DPY30 acts as an ASH2L-specific stabilizer to stimulate the enzyme activity of MLL family methyltransferases on different substrates

Summary Dumpy-30 (DPY30) is a conserved component of the mixed lineage leukemia (MLL) family complex and is essential for robust methyltransferase activity of MLL complexes. However, the biochemical role of DPY30 in stimulating methyltransferase activity of MLL complexes remains elusive. Here, we demonstrate that DPY30 plays a crucial role in regulating MLL1 activity through two complementary mechanisms: A nucleosome-independent mechanism and a nucleosome-specific mechanism. DPY30 functions as an ASH2L-specific stabilizer to increase the stability of ASH2L and enhance ASH2L-mediated interactions. As a result, DPY30 promotes the compaction and stabilization of the MLL1 complex, consequently increasing the HKMT activity of the MLL1 complex on diverse substrates. DPY30-stabilized ASH2L further acquires additional interfaces with H3 and nucleosomal DNA, thereby boosting the methyltransferase activity of the MLL1 complex on nucleosomes. These results collectively highlight the crucial and conserved roles of DPY30 in the complex assembly and activity regulation of MLL family complexes.


INTRODUCTION
H3K4 methylation is critical to the epigenetic regulation of gene transcription (Hyun et al., 2017;Shilatifard, 2008). Defects in H3K4 methylation have been closely associated with a broad spectrum of hematologic and solid malignancies (Rao and Dou, 2015;Yang and Ernst, 2017). H3K4 methylation is mainly mediated by MLL family proteins, including MLL1, MLL2, MLL3, MLL4, SET1A, and SET1B (Ansari and Mandal, 2010). Among them, MLL1 has drawn the most attention because its chromosomal translocations lead to various forms of acute lymphoid and myeloid leukemia (Krivtsov et al., 2017).
Dpy-30 was initially discovered in Caenorhabditis elegans as a regulator in X chromosome dosage compensation (Hsu and Meyer, 1994). Mammalian DPY30 can be assembled into MLL family complexes through its C-terminal 44-residue helical bundle, termed the docking and dimerization domain (DD domain), which directly interacts with the ASH2L C-terminal DPY30-binding motif (DBM) (Haddad et al., 2018;South et al., 2010). In embryonic stem cells (ESCs), knockdown of DPY30 reduces H3K4 trimethylation and impairs ESC plasticity in transcriptional reprogramming in vivo (Jiang et al., 2011). Similarly, knockdown of DPY30 by siRNA led to decreased H3K4 methylation levels and inhibited the proliferation and differentiation of hematopoietic progenitor cells (Yang et al., 2014). Complete depletion of DPY30 from conditional In this work, we reveal that DPY30 can stimulate the HKMT activity of the MLL1 complex through a nucleosome-independent mechanism and a nucleosome-specific mechanism. By combining crosslinking mass spectrometry (CX-MS), structural prediction, molecular dynamics (MD) analyses, biological small-angle X-ray scattering (SAXS), and biochemical assays, we demonstrate that DPY30 functions as an ASH2L-specific stabilizer. DPY30-stabilized ASH2L acquires functional improvement to enhance ASH2L-mediated interactions to promote the assembly and stabilization of the MLL1 complex, which explains the activity stimulation on a wide range of substrates (H3 peptide, H3/H4 tetramer, and octamer) by DPY30. DPY30-stabilized ASH2L further gains additional interfaces with nucleosomal DNA and H3, thereby specifically boosting HKMT activity on nucleosomes.

DPY30 enhances the HKMT activity of the MLL1 complex
To examine the role of DPY30 in stimulating MLL1 activity, we first characterized the HKMT activity of the MLL1 complex (MLL1-WDR5-RBBP5-ASH2L, abbreviated as M1WRA) in the presence and absence of DPY30 on different substrates by performing a western-blot-based methyltransferase assay. We found that DPY30 remarkably enhanced the HKMT activity of the MLL1 complex on NCPs but had negligible effects on octamer and H3-H4 tetramer substrates ( Figure 1A). To quantitatively dissect the roles of DPY30 on different substrates, we compared the reaction rates of MLL1 core complexes by using an MTase-Glo Methyltransferase Assay kit. DPY30 substantially boosted the methylation rate on NCP by $18-fold but only increased the reaction rate on the octamer, H3-H4 tetramer, and H3 1-9 peptide by $1.6-fold Figure 1B).
To further compare the kinetic difference of the MLL1 complex in the presence or absence of DPY30 on different substrates, we performed steady-state kinetic analyses of the MLL1 complex by fixing the concentration of AdoMet and changing the substrate concentrations of NCP or H3 1-9 . In the absence of DPY30, M1WRA exhibited relatively weak activity with a K m (NCP) of 0.16 mM and a turnover rate (k cat ) of 0.023 min À1 . In the presence of DPY30, the MLL1 complex (M1WRA + D) exhibited strikingly boosted activity on NCP with an $18-fold increase in k cat and a 1.5-fold decrease in K m ( Figure 1C). Thus, for NCP substrates, the catalytic efficiency (k cat /K m ) of the MLL1 complex with DPY30 was 27-fold higher than that of the MLL1 complex without DPY30. In contrast, the activity-stimulating effect of DPY30 was much weaker on the H3 1-9 peptide, with a 1.2-fold increase in k cat and a 1.5-fold decrease in K m ( Figure 1D). The 1.8-fold increase in the catalytic efficiency (k cat /K m ) of the MLL1 complex indicates a weak but appreciable effect of DPY30 in stimulating the activity of the MLL1 complex on H3 peptides. These data not only reveal the important role of DPY30 in ensuring optimal HKMT activity of the MLL1 complex but also suggest that DPY30 could stimulate HKMT activity through a nucleosome-independent mechanism and a nucleosome-specific mechanism. iScience Article The activity-stimulating effect of DPY30 is dependent on ASH2L We sought to explore the underlying mechanism by which DPY30 stimulates HKMT activity of the MLL1 complex. Previous studies have reported that DPY30 only interacts with ASH2L in the MLL1 complex by binding to the C-terminal DBM of ASH2L (Haddad et al., 2018;Patel et al., 2009). We first investigated whether the activity-stimulating role of DPY30 is dependent on its interaction with ASH2L. By utilizing an ASH2L mutant (L513E/L517E/V520E) (hereafter referred to as ASH2L 3E ) that completely abolished the interaction with DPY30 (Chen et al., 2012), as shown in the GST pull-down assay ( Figure S1A), we found that DPY30 could not increase the HKMT activity of the MLL1 complex reconstituted with this DPY30-binding-deficient ASH2L mutant on NCPs or H3 peptides (Figures S1B and S1C). Moreover, the DPY30 dimerization domain (DPY30 DD , 45-99), mediating the interaction with ASH2L (Haddad et al., 2018), was sufficient and necessary to stimulate the HKMT activity of the MLL1 complex ( Figure S1B). A previously-identified DPY30 mutant (L69D), which impairs DPY30 dimerization and abolishes its binding to ASH2L ( Figure S1D) (Tremblay et al., 2014), could not increase the HKMT activity of the MLL1 complex (Figures S1B). These results reveal that the stimulatory effect of DPY30 relies on its interaction with ASH2L.
Next, we investigated which ASH2L domain(s) contribute to DPY30-induced stimulation of MLL1 activity. ASH2L encompasses an N-terminal PHD-WH domain (1-177), an intrinsically disordered region (IDR) (178-229), a pre-SPRY motif (230-285), a split SPRY domain (286-499) with an SPRY-insertion (400-440), and a C-terminal DPY30-binding motif (DBM, 500-534) ( Figure 2A). The essential roles of the ASH2L SPRY domain in the regulation of MLL1 complex activity have been firmly established (Li et al., 2016), but the functions of other ASH2L domains remain elusive. We used a series of ASH2L constructs with the deletion of each domain iScience Article or motif to probe how these individual domains or motifs affect the DPY30-dependent activity stimulation of the MLL1 complex. All ASH2L domain-deletion mutants were eluted at the expected peak positions corresponding to their molecular weights on gelfiltration chromatography and could be assembled into M1WRA complexes ( Figures S2A-S2C), suggesting that these domain-deletion mutants retained the structural integrity of ASH2L. To our surprise, all ASH2L domains were required for maintaining the optimal HKMT activity of the MLL1 complex. Deleting the PHD-WH domain slightly reduced the stimulatory effect of DPY30, whereas deletion of IDR, pre-SPRY, and SPRY-insertion severely decreased the stimulatory effects of DPY30 on NCP substrates ( Figure 2B). Combined deletion of IDR, pre-SPRY, and SPRY-insertion motifs (DLL) completely abolished DPY30-dependent activity stimulation on NCP ( Figure 2B). Notably, deletion of pre-SPRY or SPRY-insertion, but not PHD-WH or IDR, also disrupted the stimulatory effect of DPY30 on  iScience Article H3 1-9 peptides ( Figure 2C). These results indicate that these previously noteless domains or motifs of ASH2L play essential roles to enhance the HKMT activity of the MLL1 complex: the pre-SPRY and SPRY-insertion are critical for both nucleosome-independent and nucleosome-specific activity stimulation, whereas PHD-WH and IDR are only required for nucleosome-specific activity stimulation by DPY30.

DPY30 interacts with multiple regions of ASH2L
The functional interplay between DPY30 and these less-characterized motifs of ASH2L intrigued us to reexamine the DPY30-mediated interactions in the MLL1 complex. We performed crosslinking mass spectrometry (CX-MS) to probe the potential differences in protein-protein interaction networks of the MLL1 complex induced by DPY30. By using a cutoff of spectrum counts of more than three and an E score value smaller than 0.02, we identified 158 crosslinked peptides in the MLL1 complex with DPY30 (M1WRAD) and 122 crosslinked peptides in the MLL1 complex without DPY30 (M1WRA) (Data S1). The majority of the crosslinked peptides were shared in the two complexes, but the addition of DPY30 induced 47 specific crosslinks identified exclusively in the M1WRAD complex ( Figure S3A and Table S1). DPY30 is intensely crosslinked to the SPRY-insertion, IDR, and PHD-WH domains of ASH2L ( Figure 2D). It should be noted that no crosslink was detected between DPY30 and ASH2L DBM because there is no lysine residue in the ASH2L DBM region to enable crosslinking. In addition to the newly found DPY30-ASH2L intermolecular crosslink, we also found that DPY30 induced a substantial enrichment of the intramolecular crosslinks in ASH2L, especially the extensive crosslinks between SPRY-insertion and other regions of ASH2L ( Figure 2D).
The widespread crosslinks between multiple regions of ASH2L and DPY30, as well as between RBBP5 and DPY30, indicate that these ASH2L or RBBP5 regions may interact with DPY30 or be in closeness with DPY30.
To distinguish these two possibilities, we performed GST pull-down assays to characterize DPY30 interactions with ASH2L or RBBP5. No interaction was detected between DPY30 and RBBP5 under our assay conditions (Figure S3B), indicating that DPY30 and RBBP5 may just be in spatial proximity but do not directly interact with each other. In sharp contrast, ASH2L was readily pulled down by GST-DPY30 ( Figure 2E). Moreover, we found that deletion of PHD-WH and IDR did not affect the amount of ASH2L pulled down by GST-DPY30, but the deletion of pre-SPRY or SPRY-insertion severely decreased the ASH2L-DPY30 interactions ( Figures 2E and S3C), suggesting that pre-SPRY and SPRY-insertion of ASH2L may be directly involved in the interaction with DPY30.

The structural basis of the interaction between DPY30 and ASH2L
Next, we sought to determine how pre-SPRY and SPRY-insertion of ASH2L contribute to DPY30 binding. After extensive but unsuccessful attempts to crystallize the ASH2L-DPY30 complex, we decided to use AlphaFold2 to predict the ASH2L-DPY30 complex structure and apo ASH2L structure (Figures 3A and S4A) (Mirdita et al., 2022). ASH2L exhibits a two-lobe structure separated by a flexible IDR: one lobe (aa 1-177) is the PHD-WH domain, and the other lobe (aa 230-534) is composed of pre-SPRY, SPRY, SPRY-insertion, and DBM motifs ( Figure S4A). Each lobe shows a compact fold with high pLDDT (prediction local distance difference test) values, indicating high confidence of structural prediction, but the ASH2L IDR loop (178-229) connecting the two lobes does not have any defined structure ( Figure S4B). As a result, the PHD-WH domains show random orientations relative to ASH2L 230-534 in five apo ASH2L models ( Figure S4C). The ASH2L-DPY30 complex also shows a two-lobe feature ( Figures S4D and S4E). Although ASH2L IDR is still unstructured, the PHD-WH domain in the ASH2L-DPY30 complex has a fixed orientation relative to ASH2L 230-534 ( Figure S4F). The PAE (predicted aligned error) plots also confirmed that the ASH2L PHD-WH domain has some inter-domain packings with ASH2L pre-SPRY, SPRY-insertion, DBM, and one DPY30 ( Figure S4G). This suggests that DPY30 binding restrains the rotational freedom between ASH2L 1-177 and ASH2L 230-534 . This notion was supported by the observation that DPY30 induced more intramolecular crosslinked peptides of ASH2L in the M1WRAD complex, especially dramatically increased crosslinking peptides between PHD-WH and SPRY-insertion ( Figure 2D).
A recent publication reported that the pre-SPRY and SPRY-insertion motifs of ASH2L are intrinsically disordered regions (IDRs), and DPY30 induces conformational changes in ASH2L IDRs to form ordered structures (Lee et al., 2021). However, the structural prediction of apo ASH2L indicates that pre-SPRY and SPRY-insertion motifs have well-defined structural features with high confidence ( Figures S4A and S4B). The structures of the apo ASH2L 230-534 and ASH2L 230-534 -DPY30 complexes can be superimposed with a root-main-square deviation (rmsd) value of 0.32 Å ( Figure S4H). The pre-SPRY and SPRY-insertion motifs in both structures are almost identical. Molecular dynamic simulation analyses further indicated that the secondary structures of pre-SPRY and SPRY-insertion motifs in both apo ASH2L and ASH2L-DPY30 remained stable during the simulation process ll OPEN ACCESS iScience 25, 104948, September 16, 2022 5 iScience Article ( Figures S5A-S5D), suggesting that DPY30 may not directly induce the conformational change of ASH2L pre-SPRY and ASH2L SPRY-insertion, as proposed by the previous publication (Lee et al., 2021) In the representative complex structure model, the main DPY30-ASH2L binding interface is established by the ASH2L DBM helix docked into the hydrophobic cleft of DPY30 dimers ( Figure 3B). The pre-SPRY and the SPRY-insertion motifs of ASH2L function as two arms to clamp the ASH2L DBM helix, thus embracing the DBM helix together with two DPY30 dimerization helices ( Figure 3B). Consistent with the structural model, deletion of pre-SPRY and SPRY-insertion decreased the interaction between ASH2L and DPY30 ( Figure 2E). In addition, the ASH2L PHD domain directly contacts ASH2L pre-SPRY and one copy of DPY30 ( Figure 3B).
The importance of pre-SPRY and SPRY-insertion in maintaining the ASH2L-DPY30 interaction can also be in-    Figure S6A), indicating that the inclusion of pre-SPRY and SPRY-insertion motifs ensures the conserved ASH2L-DPY30/Bre2-Sdc1 binding mode in different species. In addition, the predicted ASH2L-DPY30 structure can be superimposed into the M1WRAD-NCP structure without any clash with other MLL complex or NCP components ( Figure S6B), suggesting that the similar ASH2L-DPY30 configuration could be maintained in the M1WRAD-NCP complex.
A complex array of electrostatic interactions stabilizes the tetrapartite interface composed of ASH2L pre-SPRY , ASH2L SPRY-insertion , ASH2L DBM , and DPY30 DD ( Figure 3C). For example, DPY30 D58 forms three saltbridges with H257 and R280 from ASH2L pre-SPRY , and R280 is further secured by electrostatic interactions with D255, which additionally coordinates two positively charged residues, including K413 from ASH2L SPRY-insertion and H519 from ASH2L DBM . DPY30 R54 is linked to K413 from ASH2L SPRY-insertion through the D515 bridge from ASH2L DBM ( Figure 3C). In addition, the PHD domain in ASH2L PHD-WH interacts with pre-SPRY and DPY30 mainly through electrostatic and hydrogen-bonding interactions ( Figure 3D). In support of the important roles of these electrostatic interactions, mutations of ASH2L R280A, K413A, D515A/Y518A/H519A, or DPY30 R54A/D58A, which did not affect the structural integrity of ASH2L ( Figure S6C), specifically decreased the activity stimulation by DPY30 ( Figure 3E). Collectively, these results reveal some previously unrecognized interaction interfaces between ASH2L and DPY30.

DPY30 stabilizes ASH2L
We then explored how the ASH2L-DPY30 interaction affects the structures and functions of ASH2L and the MLL1 complex. The structural prediction indicated that DPY30 could restrain the turbulent motion between two ASH2L lobes, leading to stabilized ASH2L with a relatively fixed conformation ( Figure S4F). We wondered whether the structural stabilization of ASH2L by DPY30 could lead to the functional improvement of ASH2L. We first compared the thermostability of ASH2L or the ASH2L-DPY30 complex by nano differential scanning fluorimetry (nanoDSF), which monitors the intrinsic tryptophan fluorescence of proteins. Because DPY30 does not contain any tryptophan, the fluorescence signals reflect the folding status of ASH2L. The unfolding curves clearly showed that ASH2L exhibited a polyphasic unfolding transition with the first melting temperature (Tm1) at 32.3 C and the second melting temperature (Tm2) at 44.4 C, consistent with the multiple independent structural domains in ASH2L. The presence of DPY30 yielded a cooperative unfolding transition with one melting temperature at 50.0 C ( Figure 4A), indicating that the ASH2L-DPY30 complex exhibits a compact fold with a much higher thermostability than ASH2L. Disrupting the ASH2L-DPY30 interaction by introducing ASH2L 3E or DPY30 L69D abolished the DPY30-dependent Tm increase ( Figures 4B and 4C). The DPY30 R54A/D58A mutant only slightly increased the Tm of ASH2L ( Figure 4D). Deletion of pre-SPRY or SPRYinsertion of ASH2L, which impaired the ASH2L-DPY30 interaction, also severely destabilized the ASH2L-DPY30 complex with slightly increased Tm1 and unchanged Tm2 compared to ASH2L alone ( Figures 4E and 4F). These results highlight the essential role of DPY30 in improving the thermostability of ASH2L.
We then checked the ability of DPY30 to prevent ASH2L aggregation. We found that the addition of increasing amounts of DPY30 substantially reduced ASH2L aggregation at 37 C as monitored by light scattering ( Figure 4G). A stoichiometric quantity of DPY30 (DPY30:ASH2L = 2:1) effectively inhibited aggregation, and extra DPY30 did not further suppress the aggregation of ASH2L Figure 4G). The temperaturedependent protein aggregation curves measured by nanoDSF further confirmed that DPY30 substantially increased the T agg (temperature of aggregation) of ASH2L from 44.8 C to 62.2 C Figure 4H). Collectively, DPY30 can decrease the internal structural flexibility of ASH2L to maintain a compact conformation of ASH2L and stabilize ASH2L with increased thermostability and a reduced aggregation tendency.

DPY30 promotes the assembly of the MLL1 complex
We speculate that DPY30-dependent ASH2L stabilization may enhance ASH2L interaction with other proteins and facilitate the formation of a more stable MLL1 complex with increased HKMT activity. The crosslinking-MS data partially supported this speculation. DPY30 induced a substantial enrichment of the intramolecular crosslinks in ASH2L and intermolecular crosslinks in MLL1-ASH2L and RBBP5-ASH2L pairs ( Figure 2D), indicating an enhanced internal interaction network in MWRA on DPY30 binding. iScience Article ASH2L-RBBP5 interaction. For FP assays, we used a fluorescence-labeled RBBP5 AS-ABM (residues 330-363), a minimal RBBP5 fragment required for ASH2L binding (Li et al., 2016). Our FP assays showed that RBBP5 AS-ABM binds to free ASH2L with a dissociation constant (K d ) of 0.72 mM, but it binds to DPY30-bound ASH2L with a K d of 0.16 mM, a 4-fold increase in binding affinity ( Figure 5A). These results suggest that DPY30 binding to ASH2L confers positive cooperativity for the ASH2L-RBBP5 interaction. iScience Article DPY30-dependent enhancement of internal interactions in the MLL1 complex might result in a more compact M1WRAD complex than M1WRA. To check whether DPY30 contributes to the assembly of the MLL1 complex, we utilized small-angle X-ray scattering (SAXS) to characterize the conformation of the M1WRA in the presence or absence of DPY30. The Guinier regions of the scattering curves were linear at low q (range of momentum transfer), indicating that the samples were not aggregated (Figures S7A and S7B). The SAXS data were used to calculate the maximum particle dimension (d max ) and the radius of gyration (R g ). Although the molecular weight of the M1WRAD complex (203.3 kDa) is 12.4% larger than M1WRA (180.8 kDa), the M1WRAD complex has a similar Rg as M1WRA and a smaller d max (175 Å ) than that of M1WRA (185 Å ) (Figures 5B and Table S2), suggesting that DPY30 promotes the compaction of the MLL1 complex.
DPY30-induced compaction of the MLL1 complex might ensure a more stable DPY30-containing MLL1 complex. Indeed, nanoDSF analyses showed that DPY30 could elevate the Tm1 and Tm2 of the MLL1 complex from 40.3 C to 44.5 C and 44.3 C-53.5 C, respectively Figure 5C), but could not increase the Tm of the See also Figure S7 and iScience Article MLL1 complex reconstituted with DPY30-binding-deficient ASH2L (ASH2L 3E ) ( Figure 5D). Moreover, DPY30 L69D and R54A/D58A mutations impaired the stabilization ability of DPY30 because these DPY30 mutants only mildly increased the Tm of the MLL1 complex ( Figures 5E and 5F). These results indicate that DPY30dependent ASH2L stabilization promotes the assembly and stability of the MLL1 complex. We reason that this DPY30-induced structural stabilization and compaction of the MLL1 complex may account for the mild nucleosome-independent activity stimulation by DPY30 on non-nucleosome substrates, including histone octamers, H3-H4 tetramers, and H3 peptides ( Figures 1B and 1D).

The DPY30-ASH2L complex provides additional anchors on nucleosomes
Although DPY30-induced compaction of the MLL1 complex could explain the nucleosome-independent activity stimulation by DPY30, an additional mechanism must exist that determines the nucleosome-specific activity boosted by DPY30. To probe how DPY30 affects the M1WRA-NCP interaction, we performed crosslinking mass spectrometry analyses of the M1WRAD-NCP and M1WRA-NCP complexes, aiming to identify potential M1WRAD-NCP interfaces induced by DPY30. There were 102 crosslinked peptides identified in the M1WRA-NCP sample, and 10 of them were between M1WRA and NCP histones (Figures S8A, S8B and S8D and Data S2). For comparison, 20 out of 148 crosslinked peptides were identified between M1WRAD and NCP histones (Figures S8A, S8C and S8E and Data S2). The 10 crosslinked peptides between M1WRA and NCP histones identified in M1WRA-NCP were all found in M1WRAD-NCP ( Figure 6A). In the presence of DPY30, there are 10 specific crosslinks between M1WRAD and NCP ( Figure 6B), assumably providing additional anchors for M1WRAD on nucleosomes.
Notably, the N-terminal tail of H3 was extensively crosslinked to the SPRY-insertion and IDR elusively found in M1WRAD-NCP, indicating a potential direct interaction between ASH2L and the H3 tail in the presence of DPY30 Figure 6B). To provide direct evidence that DPY30 may enhance ASH2L's interaction with the H3 tail, we used a fluorescence polarization assay to characterize the ASH2L interaction with a fluorescent H3 1-36 peptide. FP assays showed that ASH2L or DPY30 alone had a negligible H3-binding ability, but the ASH2L-DPY30 complex had an appreciable interaction with the H3 tail Figure 6C), suggesting that DPY30-stabilized ASH2L could interact with the H3 tail.
In addition to the ASH2L-H3 interfaces, DPY30-stabilized ASH2L may achieve the ability to bind DNA, as indicated by the previous M1WRAD-NCP structure showing the close proximity between ASH2L and nucleosomal DNA (Park et al., 2019;Xue et al., 2019). Because our previous studies demonstrated that ASH2L had DNA-binding activity (Chen et al., 2011), we wondered whether DPY30 might modulate ASH2L's DNA-binding activity. The electrophoretic mobility shift assay (EMSA) confirmed that apo ASH2L had DNA-binding activity but mostly formed protein-DNA aggregates not migrating into the gel, as judged by the density of the shifted band ( Figure 6D). Although DPY30 itself does not have any DNA-binding activity, the inclusion of DPY30 not only increased the binding affinity between ASH2L and DNA but also facilitated the formation of a soluble protein-DNA complex running as a sharp band on EMSA gels Figure 6D).
Previous studies have shown that the PHD-WH domain of ASH2L can bind DNA (Chen et al., 2011;Sarvan et al., 2011). We found that the deletion of PHD-WH (ASH2L DPHD-WH ) indeed decreased the ASH2L-DNA association, but ASH2L DPHD-WH still responded to DPY30. DPY30 greatly increased the binding affinity between ASH2L DPHD-WH and DNA ( Figure 6E), consistent with the observation that the deletion of PHD-WH had a marginal effect on the HKMT activity on NCP ( Figure 2B). The ASH2L IDR, pre-SPRY, and SPRY-insertion motifs are more critical for DNA-binding activity of ASH2L. The deletion of these motifs (ASH2L DLL ) completely abolished the DNA-binding ability of ASH2L and severely disrupted the DPY30dependent DNA-binding activity of ASH2L ( Figure 6F). Thus, we conclude that the presence of DPY30 makes ASH2L competent for DNA binding through ASH2L IDR, pre-SPRY, and SPRY-insertion motifs. Taken together, these results support the notion that DPY30-stabilized ASH2L acquires additional interaction interfaces with histones and DNA, which may reduce the dynamics of the MLL1 complex on NCP and ensure the correct priming of the H3 substrate to boost the enzymatic activity of the MLL1 complex on nucleosomes.
The conserved role of DPY30 in stimulating HKMT activity of MLL-family complexes  Figure 7A). We then measured the reaction rates of different MLL family complexes by using the MTase-Glo Methyltransferase Assay kit. The MLL complexes with DPY30 possessed 10-to 25-fold higher methyltransferase activity than the corresponding complexes without DPY30 Figure 7B). These results demonstrate that (E) EMSA showed that ASH2L DPHD-WH had a decreased DNA binding ability, but the ASH2L DPHD-WH -DPY30 complex had a similar DNA binding ability as wild type ASH2L-DPY30. F. EMSA showed that ASH2L DLL failed to bind DNA and that the ASH2L DLL -DPY30 complex had severely decreased DNA binding ability. DLL indicates the deletion of ASH2L IDR, pre-SPRY, and SPRY-insertion. See also Figure S8 and iScience Article the DPY30-dependent activity-stimulation mechanism derived from studies of the MLL1 complex can also be applied to other MLL family methyltransferases.

DISCUSSION
DPY30 is the smallest subunit (99 amino acids) in the MLL complex and associates peripherally with the MLL complex through its interaction with ASH2L (Patel et al., 2009). Although DPY30 plays an essential role in maintaining H3K4 methylation levels in vivo (Jiang et al., 2011;Yang et al., 2014Yang et al., , 2016, its biochemical role in maintaining the HKMT activity of the MLL complex has been underestimated. Here, we demonstrate that DPY30 plays a crucial role in the activity regulation of the MLL complex through two complementary mechanisms: a nucleosome-independent mechanism and a nucleosome-specific mechanism. First, DPY30 improves the stability of ASH2L by interacting with much broader interfaces of ASH2L than previously characterized. The stabilized ASH2L gains multiple functional improvements, including increased thermal stability, less aggregation, and enhanced interaction with RBBP5. All these DPY30-dependent iScience Article properties collectively contribute to the assembly of a compact and stable MLL complex with enhanced HKMT activity Figure 7C). This DPY30-dependent compaction and stabilization of the MLL complex could explain the previously ignored nucleosome-independent activity stimulation by DPY30, as observed on histone octamer, H3-H4 tetramer, and H3 peptide ( Figure 1B).
Second, DPY30 significantly enhances the HKMT activity of the MLL complex on nucleosomes (Kwon et al., 2020;Lee et al., 2021) ( Figure 1C). This nucleosome-specific activity enhancement relies on the newly generated interfaces between DPY30-stabilized ASH2L and nucleosomes. The extra interfaces of ASH2L-H3 and ASH2L-DNA may ensure a relatively fixed configuration of the MLL complex on nucleosomes, thereby boosting the HKMT activity of the MLL complex ( Figure 7C). It should be noted that the major role of these newly generated interfaces is not to increase the binding affinity between the MLL complex and nucleosomes. Our kinetic analyses showed that DPY30 decreased the K m by only 1.5-fold ( Figure 1C). Thus, the primary roles of these newly generated ASH2L-nucleosome interfaces are to prime the MLL1 complex in a correct orientation on nucleosomes and facilitate H3 alignment into the active pocket of the SET domain to catalyze H3K4 methylation more efficiently. This can explain why DPY30 causes more of a change in k cat rather than a K m change ( Figure 1C).
A recent report from the Dou laboratory concluded that DPY30 enhanced the activity of the MLL1 complex on nucleosomes by restricting the rotational dynamics of the MLL1 complex on NCP (Lee et al., 2021). Their work and our present study complement each other to reveal how ASH2L and DPY30 interact to affect the conformation of the MLL complex on nucleosomes. Notwithstanding the similar conclusions we both held, our studies reveal some new aspects of the ASH2L-DPY30 interaction. For example, the study from the Dou lab only addressed the nucleosome-specific activation mechanism (Lee et al., 2021), but our study also reveals the nucleosome-independent mechanism, explaining the activity stimulation by DPY30 on broad-spectrum substrates. Moreover, our study provides direct biochemical evidence that DPY30 enhances ASH2L's interactions with the H3 tail and DNA (Figure 6), explaining why DPY30 reduces the rotation dynamics of the MLL1 complex on NCP.
In addition, our study provides an alternative explanation for how DPY30 affects the structures and functions of ASH2L. Dou's work emphasized the importance of ASH2L IDRs and proposed that the major role of DPY30 was to induce the conformational change of ASH2L IDRs (Lee et al., 2021). Here, we show that the so-called ASH2L IDRs in the previous publication (Lee et al., 2021), especially the pre-SPRY and SPRY-insertion motifs, have well-defined structural features and are not obviously altered by DPY30 binding (Figures S4 and S5). The major role of DPY30 is not to induce the conformational change of ASH2L but to increase the stability of ASH2L and allosterically enhance ASH2L-mediated interactions (including interactions with RBBP5, H3, and DNA). DPY30-dependent enhancement of the internal interaction networks thus facilitates the formation of a compact MLL core complex and reduces the conformational flexibility of the MLL complex on nucleosomes.
The functions of DPY30 in increasing ASH2L thermostability, preventing ASH2L aggregation, and enhancing the ASH2L-dependent methyltransferase activity of the MLL1 complex are analogous to protein chaperones in regulating the stabilities and activities of client proteins. We propose that DPY30 functions as an ASH2L-specific chaperone to stabilize ASH2L. Whether DPY30 functions as a general chaperone remains to be determined. Notwithstanding, DPY30 is the peripheral protein of the MLL complex, and the dissociation of DPY30 does not lead to the disassembly of the MLL complex (Patel et al., 2009) but rather ''turns off'' the HKMT activity of the MLL1 complex. In certain circumstances, the MLL complex binds to the target chromatin regions in the priming state (without DPY30, low activity) and waits for the signal to quickly switch to the activation state (with DPY30 binding, high activity) to ''turn on'' HKMT activity. Therefore, DPY30 may serve as a delicate on/off switch for MLL-family complexes to precisely regulate the HKMT activity.
Owing to the critical role of DPY30 in maintaining H3K4 methylation, abnormal expression of DPY30 can lead to the initiation and progression of human diseases. Extensive studies have reported that DPY30 is overexpressed in many types of cancers, accompanied by increased H3K4me3 modification in cancer cells (Dixit et al., 2022;Gu et al., 2021;He et al., 2019;Hong et al., 2020;Lee et al., 2015;Shah et al., 2019;Yang et al., 2018;Zhang et al., 2018). Overexpression of DPY30 promotes proliferation, migration, and invasion of tumor cells (Lee et al., 2015;Yang et al., 2018;Zhang et al., 2018 iScience Article DPY30-ASH2L interface might be a therapeutic target for cancer treatment. As the first proof-of-concept for targeting the DPY30-ASH2L interaction, a peptide derived from ASH2L DBM (residues 510-529) decreased the global H3K4me3 and modestly inhibited the growth of MLL-rearranged leukemia (Shah et al., 2019), demonstrating the feasibility of targeting the DPY30-ASH2L interaction for cancer treatment. The newly identified DPY30-ASH2L interface and ASH2L-NCP interface revealed in the current study provide a foundation for designing or screening inhibitors with high potency and specificity to target the ''DPY30-ASH2L-MLL-NCP'' axis, hopefully contributing to the discovery of new therapeutic drugs for certain cancers.

Limitation of the study
Here we used the ColabFold powered by AlphaFold2 to predict the structure of the ASH2L-DPY30 complex.
Although the predicted structural model looks plausible and has been supported by mutagenesis studies, the exact structure of the ASH2L-DPY30 complex still awaits experimental determination by X-ray crystallography or other structural methodologies, which will reveal more detailed interface information between ASH2L and DPY30. The structural information will provide a foundation for the rational design of small molecules or peptide mimics to inhibit the ASH2L-DPY30 interaction for potential therapeutic usage.
Our biochemical data suggest that multiple regions of ASH2L and DPY30 are essential for maintaining the HKMT activity of MLL complexes on NCP. Unfortunately, currently available cryo-EM structures of MWRAD-NCP had the lowest resolution (8-12 Å ) in the ASH2L-DPY30 region, preventing us from building a reliable model for ASH2L-DPY30 on NCP. Moreover, the conformational change of ASH2L-DPY30 on binding with NCP is expected, especially in the highly flexible ASH2L IDR region (aa 178-229). Therefore, the high-resolution cryo-EM structure of MWRAD-NCP is required to dissect how ASH2L-DPY30 impacts the methylation ability of the MLL complexes on nucleosomes.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:   Article 25 mM Tris-HCl, pH 8.0, and 150 mM NaCl. Next, capillaries filled with samples were placed on the loading tray and heated from 20 C to 85 C at a heating rate of 1 C/min. The fluorescence at 330 and 350 nm and the light scattering signals were recorded. The melting temperature (Tm) and the aggregation temperature (Tagg) were determined by the PR. ThermControl software (NanoTemper Technologies, Germany).

ASH2L aggregation assay
To monitor thermoinduced ASH2L aggregation, ASH2L was diluted into 1 mL 37 C prewarmed buffer containing 25 mM Tris-HCl pH 8.0 and 150 mM NaCl in a cuvette to a final concentration of 1.5 mM. Different ratios of DPY30 (ASH2L: DPY30 = 1:0, 1:1, 1:2, or 1:4) were then added and continuously mixed with a small magnetic stirring bar. ASH2L aggregation was monitored by light scattering at 360 nm using a Thermo LUMINA fluorescence spectrophotometer (Thermo Fisher Scientific, USA) with a Peltier temperature controller.

Fluorescence polarization assay
ASH2L only or ASH2L-DPY30 complexes were diluted to a series of concentrations from 432 mM to 13.5 mM in buffer containing 25 mM Tris, pH 8.0, and 150 mM NaCl. Various concentrations of proteins (30 mL) were mixed with 2.4 mL of 1.35 mM FAM-labeled H3 1-36 peptide (ARTKQTARKSTGGKAPRKQLATKAA RKSAPATGGVK-FAM) (final concentration 100 nM) and incubated on ice in the dark for 30 min. The fluorescence polarization values in 384-well black plates were measured using a Synergy Neo Multi-Mode Reader (Bio-Tek, USA) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Fluorescence was quantitated with GEN 5 software (Bio-Tek, USA), and data were analyzed with GraphPad Prism 8.0 (GraphPad, USA). Notably, K d values cannot be accurately determined because the binding is not saturated even at the highest protein concentration.
To measure the binding affinity between ASH2L and RBBP5, ASH2L (or the ASH2L-DPY30 complex) was diluted to a series of concentrations from 20 mM to 0.01 mM in buffer containing 25 mM Tris, pH 8.0, and 150 mM NaCl. Fifteen microliters of various concentrations of proteins were mixed with equal volumes of 200 nM RBBP5 330-363 -FAM and incubated on ice in the dark for 30 min. The following experimental operations were consistent with those mentioned above.

Electrophoretic mobility shift assay (EMSA)
ASH2L or the ASH2L-DPY30 complex was diluted to a series of concentrations ranging from 11.52 mM to 0.18 mM using buffer containing 25 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mg/mL BSA, and 5% glycerol.7 mL of various concentrations of protein was mixed with 7 mL of 80 nM 25 bp 5 0 -FAM-labeled dsDNA (FAM-TCTCTAGAGTCGACCTGCAGGCATG). After incubation on ice for 30 min, each reaction mixture was separated by electrophoresis on a 6% native polyacrylamide gel. The gels were then visualized on a Bio-Rad ChemiDoc MP Imaging System (Bio-Rad, USA).

Structural prediction by ColabFold
Structural prediction was carried out by ColabFold, which was powered by AlphaFold2 and featured sequence alignment using MMseqs2 (Mirdita et al., 2022). The parameters were the default settings, including unrelaxed, no template information used, MMseqs2 (UniRef + Environment), pair mode (unpaired + paired), and model_type (complex prediction using Alpha-Fold-multimer-v2 and single-chain prediction using AlphaFold2-ptm). The results were similar to the models predicted by AlphaFold2 v2.0.0 (Jumper et al., 2021) installed in the local workstation using the nondocker installation. The output five models were aligned and inspected in PyMOL (Schrö dinger, USA).

Molecular dynamics simulations
For MD simulation, apo ASH2L 230-534 and the ASH2L 230-534 -DPY30 DD complex were used because the turbulent motion between ASH2L 1-177 and ASH2L 230-534 because of the highly flexible ASH2L IDR (residues 178-229) leads to overall dynamics in ASH2L FL . All systems were set up using GROMACS (Kutzner et al., 2015) and the CHARMM36 force field (Huang and MacKerell, 2013). The proteins were centered in a cubic box with a buffering distance of 1.0 nm and solvated with TIP3P water molecules. Charges were neutralized by adding Na + or Cl À accordingly, and the final NaCl concentration was kept at 0.15 M, consistent with the experiments. After energy minimization with the steepest descending algorithm, we gently heated the system to 300 K under NVT conditions with the position of the protein constrained with a harmonic potential of ll OPEN ACCESS iScience 25, 104948, September 16, 2022 iScience Article iScience Article