Edit · 2026 01 11 231507 Nature S41594 024 01455 8

Saving needs a token. Log in or register to get one. Edits are public and licensed CC-BY-SA-4.0.

# A multivalent engagement of ENL with MOZ | Nature Structural & Molecular Biology

**URL:** https://www.nature.com/articles/s41594-024-01455-8

**Saved:** 2026-01-11T07:15:12.822Z

---

Article
Published: 10 January 2025
A multivalent engagement of ENL with MOZ
Dustin C. Becht, Karthik Selvam, Catherine Lachance, Valérie Côté, Kuai Li, Minh Chau Nguyen, Akshay Pareek, Xiaobing Shi, Hong Wen, M. Andres Blanco, Jacques Côté & Tatiana G. Kutateladze

Nature Structural & Molecular Biology 
volume
 32, 
pages
709–718 (2025)Cite this article

1787 Accesses

5 Citations

Metrics
details

Abstract

The epigenetic cofactor ENL (eleven-nineteen-leukemia) and the acetyltransferase MOZ (monocytic leukemia zinc finger) have vital roles in transcriptional regulation and are implicated in aggressive forms of leukemia. Here, we describe the mechanistic basis for the intertwined association of ENL and MOZ. Genomic analysis shows that ENL and MOZ co-occupy active promoters and that MOZ recruits ENL to its gene targets. Structural studies reveal a multivalent assembly of ENL at the intrinsically disordered region (IDR) of MOZ. While the extraterminal (ET) domain of ENL recognizes the canonical ET-binding motif in IDR, the YEATS domains of ENL and homologous AF9 bind to a set of acetylation sites in the MOZ IDR that are generated by the acetyltransferase CBP (CREB-binding protein). Our findings suggest a multifaceted acetylation-dependent and independent coupling of ENL, MOZ and CBP/p300, which may contribute to leukemogenic activities of the ENL–MOZ assembly and chromosomal translocations of ENL, MOZ and CBP/p300.

You have full access to this article via your institution.

Download PDF
Similar content being viewed by others
MORF and MOZ acetyltransferases target unmethylated CpG islands through the winged helix domain
Article Open access
08 February 2023
MOZ/ENL complex is a recruiting factor of leukemic AF10 fusion proteins
Article Open access
08 April 2023
The epigenetic regulators EP300/CREBBP represent promising therapeutic targets in MLL-rearranged acute myeloid leukemia
Article Open access
01 May 2024
Main

ENL (eleven-nineteen-leukemia) is a key driver of acute leukemias, the most common types of childhood cancer and a leading cause of mortality in infants. Oncogenic chromosomal translocations of ENL with mixed-lineage leukemia are frequently found in one of the deadliest hematologic malignancies, acute myelogenous leukemia (AML), and in acute lymphoblastic leukemia (ALL)1,2,3,4,5,6,7. ENL itself has been implicated in leukemic cell growth and is found to be mutated in individuals with Wilms tumors8,9,10,11. Because ENL and homologous AF9 are transcriptional coactivators that associate with transcription initiation and elongation complexes, their diverse genetic translocations perturb normal gene expression programs and tether the complexes to oncogenic targets. This results in a constitutive activation of proleukemic genes, triggering leukemic transformation2,3,4,5,6,7. Infant AML and ALL are particularly difficult to treat and are associated with poor clinical outcomes12,13.

ENL is a 559-residue protein that contains two domains, the YEATS domain (ENLYEATS) and the extraterminal domain (ENLET); AF9 has a similar domain architecture (AF9YEATS and AF9ET) (Fig. 1a). ENLYEATS recognizes acylated K18 and K27 of histone H3 (H3K18 and H3K27), particularly the acetylated and crotonylated marks8, and AF9YEATS selects for H3K9ac/cr (refs. 14,15,16). This interaction links ENL and AF9 with the acyllysine-rich promoters of their gene targets8,14,15, whereas ENLET and AF9ET bind to components of several transcription complexes. The association of ENLET or AF9ET with proteins anchored to the super elongation complex facilitates their recruitment to chromatin and the interaction with TATA-binding protein-associated factors (TAFs) of transcription factor IID aids in the release of paused RNA polymerase II for productive elongation6,7,17,18,19,20,21,22. Binding of ENLET or AF9ET to the H3K79 methyltransferase DOT1L has an important role in the targeting of DOT1L to promoters and activation of gene expression15,18. ENL has also been shown to cooperate with the histone acetyltransferase (HAT) monocytic leukemia zinc finger (MOZ) in the regulation of myeloid differentiation that drives leukemogenic gene transcription programs and has a role in coupling of leukemogenic AF10 fusions with the HOXA genes23,24,25. The ENL–MOZ association was found to depend on ENLYEATS24,25; however, the mechanism underlying this association has not been investigated.

Fig. 1: ENL and MOZ co-occupy active promoters.

a, Domain architecture of ENL and AF9. Histone H3 acetylation sites recognized by the YEATS domain are shown. b, A cartoon of MOZ and MORF complex subunits. Folded domains and their biological ligands are shown and labeled. c, Genomic distribution of MOZ and ENL cobound regions. d, Overlap between H3K23ac sites and regions cobound by MOZ and ENL. The significance of overlap was determined using ChIPseeker’s genome shuffling method with n = 1,000 shuffles and the P value was adjusted using the Benjamini–Hochberg method. e, Heat maps of MOZ, ENL and H3K23ac ChIP-seq signals at genomic regions bound by MOZ. f, Top five MSigDB hallmark gene sets most enriched in genes with proximal MOZ and ENL cobound sites. Pathway enrichment analyses were calculated using Enrichr’s variation on Fisher’s exact test; Benjamini–Hochberg-adjusted P values are shown. g, Representative tracks from ChIP-seq of MOZ, ENL and H3K23ac at the FLT3 locus (left) and MYC locus (right).

Full size image

MOZ, also known as MYST3 or KAT6A (lysine acetyltransferase 6A), is a catalytic subunit of the acetyltransferase MOZ complex (Fig. 1b). The MOZ complex is involved in transcriptional activation and developmental signaling26,27 and its aberrant activity is linked to aggressive forms of leukemia and intellectual disability28,29,30,31. The other three subunits of the native MOZ and homologous MORF complexes include BRPF1 that acts as a platform for the complex assembly and associates with H3 and DNA through the PZP domain32, ING4/5 that recognizes the methyllysine mark H3K4me3 through its plant homeodomain (PHD) finger33,34 and MEAF6 (ref. 35). The MOZ or MORF subunit of the complex contains two winged helix (WH) domains with distinct DNA-binding specificity, the double PHD finger (DPF) domain (MOZDPF), a reader of H3K14acyl and DNA, and the H3K23-specific catalytic MYST domain (MOZMYST) that is followed by an intrinsically disordered region (IDR)36,37,38,39,40,41.

In this study, we report the molecular basis for the multivalent engagement of ENL with the acetyltransferase MOZ that implicates two domains of ENL. As ENLYEATS binds to multiple acetylated sites of MOZ (YEATS-binding site, MOZYBS), particularly located in the IDR and likely produced by the acetyltransferase CBP (CREB-binding protein), ENLET binds to the canonical ET-binding motif of MOZ (MOZEBM) in the IDR. These findings provide mechanistic details essential to our understanding of the physiological and pathological activities of the ENL–MOZ complex and chromosomal translocations of ENL, MOZ and CBP/p300, the drivers of acute leukemia. Our study reveals a mechanism for the assembly of a chromatin-modifying complex through post-translational modifications (PTMs) in the subunits of the complex.

Results and discussion
ENL and MOZ co-occupy active promoters

The transcriptional regulator ENL has a critical role in maintaining the expression of proleukemic genes and this function depends on ENLYEATS that binds to the acyllysine epigenetic marks, selecting for acylated H3K18 and H3K27. We previously reported that acetylation of H3K18, H3K14 and H3K23 represents the most abundant coexisting combination of acetylated marks on histone H3 (ref. 37). This finding suggested a potential functional link between ENL and MOZ, as these marks are either recognized by ENLYEATS and MOZDPF or generated by MOZMYST. To explore this potential link, we analyzed chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) datasets previously generated by us from human acute myeloid leukemia MOLM13 cells and assessed the genomic occupancies of ENL and MOZ23. We found that ENL and MOZ cobind almost exclusively promoters (87%) and have notable overlap with H3K23ac, a product of enzymatic activity of MOZ (Fig. 1c,d). Among the 674 ENL–MOZ cobound regions, 58% (390) overlapped with the H3K23ac-rich regions (Fig. 1d). At MOZ-binding genomic targets, the association of ENL with chromatin and deposition of H3K23ac were both markedly reduced in MOZ-knockout cells, suggesting that ENL recruitment to the MOZ target sites depends on MOZ (Fig. 1e). A high degree of colocalization of ENL and MOZ was observed on promoters of genes associated with the regulation of cell-cycle progression and critical oncogenic drivers, such as MYC and FLT3 (Fig. 1f,g). Together these results suggested a cooperativity between ENL and MOZ that co-occupy transcriptionally active promoters.

ENLYEATS binds to MOZYBS

The MOZ-rearranged leukemia studies showed that two regions in the IDR of MOZ (aa 781–1013 and aa 1014–1116) and functional ENLYEATS are necessary for the association of ENL with MOZ25. On the other hand, three acetylation sites at K815, K1007 and K1014 in the IDR of MOZ were identified by proteomic analyses42,43. These observations prompted us to test the idea of whether the association of ENL with MOZ is acetylation dependent (Fig. 2a). We treated the K562 cell line expressing epitope-tagged MOZ–3FLAG–2Strep from the AAVS1 safe harbor with the histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and nicotinamide to block deacetylation (Fig. 2b and Extended Data Fig. 1). Immunoprecipitation with anti-FLAG beads followed by western blot with anti-ENL antibodies showed an increase in binding of ENL to FLAG–MOZ after the treatment of cells with the HDAC inhibitors. These data indicate that deacetylation of MOZ reduces binding and support the idea of the acetylation-dependent association of ENL.

Fig. 2: ENLYEATS binds to MOZYBS.

a, Domain architecture of MOZ. Acetylation sites K815ac, K1007ac and K1014ac in the IDR of MOZ, detected by mass spectrometry and reported in PTM databases, are indicated by orange circles. b, Western analysis of the FLAG eluted fractions from cells expressing MOZ–3FLAG from the AAVS1 safe harbor, untreated or treated with HDAC inhibitors TSA and nicotinamide probed by indicated antibodies. The experiment was performed two times. c, A zoomed-in view of the AlphaFold-generated model of MOZ from UniProt Q92794. The MOZ folded domains and IDR are labeled. d–f, Overlaid 1H,15N HSQC spectra of 15N-labeled ENLYEATS collected in the absence (black) and presence of increasing amounts of indicated monoacetylated and unmodified peptides: MOZYBSK1007ac (d; aa 1004–1012 of MOZ), MOZYBSK1014ac (e; aa 1010–1017 of MOZ with C-terminal RHR) and unmodified MOZ (f; aa 1005–1017 of MOZ). Spectra are color-coded according to the protein:peptide molar ratio. g,h, Peptide pulldown assays of GST–ENLYEATS (g) and His–ENLYEATS (h) with biotinylated unmodified MOZ and diacetylated K1007ac and K1014ac MOZ peptides (aa 998–1022 of MOZ). The experiments were performed at least three times. i, Peptide pulldown assays of full-length FLAG–ENL in whole-cell lysate from transiently transfected 293T cells with biotinylated unmodified MOZ and acetylated MOZ peptides. The experiment was performed three times. j,k, Normalized binding curves used to determine the Kd values for the interaction of ENLYEATS with MOZYBSK1007ac (j) or MOZYBSK1014ac (k) by tryptophan fluorescence. Data and the Kd values are the average ± s.e.m. of three independent experiments. l, Normalized binding curve used to determine the Kd value by MST. Kd represents the average of three independent measurements ± s.d. Point errors represent the s.d. m, Liquid HAT assays using 55 ng and 35 ng of recombinant CBP and unmodified MOZYBS and diacetylated MOZYBSK1007acK1014ac peptides as substrates (300 ng). CPM, counts per minute. HAT assays were performed in triplicate. Data are represented as the mean ± s.d. of three replicates. n, Table summarizing the acetyltransferase activities of indicated KATs toward the MOZ substrate.

Source data

Full size image

The AlphaFold2 modeling of MOZ (UniProt Q92794) suggested that the acetylated lysine sites are located in the IDR following the catalytic MYST domain and, therefore, are amenable to PTMs (Fig. 2c). To determine whether ENLYEATS recognizes the K1007 or K1014 acetylation sites of MOZ, we carried out nuclear magnetic resonance (NMR) titration and pulldown experiments. We generated 15N-labeled ENLYEATS and collected its 1H,15N heteronuclear single quantum coherence (HSQC) spectra while unlabeled MOZ peptides harboring acetylated K1007 or acetylated K1014 were titrated (Fig. 2d,e and Extended Data Fig. 2). Addition of either acetylated MOZ peptide (from here on referred to as MOZYBSK1007ac and MOZYBSK1014ac; YBS: YEATS-binding site) led to chemical shift perturbations (CSPs) indicative of the formation of complexes with ENLYEATS. This interaction was acetylation dependent, as no CSPs in ENLYEATS were induced by a 25-fold excess of the MOZ peptide containing both unmodified K1007 and K1014 (Fig. 2f and Extended Data Fig. 2). These results were corroborated by pulldown assays using the 25-residue-long unmodified and diacetylated K1007ac and K1014ac biotinylated MOZ peptides. Recombinant ENLYEATS and full-length FLAG–ENL expressed in 293T cells were pulled down by the acetylated MOZ peptide but not the corresponding unmodified peptide (Fig. 2g–i). Binding affinity of ENLYEATS to MOZYBSK1007ac and MOZYBSK1014ac, measured by fluorescence (Kd of ~30–32 µM; Fig. 2j,k) and microscale thermophoresis (MST) (Kd of 23 µM to MOZYBSK1014ac; Fig. 2l), revealed that ENLYEATS binds to acetylated MOZ as strongly as it binds to the histone mark H3K27ac (Kd of 31 µM)8.

MOZ is acetylated by CBP

To identify the acetyltransferase that acetylates MOZ, we performed in vitro acetyltransferase assays using a set of human acetyltransferases known to acetylate histone and nonhistone proteins (Fig. 2m,n and Extended Data Fig. 3). As shown in Fig. 2m, recombinant CBP efficiently acetylated the unmodified MOZ peptide in a concentration-dependent manner, corroborating previous findings that CBP/p300 acetylates MOZ and MOZ acetylation is lost when CBP/p300 is knocked out or inhibited42,43. When diacetylated K1007ac and K1014ac MOZ peptide was used as a substrate, CBP acetylated other lysine residues present in this sequence, K1011 and K1013, although the HAT activity on the diacetylated peptide was lower than that on the unmodified peptide. CBP was the only enzyme capable of acetylating MOZ, whereas the enzymatic activity of other acetyltransferases and complexes tested was undetectable (Fig. 2n).

Molecular mechanism of MOZYBS recognition by AF9YEATS

To gain insight into the molecular basis for the recognition of acetylated MOZ, we set up crystallization trials mixing ENLYEATS or homologous AF9YEATS with acetylated MOZ peptides. Although we were unable to crystallize ENLYEATS, we obtained diffracting crystals of AF9YEATS bound to MOZYBSK1007ac (aa 1005–1008 of MOZ) and refined the structure of the complex to 2.3-Å resolution (Table 1). CSPs caused by this peptide in the 1H,15N HSQC spectrum of AF9YEATS confirmed that, similarly to ENLYEATS, AF9YEATS binds to acetylated MOZ in solution (Fig. 3a and Extended Data Fig. 4). In the complex, AF9YEATS folds into a characteristic β-sandwich consisting of eight antiparallel β-strands and two short α-helices positioned at one of the open ends of the domain. The loops at the opposite end of the β-sandwich create the binding groove for MOZYBSK1007ac that is mainly hydrophobic in nature (Fig. 3b). The complex is stabilized through several intermolecular hydrogen bonds, including the hydrogen bonds formed between the carbonyl group of G80 of AF9YEATS and the backbone amide of K1007 and between the amide group of G80 of AF9YEATS and the carbonyl group of L1005 of the peptide (Fig. 3c). The imidazole nitrogen atom of H56 of AF9YEATS donates a hydrogen bond to the oxygen atom of the carbonyl group of P1008 of MOZ.

Table 1 Data collection and refinement statistics
Full size table
Fig. 3: Structural basis for the recognition of MOZYBS by AF9YEATS.

a, Superimposed 1H,15N HSQC spectra of 15N-labeled AF9YEATS recorded in the presence of increasing amount of MOZYBSK1007ac (aa 1005–1008 of MOZ) peptide. Spectra are color-coded according to the protein:peptide molar ratio. b, The crystal structure of AF9YEATS in complex with the MOZYBSK1007ac peptide. AF9YEATS (blue) is shown as a ribbon diagram and MOZYBSK1007ac (yellow) is shown in stick representation. Dashed lines indicate hydrogen bonds. c, Close-up view of the MOZYBSK1007ac-binding site of AF9YEATS. d, Overlay of the crystal structures of the AF9YEATS–MOZYBSK1007ac (colored as in b) and AF9YEATS–H3K9ac (wheat and salmon, PDB 4TMP) complexes.

Full size image

Acetylated K1007 of MOZ is bound in a tunnel created by three aromatic residues of AF9YEATS. The side chain of K1007ac is in a fully extended conformation and inserts between the aromatic rings of F28, F59 and Y78 of AF9YEATS. The acetyl group of K1007ac is affixed by two hydrogen bonds; the carbonyl group forms a hydrogen bond with the backbone amide of Y78 of AF9YEATS and the side chain ε nitrogen atom of K1007ac is engaged with the hydroxyl group of S58 of AF9YEATS. This mode of MOZYBSK1007ac recognition by AF9YEATS is reminiscent of that seen in the complex of AF9YEATS with H3K9ac (ref. 14). The two structures are superimposed with a root-mean-square deviation (r.m.s.d.) of 0.3 Å, indicating overall conservation of the binding mode, even though there is little similarity between the two sequences beyond the presence of acetylated lysine and the ability to form main-chain hydrogen bonds (Fig. 3d).

Two AF9YEATS concurrently bind two acetylation sites of MOZ

The K1007ac and K1014ac sites of MOZ are in close proximity; therefore, we tested whether ENL or AF9YEATS binding to one site would preclude binding of ENL or AF9YEATS to another site. Addition of the diacetylated MOZYBSK1007acK1014ac peptide to 15N-labeled ENLYEATS led to CSPs in 1H,15N HSQC spectra of ENLYEATS that were on par with CSPs observed upon addition of monoacetylated MOZ peptides (Fig. 4a and Extended Data Fig. 5). The binding affinity of ENLYEATS to the MOZYBSK1007acK1014ac peptide indicated that ENLYEATS can independently engage the K1007ac and K1014ac sites of MOZ (Fig. 4b,c,e). Crotonylation slightly augmented the interaction of ENLYEATS with MOZYBSK1007crK1014cr peptide (Fig. 4d–f and Extended Data Figs. 5 and 6). Similarly, the MOZYBSK1007crK1014cr peptide induced CSPs in 15N-labeled AF9YEATS and associated with AF9YEATS with a binding affinity of 19 μM (Fig. 4g, h and Extended Data Fig. 5).

Fig. 4: The avidity-based mechanism for the accumulation of ENL and AF9 at acylated MOZ.

a, Overlaid 1H,15N HSQC spectra of 15N-labeled ENLYEATS collected in the absence (black) and presence of increasing amounts of MOZYBSK1007acK1014ac peptide. Spectra are color-coded according to the protein:peptide molar ratio. b, Normalized binding curve used to determine the apparent Kd values by tryptophan fluorescence. Data and the apparent Kd values are the average ± s.e.m. of five separate experiments. c, Normalized binding curve used to determine the apparent Kd value by MST. The apparent Kd represents the average of three independent measurements ± s.d. Point errors represent the s.d. d, Normalized binding curve used to determine the apparent Kd values by tryptophan fluorescence. Data and the apparent Kd values are the average ± s.e.m. of three separate experiments. e, Summary of binding affinities of ENLYEATS and AF9YEATS to the indicated MOZYBS peptides. f, Overlaid 1H,15N HSQC spectra of 15N-labeled ENLYEATS collected in the absence (black) and presence of increasing amounts of MOZYBSK1007crK1014cr peptide. Spectra are color-coded according to the protein:peptide molar ratio. g, Overlaid 1H,15N HSQC spectra of 15N-labeled AF9YEATS collected in the absence (black) and presence of increasing amounts of MOZYBSK1007crK1014cr peptide. Spectra are color-coded according to the protein:peptide molar ratio. h, Normalized binding curve used to determine the apparent Kd values by tryptophan fluorescence. Data and the apparent Kd values are the average ± s.e.m. of five separate experiments. i, The crystal structure of two molecules of AF9YEATS (wheat and pink) bound to the MOZYBSK1007crK1014cr peptide (green). The side chains of K1007cr and K1014cr are shown in stick representation. j, Electrostatic surface potential of the two AF9YEATS molecules in complex with MOZYBSK1007crK1014cr generated in PyMOL. Blue and red colors represent positive and negative charges, respectively. k, Close-up view of the MOZYBSK1007cr-binding and MOZYBSK1014cr-binding sites of AF9YEATS. Dashed lines represent short distances indicative of the aromatic–amide and aromatic–alkene π–π stacking.

Source data

Full size image

To understand the mechanism of the crotonyllysine recognition, we cocrystallized AF9YEATS with the dicrotonylated MOZYBSK1007crK1014cr peptide and determined the crystal structure of the complex. In the structure, two AF9YEATS molecules concomitantly bind two acylated sites of MOZYBSK1007crK1014cr (Fig. 4i). The three-molecule complex shows that the distance between K1007cr and K1014cr of MOZ is ideal to accommodate two YEATS domains without steric hindrance or electrostatic repulsion (Fig. 4j). This avidity-based mechanism could be important for efficient accumulation of ENL or AF9 at acylated MOZ.

The structure of the MOZYBSK1007crK1014cr-bound AF9YEATS also explains the inability of ENL and AF9YEATS to strongly favor the crotonyl modification. Short (less than 4 Å) distances observed between the Cδ and Cε atoms of Y78 and the amide group of crotonyllysine and between the Cε and Cζ atoms of F59 and the alkene group of crotonyllysine indicated distinct aromatic–amide and aromatic–alkene π–π stacking contacts (Fig. 4k). Although the crotonyl C=C double bond and the amide form a conjugated system, neither is engaged in π–π–π stacking44 with both aromatic residues Y78 and F59 of AF9YEATS that would provide additional energetically favorable contacts. In contrast, the aromatic ring of F28 is not parallel to the aromatic rings of Y78 and F59 and not involved in π–π stacking but provides a hydrophobic surface for the methyl group of crotonyllysine.

Acetylation-independent association of ENL with MOZ

Mass spectrometry data showed that full-length ENL harboring the Y78A substitution in ENLYEATS (impaired acyllysine binding) retains MOZ-binding ability25 and treatment of K562 cells expressing epitope-tagged MOZ–3FLAG–2Strep from the AAVS1 safe harbor with the CBP/p300 inhibitor A485 did not disrupt the interaction with ENL (Extended Data Fig. 1b). As AF9ET was shown to recognize a hydrophobic LxI/VxI/V motif21,22, we explored the possibility that the MOZ IDR might have a binding site for ENLET and identified the LEISV sequence in IDR (aa 809–813 of MOZ) (Fig. 5a,b). The MOZ peptide containing this sequence (aa 806–816 of MOZ) induced CSPs in 15N-labeled ENLET, indicating binding, and measurements of binding affinity of ENLET to the peptide (from here on referred to as MOZEBM; EBM: ET-binding motif) yielded a Kd of 4 μM, which was in the range of binding affinities reported for binding of the ET domains of AF9 and Taf14 to their biological ligands22,45 (Fig. 5c,d and Extended Data Fig. 7).

Fig. 5: Acetylation-independent association of ENL with MOZ.

a, Domain architecture of ENL and AF9. b, Alignment of EBM amino acid sequences. c, Overlaid 1H,15N HSQC spectra of 15N-labeled ENLET (the ET domain of ENL) collected in the absence (black) and presence of increasing amounts of MOZEBM peptide (inset). d, Binding curves used to determine the Kd value by tryptophan fluorescence. Kd values represent the average ± s.d. of three separate experiments. e, A zoomed-in view of the AlphaFold-generated model of MOZ from UniProt Q92794. MOZEBM is outlined by red dotted oval. Residues in MOZEBM are shown in stick representation and labeled. f, Overlaid 1H,15N HSQC spectra of 15N-labeled ENLYEATS collected in the absence (black) and presence of increasing amounts of MOZEBMK815ac peptide. Spectra are color-coded according to the protein:peptide molar ratio. g, Binding curves used to determine the Kd value by tryptophan fluorescence. Kd values represent the average ± s.d. of three separate experiments. h, Models of the multivalent association of ENL and MOZ.

Source data

Full size image

MOZEBM is located next to the K815 acetylation site that may serve as a docking site for ENLYEATS (Fig. 5e). Addition of MOZEBMK815ac peptide induced large CSPs in 1H,15N HSQC spectra of 15N-labeled ENLYEATS, confirming that ENLYEATS recognizes this acetylation site of MOZ (Fig. 5f and Extended Data Fig. 8). The presence of hydrophobic EBM did not affect binding of ENLYEATS to MOZEBMK815ac but acetylation of K815 moderately decreased binding of ENLET to MOZEBM (Fig. 5g). As anticipated, ENLYEATS did not recognize nonacetylated MOZEBM (Extended Data Fig. 8). Collectively, these data suggest a multifaceted mechanism for the association of ENL with MOZ that involves both acetylation-dependent (through ENLYEATS) and acetylation-independent (through ENLET) modes. The former mode would allow for tethering transcription cofactors, such as AF4 and DOT1L, whereas the latter mode would allow for targeting the chromatin sites acylated at H3K27, K18 or K9 (Figs. 5h and 6a). Because the EBM and the K1007 and K1014 acetylation sites in MOZ are far apart, we also cannot exclude the possibility that both ENLYEATS and ENLET associate with the same IDR to further fine-tune the MOZ–ENL assembly at chromatin.

Fig. 6: BDs provide additional contacts to acetylated MOZ.

a, Overlaid 1H,15N HSQC spectra of 15N-labeled ENLYEATS collected in the absence (black) and presence of increasing amounts of H3K27cr peptide. b,c, Overlaid 1H,15N HSQC spectra of 15N-labeled BDs of p300 (b) and BRD4 (c) collected in the absence (black) and presence of increasing amounts of diacetylated MOZEBM peptide. Spectra are color-coded according to the protein:peptide molar ratio. d, A model of the feedforward mechanism for the MOZ–CBP/p300 fusion as a result of uncontrolled acetylation and generation of additional binding sites within the MOZ IDR. e, A model of functional crosstalk involving ENL, MOZ and CBP/p300. f, Lysine acetylation sites in histone H3 recognized by ENL and AF9YEATS and lysine acetylation sites within MOZIDR reported in PTM databases.

Full size image
Bromodomains (BDs) provide additional contacts to acetylated MOZ

Because CBP/p300 contains a BD, which is a well-characterized acetyllysine reader, we tested whether the BD (p300BD) itself can bind acetylated MOZ, which in turn is acetylated by CBP. For comparison, we also tested the first BD of BD-containing protein 4 (BRD4), a transcriptional coactivator known to cooperate with AF9. The MOZYBSK1007acK1014ac peptide caused small but evident CSPs in 1H,15N HSQC spectra of 15N-labeled p300BD and BRD4BD, indicating formation of the respective complexes (Fig. 6b,c). Although the pattern of CSPs (fast exchange regime on the NMR timescale) pointed to a relatively weak association of the isolated BDs, the binding affinity should be augmented for linked BDs in the case of BRD4 or for the BD of CBP/p300 covalently coupled to MOZ in leukemic MOZ–CBP/p300 fusions (Fig. 6d). The chromosomal MOZ–CBP/p300 translocations have been shown to transform hematopoietic progenitors, interfere with the functions of p53, Runx and other transcription factors and nuclear receptors and be directly linked to AML46,47,48,49,50,51. In the leukemic MOZ–CBP/p300 fusion protein, the MOZ fragment retains all folded domains and IDR up to K1013, whereas the CBP/p300 fragment contains nearly the entire protein just lacking the first 243 residues (Fig. 6d). The resulting chimera possesses two catalytic acetyltransferase domains, one from the MOZ fragment and another from the CBP/p300 fragment. The formation of such a ‘super-HAT’ fusion causes aberrant acetylation, subsequently leading to the rapid transcriptional activation and overexpression of HOX and MEIS genes and induction of acute leukemias52,53,54. Our finding that CBP acetylates MOZ, creating binding sites for ENLYEATS and, to some extent, p300BD, suggests a feedforward mechanism that could have a role in the aberrant expression of proleukemic genes.

In conclusion, in this study, we characterized the multifaceted relationship between the major epigenetic and transcriptional regulators ENL and MOZ that have critical roles in normal cellular processes and are implicated in leukemias. We found that both folded domains of ENL, ENLYEATS and ENLET, are capable of binding to the IDR of MOZ. Binding of ENLYEATS to the acetylation sites of MOZ can subsequently recruit transcription cofactors through their association with ENLET, whereas binding ENLET to MOZEBM can bridge the ENL–MOZ assembly with chromatin through the interaction of ENLYEATS with histone H3K27 and H3K18 acylation marks. Both acetylation-dependent and acetylation-independent modes for the MOZ–ENL association and our previous finding that the first WH of MOZ or MORF binds exclusively unmethylated CpG islands and the TAZ2 domain of p300 (refs. 36,55) would facilitate the expression of MOZ–ENL target genes, including proleukemic genes23,24,25 (Fig. 6e).

A comprehensive study of the CBP/p300 acetylome showed that up to one third of the nuclear protein acetylome is regulated by CBP/p300 and that CBP/p300 displays a tendency to acetylate lysine clusters43. In addition to the three experimentally confirmed MOZ acetylation sites originally reported in PTM databases and studied here, a number of other lysine residues in the IDR of MOZ have more recently been found to undergo acetylation42,43 (Fig. 6f and Extended Data Fig. 9). The presence of multiple acetylation sites in MOZ suggests the avidity mechanism for the accumulation of ENL and the number and half-life of these acetylation sites can dictate how fast and how many ENL molecules can be accumulated. The finding that CBP acetylates MOZ, producing binding sites for ENLYEATS, adds another layer of complexity to the functional link between MOZ and ENL, as CBP/p300 specifically generates H3K27 and H3K18 acetylation marks that are also recognized by ENLYEATS. The recognition of acetylated MOZ by ENLYEATS represents an intriguing example of the assembly of a chromatin-modifying complex through PTMs in the complex subunits. Interestingly, p300 has been shown to acetylate another AML leukemogenic translocation, AML1–ETO, and this acetylation is required for leukemogenicity in human preleukemia and mouse AML models56. Future studies are needed to better understand the functional link coupling MOZ, ENL and CBP/p300 and to explore the feedforward mechanism that can lead to uncontrolled acetylation in leukemic MOZ–CBP/p300 translocations.

Methods
Protein purification

ENLYEATS (aa 1–148, pNIC-CH vector), ENLET (aa 483–559, pGEX-6P-1) and AF9YEATS (aa 1–138, pET28b vector) were expressed in Rosetta 2 (DE3) pLysS cells grown in Luria broth, Terrific broth or M9 minimal medium supplemented with 15NH4Cl (Sigma-Aldrich). A tryptophan was introduced to the C terminus of ENLET and was generated using a QuikChange Lightning site-directed mutagenesis kit (Agilent). The cells were induced with IPTG, harvested by centrifugation and lysed by sonication in a buffer consisting of 25–50 mM Tris pH 7.5, 500 mM NaCl, 2 mM β-mercaptoethanol or 5 mM DTT, 0–1% Triton X-100, PMSF and DNase I. His-tag fusion proteins were purified on a HisTrap HP affinity column and eluted by a gradient of 1 M imidazole. The His-tag of AF9YEATS was cleaved with thrombin protease while the His-tag of ENLYEATS was left intact. Glutathione S-transferase (GST)-tagged ENLET was purified on glutathione agarose 4B beads (Thermo Fisher Scientific). The GST-tag was cleaved on resin with tobacco etch virus protease. Proteins were further purified by size-exclusion chromatography using HiPrep 16/600 Superdex 75 columns (GE Healthcare). The sizing buffer was 50 mM Tris pH 7.5, 150 mM NaCl and 2 mM BME or 5 mM DTT. The proteins were concentrated in Millipore concentrators and stored at −80 °C.

MOZ peptides

The following peptides were synthesized by Synpeptide and used in NMR, tryptophan fluorescence and X-ray crystallography experiments: Ac-ILTK(ac)PTLKR-NH2 (aa 1004–1012 of MOZ), Ac-LKRKK(ac)PFL[RHR]-NH2 (aa 1010–1017 of MOZ with C-terminal RHR), Ac-ILTK(ac)PTLKRKK(ac)PFL[RHR]-NH2 (aa 1004–1017 of MOZ with C-terminal RHR), Ac-LTK(cr)PTLKRKK(cr)PFL-NH2 (aa 1005–1017 of MOZ), Ac-LTK(ac)P-NH2 (aa 1005–1008 of MOZ), Ac-LTKPTLKRKKPFL-NH2 (aa 1005–1017 of MOZ), Ac-ERELEISVGKS-NH2 (aa 806–816 of MOZ) and Ac-ERELEISVGK(ac)SVS-NH2 (aa 806–818 of MOZ).

The following peptides were synthesized by CPC Scientific and used in pulldown assays: RSSSPPILTKPTLKRKKPFLHRRRR (aa 998–1022 of MOZ) and RSSSPPILTKacPTLKRKKacPFLHRRRR (aa 998–1022 of acetylated MOZ).

ChIP-seq data analysis

HA–MOZ, ENL and H3K23ac ChIP-seq datasets were obtained from published Gene Expression Omnibus datasets GSE157039, GSE157041 and GSE165076 (ref. 23). Genomic distributions of MOZ and ENL and overlaps of MOZ, ENL and H3K23ac were obtained with the ChIPseeker R package. ChIP-seq signal heat maps were generated with the deepTools R package. Peak-to-gene mapping was performed with ChIPseeker and pathway enrichment analyses of gene lists were carried out with Enrichr57. ChIP-seq tracks were visualized with Integrated Genome Viewer58.

Fractionation of the native MOZ complex

The K562 cell line expressing MOZ–3FLAG–2Strep from the AAVS1 locus was described previously37. Cells were treated with 40 ng ml−1 TSA and 10 µM nicotinamide or with 5 µM CBP/p300 inhibitor A485 overnight and then harvested and lysed in high-salt buffer (450 mM NaCl, 50 mM Tris-HCl pH 8.0, 1% Triton X-100, 2 mM MgCl2, 0.1 mM ZnCl2, 2 mM EDTA, 1 mM DTT and 10% glycerol) supplemented with a cocktail of protease inhibitors. The NaCl concentration was reduced to 225 mM and the whole-cell extract was centrifuged at 14,000 rpm for 30 min. The native MOZ complex was purified from the soluble fraction using anti-FLAG M2 affinity beads (Sigma) overnight at 4 °C on a rotating wheel. FLAG beads were next washed with 225 mM NaCl buffer and eluted with 400 μg ml−1 3xFLAG peptide (Sigma). For immunoblotting, anti-ENL (Cell Signaling, 14893S) was used at a 1:1,000 dilution and the immunoblots were visualized using a Western Lightning Plus enhanced chemiluminescence reagent (PerkinElmer Life Sciences). Horseradish peroxidase (HRP)-conjugated anti-FLAG M2 (Sigma, A8592) antibody was used at a 1:10,000 dilution.

HAT assays

The HAT reactions were performed in a volume of 15 μl using 300 ng of unmodified or K1007acK1014ac MOZYBS or H3 (aa 1–29 of H3) peptides in HAT buffer (50 mM Tris-HCl pH 8, 10 mM sodium butyrate, 5% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol and 1 mM PMSF) in the presence of 20 mM NaCl and 30 mM KCl. MOZYBS peptide acetylation by 35 ng or 55 ng of recombinant CBP (EP300 catalytic domain; Active Motif, 81093) was measured with 0.125 μCi of 3H-labeled Ac-CoA (2.1 Ci mmol−1; PerkinElmer Life Sciences). After incubation for 30 min at 30 °C, the reactions were captured on P81 filter paper, the free 3H-labeled Ac-CoA was washed away and the paper was analyzed using liquid scintillation. The 3H counts were used to evaluate the level of acetylation and CBP autoacetylation was assessed in the absence of MOZ peptides.

Peptide pulldown assay

Human ENL complementary DNA was cloned into pCMV-3FLAG vector to express FLAG-tagged full-length ENL protein in mammalian cells. The GST–ENLYEATS and His–ENLYEATS proteins59 were expressed in Escherichia coli strain Rosetta 2 (DE3) pLysS in the presence of 0.1 mM IPTG at 16 °C for 18 h. The GST–ENLYEATS and His–ENLYEATS proteins were purified using glutathione Sepharose (Cytiva) and Ni-NTA agarose (Qiagen) beads, respectively. For the ENLYEATS–MOZ peptide pulldown assay, 1 µg of biotinylated unmodified or diacetylated MOZ peptides were incubated with 1 µg of purified GST–ENLYEATS or His–ENLYEATS proteins in binding buffer (50 mM Tris pH 7.5, 250 mM NaCl, 0.05% NP-40 and 1 mM PMSF) overnight at 4 °C. Streptavidin magnetic beads (Pierce) were added and incubated for 1 h with rotation. The beads were then washed three times and analyzed using SDS–PAGE and western blotting. The MOZ peptide pulldown assay with full-length FLAG–ENL was performed similarly using whole-cell lysate from 293T cells transiently transfected with pCMV-3FLAG-ENL. The following antibodies were used: anti-His-tag antibody (ZSGB Bio, TA-02), anti-GST-tag antibody (Santa Cruz, sc-459), anti-FLAG-tag antibody (Sigma, F3165) and HRP-conjugated anti-streptavidin antibody (Pierce, 21130).

X-ray crystallography

AF9YEATS was buffer-exchanged and concentrated in 50 mM Bis–Tris pH 6.5 and 5 mM DTT. AF9YEATS was incubated at 5 mg ml−1 with a three-molar equivalence of MOZYBS peptides at 277 K overnight before crystallization. Crystals of AF9YEATS in complex with MOZYBSK1007crK1014cr peptide were grown at 291 K, using the microbatch under oil method against 0.2 M lithium nitrate pH 7.1 and 20% w/v PEG 3350 reservoir solution. The 2.0-µl crystallization drop contained a 1:1 ratio of protein–ligand to reservoir solution. Crystals were cryoprotected with 30% (v/v) glycerol. Crystals of AF9YEATS in complex with MOZYBSK1007ac were grown at 291 K, using the microbatch under oil method in a 1:1 ratio of protein–ligand to reservoir solution (0.2 M sodium formate pH 7.2 and 20% (w/v) PEG 3350) in a 2.0-µl crystallization drop and were cryoprotected with 30% (v/v) glycerol. X-ray diffraction data were collected at a wavelength of 1.07 Å and a temperature of 100 K on beamline 4.2.2 at the Advanced Light Source administrated by the Molecular Biology Consortium. Diffraction images were indexed and integrated using XDS and scaled using Aimless. The phase solution was obtained by the molecular replacement method with Phaser60 using Protein Data Bank (PDB) 6MIL as the search model. Model building was carried out with Coot61 and many iterations of refinement was performed with Refmac5. Coot and MolProbity62 were used to verify the model fit to electron density and overall model quality. The peptide ligands were modeled unambiguously on the basis of the initial Fo − Fc maps and subsequently refined on the basis of the 2Fo − Fc maps. The stereochemical quality of the final models was evaluated using Procheck. The data processing and refinement statistics are shown in Table 1.

NMR experiments

NMR experiments were performed at 298 K on a Varian INOVA 600-MHz spectrometer equipped with a cryoprobe. 1H,15N HSQC spectra of 50–100 µM 15N-labeled ENLYEATS, ENLET or AF9YEATS (in 25 mM Tris pH 6.8–7.0, 150 mM NaCl, 0.2–1 mM TCEP or 5 mM DTT and 10% D2O) were recorded in the presence of increasing concentrations of MOZ and histone peptides. NMR data were processed and analyzed with NMRPipe and NMRDraw as previously described63.

Tryptophan fluorescence

Spectra were recorded at 25 °C on a Fluoromax-3 spectrofluorometer (HORIBA) as previously described64 with some modifications. Briefly, the samples containing 1 µM ENLYEATS, AF9YEATS or tryptophan-containing ENLET (in 50 mM Tris pH 6.8, 150 mM NaCl and 0.2 mM TCEP or 1 mM DTT) and progressively increasing concentrations of peptides were excited at 295 nm. Emission spectra were recorded between 330 and 380 nm with a 0.5-nm step size and a 0.3–1-s integration time. The Kd values, including apparent Kd values estimated for diacylated peptides65, were determined by a nonlinear least-squares analysis and a 1:1 stoichiometry using the following equation:

$$\Delta I={\Delta I}_{\max }\left(\left(\left[L\right]\right)+\,\left[P\right]+{K}_{\rm{d}}\left.\right)-\,\sqrt{{\left(\left[L\right]+\left[P\right]+{K}_{\rm{d}}\right)}^{2}\,-\,4\left[P\right]\left[L\right]\left.\right)}\right)/\,2\left[P\right]$$

where [L] is concentration of the peptide, [P] is concentration of the protein, ΔI is the observed change in signal intensity and ΔImax is the difference in signal intensity between the free and bound states of the protein. Kd values were the average of at least three separate experiments with the error reported as the s.e.m. or s.d.

MST

MST experiments were performed on a Monolith NT.115 instrument (NanoTemper). Experiments were performed using purified ENLYEATS–His protein in a buffer containing 50 mM Tris pH 7.5, 150 mM NaCl. ENLYEATS–His was labeled using a His-Tag labeling kit RED-tris-NTA (second generation, NanoTemper) and kept constant at 20 nM. The dissociation constant was determined using a direct binding assay in which unlabeled MOZYBSK1014ac, MOZYBSK1007acK1014ac or MOZYBSK1007crK1014cr (SynPeptide) was varied in concentration by serial dilution of discrete samples. The measurements were performed at 50% light-emitting diode and medium MST power with 3 s of prelaser time, 20 s of laser on time and 1 s of laser off time. The Kd values, including apparent Kd values estimated for diacylated peptides, were calculated in MO Affinity Analysis software (NanoTemper) using a 1:1 stoichiometry and averaged over three separate experiments with the error reported as the s.d. Plots were generated in GraphPad Prism.

Isothermal titration calorimetry (ITC)

ITC experiments were carried out on a MicroCal Auto-iTC200 instrument at 25 °C while stirring at 750 rpm in ITC buffer consisting of 50 mM Tris pH 7.5 and 150 mM NaCl. Peptide concentration was determined by mass and protein concentration was determined by absorbance at 280 nm. Then, 300-μl samples of 50–100 μM ENLYEATS were placed in the cell and 1–2 mM MOZYBSK1007crK1014cr peptide was loaded in the syringe. Both protein and peptide were diluted in fresh ITC buffer immediately before each experiment. Titrations were conducted using successive injections of 2.5 μl each with 180-s spacing between injections following an initial 0.4-μl injection that was not included in the analysis. Collected data were processed using the Origin 7.0 software program (OriginLab) supplied with the instrument according to the ‘one set of sites’ fitting model.

Statistics and reproducibility

Statistical analysis was performed using GraphPad Prism 8 and Microsoft Excel software. Data were presented as the average ± s.e.m. or s.d. Tryptophan fluorescence, MST, ITC and HAT assays were independently performed at least in triplicate to confirm reproducibility.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Coordinates and structure factors were deposited to the PDB under accession numbers 9ARO and 9ARR. All other relevant data supporting the key findings of this study are available within the article or from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

This paper does not report original code.

References

Tkachuk, D. C., Kohler, S. & Cleary, M. L. Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias. Cell 71, 691–700 (1992).

Article
 
CAS
 
PubMed
 
Google Scholar

Krivtsov, A. V. et al. H3K79 methylation profiles define murine and human MLL-AF4 leukemias. Cancer Cell 14, 355–368 (2008).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Bernt, K. M. et al. MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L. Cancer Cell 20, 66–78 (2011).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Nguyen, A. T., Taranova, O., He, J. & Zhang, Y. DOT1L, the H3K79 methyltransferase, is required for MLL-AF9-mediated leukemogenesis. Blood 117, 6912–6922 (2011).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Mueller, D. et al. A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification. Blood 110, 4445–4454 (2007).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Yokoyama, A., Lin, M., Naresh, A., Kitabayashi, I. & Cleary, M. L. A higher-order complex containing AF4 and ENL family proteins with P-TEFb facilitates oncogenic and physiologic MLL-dependent transcription. Cancer Cell 17, 198–212 (2010).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Monroe, S. C. et al. MLL-AF9 and MLL-ENL alter the dynamic association of transcriptional regulators with genes critical for leukemia. Exp. Hematol. 39, 77–86.e5 (2011).

Article
 
CAS
 
PubMed
 
Google Scholar

Wan, L. et al. ENL links histone acetylation to oncogenic gene expression in acute myeloid leukaemia. Nature 543, 265–269 (2017).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Erb, M. A. et al. Transcription control by the ENL YEATS domain in acute leukaemia. Nature 543, 270–274 (2017).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Perlman, E. J. et al. MLLT1 YEATS domain mutations in clinically distinctive favourable histology Wilms tumours. Nat. Commun. 6, 10013 (2015).

Article
 
CAS
 
PubMed
 
Google Scholar

Wan, L. et al. Impaired cell fate through gain-of-function mutations in a chromatin reader. Nature 577, 121–126 (2020).

Article
 
CAS
 
PubMed
 
Google Scholar

Biondi, A., Cimino, G., Pieters, R. & Pui, C. H. Biological and therapeutic aspects of infant leukemia. Blood 96, 24–33 (2000).

Article
 
CAS
 
PubMed
 
Google Scholar

Salzer, W. L. et al. Modifications to induction therapy decrease risk of early death in infants with acute lymphoblastic leukemia treated on Children’s Oncology Group P9407. Pediatr. Blood Cancer 59, 834–839 (2012).

Article
 
PubMed
 
PubMed Central
 
Google Scholar

Li, Y. et al. Molecular coupling of histone crotonylation and active transcription by AF9 YEATS domain. Mol. Cell 62, 181–193 (2016).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Li, Y. et al. AF9 YEATS domain links histone acetylation to DOT1L-mediated H3K79 methylation. Cell 159, 558–571 (2014).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Li, X. et al. Structure-guided development of YEATS domain inhibitors by targeting π–π–π stacking. Nat. Chem. Biol. 14, 1140–1149 (2018).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Yadav, D., Ghosh, K., Basu, S., Roeder, R. G. & Biswas, D. Multivalent role of human TFIID in recruiting elongation components at the promoter-proximal region for transcriptional control. Cell Rep. 26, 1303–1317.e7 (2019).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Shen, C., Jo, S. Y., Liao, C., Hess, J. L. & Nikolovska-Coleska, Z. Targeting recruitment of disruptor of telomeric silencing 1-like (DOT1L): characterizing the interactions between DOT1L and mixed lineage leukemia (MLL) fusion proteins. J. Biol. Chem. 288, 30585–30596 (2013).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Biswas, D. et al. Function of leukemogenic mixed lineage leukemia 1 (MLL) fusion proteins through distinct partner protein complexes. Proc. Natl Acad. Sci. USA 108, 15751–15756 (2011).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Maethner, E. et al. MLL-ENL inhibits Polycomb repressive complex 1 to achieve efficient transformation of hematopoietic cells. Cell Rep. 3, 1553–1566 (2013).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Leach, B. I. et al. Leukemia fusion target AF9 is an intrinsically disordered transcriptional regulator that recruits multiple partners via coupled folding and binding. Structure 21, 176–183 (2013).

Article
 
CAS
 
PubMed
 
Google Scholar

Schmidt, C. R. et al. BCOR binding to MLL-AF9 is essential for leukemia via altered EYA1, SIX, and MYC activity. Blood Cancer Discov. 1, 162–177 (2020).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Yan, F. et al. KAT6A and ENL form an epigenetic transcriptional control module to drive critical leukemogenic gene-expression programs. Cancer Discov. 12, 792–811 (2022).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Komata, Y., Kanai, A., Maeda, T., Inaba, T. & Yokoyama, A. MOZ/ENL complex is a recruiting factor of leukemic AF10 fusion proteins. Nat. Commun. 14, 1979 (2023).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Miyamoto, R. et al. Activation of CpG-rich promoters mediated by MLL drives MOZ-rearranged leukemia. Cell Rep. 32, 108200 (2020).

Article
 
CAS
 
PubMed
 
Google Scholar

Sapountzi, V. & Cote, J. MYST-family histone acetyltransferases: beyond chromatin. Cell. Mol. Life Sci. 68, 1147–1156 (2011).

Article
 
CAS
 
PubMed
 
Google Scholar

Avvakumov, N. & Cote, J. The MYST family of histone acetyltransferases and their intimate links to cancer. Oncogene 26, 5395–5407 (2007).

Article
 
CAS
 
PubMed
 
Google Scholar

Arboleda, V. A. et al. De novo nonsense mutations in KAT6A, a lysine acetyl-transferase gene, cause a syndrome including microcephaly and global developmental delay. Am. J. Hum. Genet 96, 498–506 (2015).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Tham, E. et al. Dominant mutations in KAT6A cause intellectual disability with recognizable syndromic features. Am. J. Hum. Genet 96, 507–513 (2015).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Sheikh, B. N. et al. MOZ (MYST3, KAT6A) inhibits senescence via the INK4A–ARF pathway. Oncogene 34, 5807–5820 (2015).

Article
 
CAS
 
PubMed
 
Google Scholar

Yang, X. J. MOZ and MORF acetyltransferases: molecular interaction, animal development and human disease. Biochim. Biophys. Acta 1853, 1818–1826 (2015).

Article
 
CAS
 
PubMed
 
Google Scholar

Klein, B. J. et al. Bivalent interaction of the PZP domain of BRPF1 with the nucleosome impacts chromatin dynamics and acetylation. Nucleic Acids Res. 44, 472–484 (2016).

Article
 
CAS
 
PubMed
 
Google Scholar

Peña, P. V. et al. Molecular mechanism of histone H3K4me3 recognition by plant homeodomain of ING2. Nature 442, 100–103 (2006).

Article
 
PubMed
 
PubMed Central
 
Google Scholar

Champagne, K. S. et al. The crystal structure of the ING5 PHD finger in complex with an H3K4me3 histone peptide. Proteins 72, 1371–1376 (2008).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Klein, B. J., Lalonde, M. E., Cote, J., Yang, X. J. & Kutateladze, T. G. Crosstalk between epigenetic readers regulates the MOZ/MORF HAT complexes. Epigenetics 9, 186–193 (2014).

Article
 
CAS
 
PubMed
 
Google Scholar

Becht, D. C. et al. MORF and MOZ acetyltransferases target unmethylated CpG islands through the winged helix domain. Nat. Commun. 14, 697 (2023).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Klein, B. J. et al. Histone H3K23-specific acetylation by MORF is coupled to H3K14 acylation. Nat. Commun. 10, 4724 (2019).

Article
 
PubMed
 
PubMed Central
 
Google Scholar

Klein, B. J. et al. Recognition of histone H3K14 acylation by MORF. Structure 25, 650–654.e2 (2017).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Ali, M. et al. Tandem PHD fingers of MORF/MOZ acetyltransferases display selectivity for acetylated histone H3 and are required for the association with chromatin. J. Mol. Biol. 424, 328–338 (2012).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Weber, L. M. et al. The histone acetyltransferase KAT6A is recruited to unmethylated CpG islands via a DNA binding winged helix domain. Nucleic Acids Res. 51, 574–594 (2023).

Article
 
CAS
 
PubMed
 
Google Scholar

Lv, D. et al. Histone acetyltransferase KAT6A upregulates PI3K/AKT signaling through TRIM24 binding. Cancer Res. 77, 6190–6201 (2017).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Choudhary, C. et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325, 834–840 (2009).

Article
 
CAS
 
PubMed
 
Google Scholar

Weinert, B. T. et al. Time-resolved analysis reveals rapid dynamics and broad scope of the CBP/p300 acetylome. Cell 174, 231–244.e12 (2018).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Andrews, F. H. et al. The Taf14 YEATS domain is a reader of histone crotonylation. Nat. Chem. Biol. 12, 396–398 (2016).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Klein, B. J. et al. Taf2 mediates DNA binding of Taf14. Nat. Commun. 13, 3177 (2022).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Yokoyama A. Role of the MOZ/MLL-mediated transcriptional activation system for self-renewal in normal hematopoiesis and leukemogenesis. FEBS J. 289, 7987–8002 (2022).

Article
 
CAS
 
PubMed
 
Google Scholar

Troke, P. J., Kindle, K. B., Collins, H. M. & Heery, D. M. MOZ fusion proteins in acute myeloid leukaemia. Biochem. Soc. Symp. 73, 23–39 (2006).

Article
 
CAS
 
Google Scholar

Borrow, J. et al. The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein. Nat. Genet. 14, 33–41 (1996).

Article
 
CAS
 
PubMed
 
Google Scholar

Kitabayashi, I., Aikawa, Y., Nguyen, L. A., Yokoyama, A. & Ohki, M. Activation of AML1-mediated transcription by MOZ and inhibition by the MOZ–CBP fusion protein. EMBO J. 20, 7184–7196 (2001).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Chaffanet, M. et al. MOZ is fused to p300 in an acute monocytic leukemia with t(8;22). Genes Chromosomes Cancer 28, 138–144 (2000).

Article
 
CAS
 
PubMed
 
Google Scholar

Panagopoulos, I. et al. Fusion of the MORF and CBP genes in acute myeloid leukemia with the t(10;16)(q22;p13). Hum. Mol. Genet. 10, 395–404 (2001).

Article
 
CAS
 
PubMed
 
Google Scholar

Champagne, N., Pelletier, N. & Yang, X. J. The monocytic leukemia zinc finger protein MOZ is a histone acetyltransferase. Oncogene 20, 404–409 (2001).

Article
 
CAS
 
PubMed
 
Google Scholar

Camós, M. et al. Gene expression profiling of acute myeloid leukemia with translocation t(8;16)(p11;p13) and MYST3-CREBBP rearrangement reveals a distinctive signature with a specific pattern of HOX gene expression. Cancer Res. 66, 6947–6954 (2006).

Article
 
PubMed
 
Google Scholar

Grier, D. G. et al. The pathophysiology of HOX genes and their role in cancer. J. Pathol. 205, 154–171 (2005).

Article
 
CAS
 
PubMed
 
Google Scholar

Becht, D. C. et al. The winged helix domain of MORF binds CpG islands and the TAZ2 domain of p300. iScience 27, 109367 (2024).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Wang, L. et al. The leukemogenicity of AML1-ETO is dependent on site-specific lysine acetylation. Science 333, 765–769 (2011).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Chen, E. Y. et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics 14, 128 (2013).

Article
 
PubMed
 
PubMed Central
 
Google Scholar

Robinson, J. T. et al. Integrative Genomics Viewer. Nat. Biotechnol. 29, 24–26 (2011).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Ma, X. R. et al. Discovery of selective small-molecule inhibitors for the ENL YEATS domain. J. Med. Chem. 64, 10997–11013 (2021).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

Article
 
CAS
 
PubMed
 
Google Scholar

Klein, B. J. et al. The Histone-H3K4-specific demethylase KDM5B binds to its substrate and product through distinct PHD fingers. Cell Rep. 6, 325–335 (2014).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Gatchalian, J. et al. Dido3 PHD modulates cell differentiation and division. Cell Rep. 4, 148–158 (2013).

Article
 
CAS
 
PubMed
 
PubMed Central
 
Google Scholar

Erlendsson, S. & Teilum, K. Binding revisited—avidity in cellular function and signaling. Front. Mol. Biosci. 7, 615565 (2021).

Article
 
PubMed
 
PubMed Central
 
Google Scholar

Download references

Acknowledgements

We thank J. Nix at beamline 4.2.2 of the ALS in Berkeley for help with X-ray crystallographic data collection. This work was supported in part by grants from the National Institutes of Health (HL151334, GM135671, CA252707 and AG067664 to T.G.K., CA279317 to M.A.B. and CA255506 to H.W.) and the Canadian Institutes of Health Research (FDN-143314 and PJT-178367 to J.C.). H.W. is a Leukemia & Lymphoma Society scholar.

Author information
Author notes

These authors contributed equally: Dustin C. Becht, Karthik Selvam, Catherine Lachance.

Authors and Affiliations

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, USA

Dustin C. Becht, Karthik Selvam, Minh Chau Nguyen, Akshay Pareek & Tatiana G. Kutateladze

St-Patrick Research Group in Basic Oncology, Oncology Division of CHU de Québec-Université Laval Research, Laval University Cancer Research Center, Québec City, Québec, Canada

Catherine Lachance, Valérie Côté & Jacques Côté

Department of Epigenetics, Van Andel Institute, Grand Rapids, MI, USA

Kuai Li, Xiaobing Shi & Hong Wen

Department of Biomedical Sciences, University of Pennsylvania, School of Veterinary Medicine, Philadelphia, PA, USA

M. Andres Blanco

Contributions

D.C.B., K.S., C.L., V.C., K.L., M.C.N. and A.P. performed the experiments and, together with X.S., H.W., M.A.B., J.C. and T.G.K., analyzed the data. T.G.K. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Tatiana G. Kutateladze.

Ethics declarations
Competing interests

The authors declare no competing interests.

Peer review
Peer review information

Nature Structural & Molecular Biology thanks Elisar Barbar and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data
Extended Data Fig. 1 Western analysis of MOZ-3FLAG.

Western analysis of the FLAG eluted fractions from cells expressing MOZ-3FLAG from the AAVS1 safe harbour, untreated or treated (a) with HDAC inhibitors TSA (40 ng/ml) and nicotinamide (10 µM) and (b) with HDAC inhibitors TSA (40 ng/ml) and nicotinamide (10 µM) or with CBP/p300 inhibitor A485 (5 µM) and probed by indicated antibodies (anti-Flag–HRP, Sigma A8592 1/10000 RT) and anti-ENL/MLLT1, Cell signaling 1D9M4B #148935 1/1000 o/n 4).

Source data

Extended Data Fig. 2 NMR titration experiments using ENLYEATS and monoacetylated and unmodified peptides.

Overlaid 1H,15N HSQC spectra of 15N-labeled ENLYEATS collected in the absence (black) and presence of increasing amounts of indicated monoacetylated and unmodified peptides. Spectra are color coded according to the protein:peptide molar ratio. MOZYBSK1007ac (aa 1004-1012 of MOZ), MOZYBSK1014ac (aa 1010-1017 of MOZ with C-terminal RHR), unmod MOZ (aa 1005-1017 of MOZ).

Extended Data Fig. 3 Liquid HAT assays.

Liquid HAT assays using 35 ng recombinant CBP and 300 ng of unmodified MOZYBS or 300 ng of H3 (aa 1-29) peptides as substrates. CPM, count per minute. HAT assays were performed in triplicate. Data are represented as mean ± SD between three replicates.

Source data

Extended Data Fig. 4 Binding of AF9YEATS to MOZYBSK1007ac.

Superimposed 1H,15N HSQC spectra of 15N-labeled AF9YEATS recorded in the presence of increasing amount of MOZYBSK1007ac (aa 1005-1008 of MOZ) peptide. Spectra are color coded according to the protein:peptide molar ratio.

Extended Data Fig. 5 Binding of ENLYEATS and AF9YEATS to diacylated peptides.

(top) Overlaid 1H,15N HSQC spectra of 15N-labeled ENLYEATS collected in the absence (black) and presence of increasing amounts of MOZYBSK1007acK1014ac peptide. (middle) Overlaid 1H,15N HSQC spectra of 15N-labeled ENLYEATS collected in the absence (black) and presence of increasing amounts of MOZYBSK1007crK1014cr peptide. (bottom) Overlaid 1H,15N HSQC spectra of 15N-labeled AF9YEATS collected in the absence (black) and presence of increasing amounts of MOZYBSK1007crK1014cr peptide. Spectra are color coded according to the protein:peptide molar ratio.

Extended Data Fig. 6 Binding affinity of ENLYEATS.

Binding affinity of ENLYEATS to MOZYBSK1007crK1014cr peptide measured by MST (top) and ITC (bottom). Apparent Kd from MST data represents the average of three independent measurements ± SD. Point errors represent SD. Representative ITC experiment is shown below. Three ITC experiments were performed, and apparent Kd from ITC data represents the average of three independent measurements ± SD.

Source data

Extended Data Fig. 7 Binding of ENLET to MOZEBM.

Overlaid 1H,15N HSQC spectra of 15N-labeled ENLET collected in the absence (black) and presence of increasing amounts of MOZEBM peptide. Spectra are color coded according to the protein:peptide molar ratio.

Extended Data Fig. 8 Binding of ENLYEATS to MOZEBMK815ac and MOZEBM.

Overlaid 1H,15N HSQC spectra of 15N-labeled ENLYEATS collected in the absence (black) and presence of increasing amounts of MOZEBMK815ac peptide (top) and MOZEBM peptide (bottom). Spectra are color coded according to the protein:peptide molar ratio.

Extended Data Fig. 9 AlphaFold (AF)-generated model of MOZ.

A zoom-in view of the AF-generated model of MOZ from UniProt, Q92794. MOZ folded domains and identified lysine acetylation sites are labeled.

Supplementary information
Reporting Summary
Peer Review File
Source data
Source Data Figs. 2, 4 and 5 and Extended Data Figs. 3 and 6

Binding and HAT data.

Source Data Fig. 2 and Extended Data Fig. 1

Uncropped blots.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article
Cite this article

Becht, D.C., Selvam, K., Lachance, C. et al. A multivalent engagement of ENL with MOZ. Nat Struct Mol Biol 32, 709–718 (2025). https://doi.org/10.1038/s41594-024-01455-8

Download citation

Received
26 February 2024

Accepted
19 November 2024

Published
10 January 2025

Version of record
10 January 2025

Issue date
April 2025

DOI
https://doi.org/10.1038/s41594-024-01455-8

Share this article

Anyone you share the following link with will be able to read this content:

Get shareable link

Provided by the Springer Nature SharedIt content-sharing initiative

Subjects
NMR spectroscopy
X-ray crystallography

Cancel

Preview · live