1L interactions with the H4 tail. Dot1L is depicted as a green cartoon surrounded by a semi-transparent green surface. (f) The conformational change of H3K79. The position of H3K79 in the poised state is depicted with yellow sticks and the active state H3K79 is shown with blue sticks. (g) The Dot1L hydrophobic lysine binding channel, with residues that compose the channel shown as sticks.

Source: Adapted from E. Worden et. al, [25]**

The structure of Dot1L bound to an H2B-Ub nucleosome in a catalytically competent, “active” state was determined at 3.0 Å resolution by cryo-EM (Figure 1a, b) [25]**. This active conformation was trapped by replacing H3K79 with norleucine, a non-native amino acid lacking the ε-amino group. Peptides containing norleucine in place of the substrate lysine have been shown to bind tightly to SET domain methyltransferases, mimicking the tight binding of Lysine-to-methionine mutations seen in pontine gliomas [43-45]. As compared to the poised state, the N-terminal domain of Dot1L moves toward the nucleosome by ~25Å and rotates by 22° to adopt an active state (Figure 1 a, b) in which the SAM methyl group is within 3 Å of the attacking lysine (Figure 1d). In addition to providing details on the catalytically relevant complex, the structure revealed an unexpected role for the tail of histone H4 and explained how Dot1L modifies the relatively inaccessibly side chain of H3K79.

In the active state structure, the tail of histone H4 inserts into a groove formed in the Dot1L N-terminal domain (Figure 1 e), thus explaining previous observations about the critical role of a basic patch in this histone for Dot1L and Dot1p activity [38-40]. The dual interactions at the Dot1L N-terminus with the H4 tail and the C-terminus with ubiquitin orient Dot1L over the H3K79 substrate lysine [25]**. In addition to binding to Dot1L, the H4 tail assists Dot1L in inducing a conformational change in histone H3 that alters the backbone flanking H3K79 and reorients the lysine sidechain by 90°, inserting it into the hydrophobic Dot1L lysine binding channel (Figure 1e - g). This unprecedented conformational distortion in the globular core histone is stabilized by a tripartite set of interactions involving Dot1L residues F131 and W305 and the H4 tail residue R19, which contacts backbone of H3 residues 77, 79 and 80 (Figure 1d). Together, these contacts by Dot1L and histone H4 “pinch” H3K79 from all sides and force it from its inaccessible position into the Dot1L active site. The conformational change in core histone fold of H3 and the participation of the H4 tail in stabilizing the altered conformation of H3 have never been observed previously, although recent studies have suggested that the core histone fold can be deformed due to the action of chromatin remodeling enzymes [46]* and during DNA unwrapping and translocation [47]*, [48]*. The intriguing implication of the conformational distortion induced by Dot1L is that there may be other histone-modifying enzymes that similarly deform the core histone fold to access their substrate sidechain in the context of the fully formed nucleosome.

The hydrophobic nature of the lysine binding channel may also explain how H3K79 is deprotonated for the attack on the SAM methyl group during catalysis. Dot1L lacks ionizable residues in the active site that could potentially abstract a proton from the charged lysine side chain (Figure 1g) [19], [25]**. Burying the charged H3K79 sidechain within the hydrophobic environment of the lysine binding channel would lower the pKa of the ε-amino group and favor deprotonation [49].

Recruitment of Dot1L to genomic loci

Dot1L is recruited to specific genomic loci by the partner proteins AF9, ENL, AF10 and AF17, which contain C-terminal Dot1L interaction domains and N-terminal chromatin binding domains [50]*, [51]*, [52]*, [53]*, (Figure 2b-i). The precise composition of Dot1L complexes with its various partner proteins, as well as the role each partner proteins plays in recruiting Dot1L to different genomic loci, remains an open question. Chromosomal translocations that fuse Dot1L binding proteins to the human Set1 methyltransferase homolog, MLL, lead to aberrant recruitment of Dot1L to MLL target genes, which is a causative factor in many leukemias [54] and other cancers [55,56].

Figure 2. Dot1L partner proteins.

Figure 2.

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(a) Domain architecture schematic of Dot1L. (b) Domain architecture schematic for Dot1L partners, AF9 and ENL. (c) Structure of the AF9 YEATS domain (PDB: 4TMP, gray) bound to acetylated H3K9 (yellow sticks). (d) Close up view of the acetylated H3K9 binding site. Crotonylated H3K9 (PDB: 6MIM) is shown in cyan. (e) Structure of the AF9 AHD (PDB: 2MV7, gray) domain bound to Dot1L site3 (green). Buried hydrophobic residues are colored red and shown as sticks. (f) Generalized domain architecture schematic for AF10 and AF17. (g) Structure of the AF10 PZP domain (PDB: 5DAH, gray) bound to an unmodified H3 peptide (yellow sticks). (h) Close up view of the AF10 PZP H3 binding groove. The surface of AF10 is colored according to electrostatic potential. (i) Structure of the AF10 OM-LZ motif (PDB: 6CKO, gray) bound to the CC2 of Dot1L (green). Buried hydrophobic residues are colored red and shown as sticks.

While there are, as yet, no structures of complete complexes containing intact Dot1L or its partner proteins, structural studies of several Dot1L and chromatin-interacting domains have shed light on key aspects of these complexes [50]*, [51]*, [52]*, [53]* . Dot1L partners, AF9 and ENL, contain an N-terminal YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domain that binds to acetylated or crotonylated H3K9, H3K27 and H3K18 (Figure 2b-d) [50]*, [57]*. The YEATS domain recognizes the acetyl-,[50]* or crotonyl-lysine [57]* in a surface tunnel that terminates in an aromatic cage composed of F28, F59 and Y78 (Figure 2c,d). AF9 and ENL also contain a C-terminal ANC1 homology domain (AHD) that interact with one of three binding sites (Site1-Site3) in the C-terminal part of Dot1L and recruit the enzyme to specific genomic loci (Figure 2a,e) [51]*. Mutations in the AHD that disrupt Dot1L binding decrease H3K79 methylation at targets of the MLL-AF9 fusion protein [51]*.

AF10 and AF17 have an N-terminal PDH finger-Zn knuckle-PDH finger (PZP) domain that binds to the unmodified tail of histone H3 between residues 22-28 (Figure 2f-h) [52]*. The structure of the AF10 PZP domain shows how the H3 tail binds in an acidic groove and makes specific contacts with multiple charged residues (Figure 2h). Binding of the H3 tail to the PZP domain is abolished upon methylation of H3K27, which is a hallmark of silenced chromatin [58], consistent with the role of Dot1L in transcription activation [30]. AF10 and AF17 also contain an octapeptide motif leucine zipper (OM-LZ) domain which can form a coiled-coil with any one of three sequences (CC1-CC3) in C-terminal part of Dot1L (Figure 2a,i) [53]*. Mutations in the Dot1L binding interface of AF10 disrupt Dot1L binding and lead to a reduction in the leukemogenic transformation potential of MLL-AF10 cell lines [53]*.

Architecture and activation of COMPASS

Methylation of H3K4 is highly enriched at gene promotors and transcription start sites and is dependent on prior ubiquitination of H2B [13,14,58]. H3K4 methylation is catalyzed by COMPASS, which is composed of 8 subunits: the Set1 methyltransferase, Cps60, Cps50, Cps40, Cps35, Cps30, Cps25 and Cps15 [18,59]. The H3K4 methyltransferase activity of the Set1 catalytic domain on its own is low, but is significantly stimulated when incorporated into COMPASS [60,61]. Studies of the contribution of each subunit to the activity of the catalytic subunit Set1 have identified a minimal sub-complex of COMPASS composed of Set1, Cps60, Cps50, Cps40, Cps30, and Cps25 that is sufficient for H2B-Ub dependent stimulation of Set1 [62].

Two structures of the minimal COMPASS subcomplex were recently determined [23]**, [24]**. One structure was determined by cryo-EM and comprises all 6 subunits of the minimal ubiquitin-sensing complex [23]** and the other was determined by X-ray crystallography and comprises a smaller complex lacking Cps40, which is referred to as the WRAD complex, after its human homologs (WDR5/RbBP5/ASH2L/DPY30) (Figure 3a) [24]**. The structures show that COMPASS forms a Y-shaped, highly intertwined, structure with the Set1 catalytic domain at its center (Figure 3a). An interesting feature of these structures is the C-terminal extension of Cps50, which winds around the complex and contacts almost every other subunit except the Cps25 dimer (Figure 3a and 3b, contacts 1-5). Disruption of many of these contacts greatly decreases the activity of Set1, explaining the role of Cps50 in organizing the complex and in activating the catalytic activity of Set1 [23]**.

Figure 3. Architecture of yeast COMPASS.

Figure 3.

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(a) Structures of the yeast COMPASS complex determined by X-ray crystallography (left) and Cryo-EM (right). The C-terminal extension of Cps50 is shown as a blue surface. (b) Detailed view of the contacts made by the Cps50 C-terminal extension. Distinct contacts are labeled 1-5. (c) View of the N-set motif in Set1.

The N-Set domain of Set1 is required for the ubiquitin-depended activation of H3K4 methylation [62]. The cryo-EM structure of COMPASS [23]** shows that a single lysine of the WIN motif within the N-Set domain of Set1 docks into the WD40 domain of Cps30 in a similar arrangement to the WIN motif from human homologs of Set1 (Figure 3c) [63]. In addition, the N-set domain of Set1 forms a long, helix that inserts between Cps40 and Cps30 (Figure 3c). Flowever, it is still not clear how the N-set motif of Set1 can sense ubiquitinated H2B and the location of this motif within COMPASS does not clarify this mechanism.

Crystal structures of the human homologs of Set1, MLL1 [64], [65]* and MLL3 [65]*, have established that the critical SET-I helix in these enzymes is mobile and can interconvert between “open” and “closed” states and that this interconversion can be suppressed by binding to proteins in the WRAD complex, leading to activation of the SET domain (Figure 4a,b) [64], [65]*. In both recent structures of COMPASS [23]**, [24]**, the SET-I helix is stabilized in the closed state, which explains how incorporation into COMPASS activates the enzyme (Figure 4a,b). Furthermore, the closed state of the Set-I helix in COMPASS explains the previous observation that C3882 in MLL1 can be auto-methylated in the absence of H3 peptide: closure of the Set-I helix would position C3882 close to the SAM cofactor. Interestingly the Set1 methyltransferase activity regulator (SMART) loop of Cps30 directly contacts the SET-I helix of Set1 in both structures (Figure 4c). However, the two structures differ with respect to a tryptophan residue (W183 in S. cerevisiae) on the tip of the SMART motif that points away from Set1 in the Cryo-EM structure [23]** and is completely buried in the crystal structure within a hydrophobic pocket termed the “kabuki pocket” (Figure 4c) [24]**. Because the cryo-EM structure was determined in the absence of SAM cofactor and H3 peptide [23]**, which are present in the crystal structure [24]**, the different conformations of the SMART loop may correspond to important conformational changes that occur during substrate binding and release. In agreement with its location next to the critical Set-I helix, mutations in the SMART loop of Cps30 lead to defects in H3K4 methylation by COMPASS [23]**, [24]**. Furthermore, the close contacts between the Set-I helix of Set1, the Cps30 SMART loop and the Cps50 C-terminal extension likely help determine the product specificity of different Set1 homologs for mono-, di-, or tri-methylation of H3K4 by restricting motion of the Set-I helix to varying degrees [24]**. Remarkably, re-engineering the “door loop” C-terminal to the Set-I motif in MLL3 to more closely resemble yeast Set1 converted MLL3 from a mono-methylase into a multi-methylase [24]**.

Figure 4. Activation of Set1 in COMPASS.

Figure 4.

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(a) Superposition of the Set1 catalytic domain (PDB: 6CHG, magenta) with MLL1 in different states (PDB: 5F6L, 5F5E, 2W5Y) showing different conformations of the SET-I helix. (b) Superposition of the Set1 catalytic domain (PDB: 6CHG, magenta) with MLL3 in different states (PDB: 5F6K chain C, E and 5F59) showing different conformations of the SET-I helix. (c) details of the SMART loop interactions with the SET-I helix of Set1 in COMPASS. The SMART loop in the crystal structure is colored orange and the SMART loop in the cryo-EM structure is colored tan.

Conclusions

While great strides have been made in understanding the mechanistic underpinnings of cross-talk between H2B monoubiquitination and histone methylation, several outstanding questions remain to be addressed. It is still not clear how COMPASS binds to the nucleosome or is able to sense ubiquitinated H2B. A structure of the complex between COMPASS and a ubiquitinated nucleosome will be required to understand the crosstalk between H3K4 methylation and H2B-ubiquitination. In addition, the specific mechanism by which H3K79 methylation stimulates transcription is unknown, nor have any bona fide H3K79-methyl binding proteins been identified. Because of the relatively inaccessible position of H3K79 on the nucleosome, it is likely that any protein that recognizes modifications of this side chain would need to deform the structure of the nucleosome in a fashion similar to Dot1L. Furthermore, it is not clear how Dot1L establishes the correct mono-, di- or tri-methylation state of H3K79 in vivo. Recruitment by Dot1L binding partners clearly plays a role in governing the methylation state [51], while in vitro methylation assays have shown that Dot1L preferentially mono- and di-methylates of H3K79 [35]. An interesting possibility is that Dot1L-interacting proteins may activate the enzyme for tri-methylation or make Dot1L processive through multivalent interactions with chromatin. Multivalent binding of chromatin marks has, for example, been shown to promote processive hyperacetylation by the SAGA HAT module [66]. We can look to a future of exciting structures and biochemical studies to sort out these fascinating and important questions.

Highlights.

  • Structures of Dot1L bound to ubiquitinated nucleosomes show the mechanism of crosstalk between histone H3K79 methylation and H2B monoubiquitination

  • Structural insights into how Dot1L-interacting proteins recruit Dot1L to genomic loci through bivalent contacts with Dot1L and chromatin

  • Architecture of the COMPASS complex shows how the H3 K4 methyltransferase subunit, Set1, is activated

Acknowledgements

Supported by a grant from the National Institute of General Medical Sciences (R35 GM130393). E.J.W. is supported by a fellowship from the Damon Runyon Cancer Research Foundation.

Footnotes

Conflict of Interest Statement

C.W. is a member of the scientific advisory board of Thermo Fisher Scientific.

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