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An Ash2L/RbBP5 heterodimer stimulates the MLL1 methyltransferase activity through coordinated substrate interactions with the MLL1 SET domain.

Cao F, Chen Y, Cierpicki T, Liu Y, Basrur V, Lei M, Dou Y - PLoS ONE (2010)

Bottom Line: Histone H3 lysine 4 (K4) methylation is a prevalent mark associated with transcription activation and is mainly catalyzed by the MLL/SET1 family histone methyltransferases.Taken together, our results show that the Ash2L/RbBP5 heterodimer plays a critical role in the overall catalysis of MLL1 mediated H3 K4 methylation.The results we describe here provide mechanistic insights for unique regulation of the MLL1 methyltransferase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT
Histone H3 lysine 4 (K4) methylation is a prevalent mark associated with transcription activation and is mainly catalyzed by the MLL/SET1 family histone methyltransferases. A common feature of the mammalian MLL/SET1 complexes is the presence of three core components (RbBP5, Ash2L and WDR5) and a catalytic subunit containing a SET domain. Unlike most other histone lysine methyltransferases, all four proteins are required for efficient H3 K4 methylation. Despite extensive efforts, mechanisms for how three core components regulate MLL/SET1 methyltransferase activity remain elusive. Here we show that a heterodimer of Ash2L and RbBP5 has intrinsic histone methyltransferase activity. This activity requires the highly conserved SPRY domain of Ash2L and a short peptide of RbBP5. We demonstrate that both Ash2L and the MLL1 SET domain are capable of binding to S-adenosyl-L- [methyl-(3)H] methionine in the MLL1 core complex. Mutations in the MLL1 SET domain that fail to support overall H3 K4 methylation also compromise SAM binding by Ash2L. Taken together, our results show that the Ash2L/RbBP5 heterodimer plays a critical role in the overall catalysis of MLL1 mediated H3 K4 methylation. The results we describe here provide mechanistic insights for unique regulation of the MLL1 methyltransferase activity. It suggests that both Ash2L/RbBP5 and the MLL1 SET domain make direct contacts with the substrates and contribute to the formation of a joint catalytic center. Given the shared core configuration among all MLL/SET1 family HMTs, it will be interesting to test whether the mechanism we describe here can be generalized to other MLL/SET1 family members in the future.

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H3 and SAM mediate Ash2L/RbBP5 and MLL1SET interaction.(A) 150 nM recombinant proteins Flag-Ash2L, His-RbBP5 and His-MLL1SET were mixed in the presence of ∼7.5 µM histone H3, ∼10 µM methylation product AdoHcy or both as indicated on top. After Flag-IP, eluates from M2 agarose beads were separated on SDS-PAGE and immunoblotted for MLL1SET, Ash2L and RbBP5 (indicated on right). 25% input was loaded as the control. (B) “Single catalytic center” model for the regulation of the MLL1 core complex. In this model, Ash2L/RbBP5 and MLL1SET interact with the same H3 substrate and substrate SAM and form a shared active center for catalysis. This structure is further stabilized by WDR5, which simultaneously interacts with RbBP5 and MLL1SET.
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pone-0014102-g006: H3 and SAM mediate Ash2L/RbBP5 and MLL1SET interaction.(A) 150 nM recombinant proteins Flag-Ash2L, His-RbBP5 and His-MLL1SET were mixed in the presence of ∼7.5 µM histone H3, ∼10 µM methylation product AdoHcy or both as indicated on top. After Flag-IP, eluates from M2 agarose beads were separated on SDS-PAGE and immunoblotted for MLL1SET, Ash2L and RbBP5 (indicated on right). 25% input was loaded as the control. (B) “Single catalytic center” model for the regulation of the MLL1 core complex. In this model, Ash2L/RbBP5 and MLL1SET interact with the same H3 substrate and substrate SAM and form a shared active center for catalysis. This structure is further stabilized by WDR5, which simultaneously interacts with RbBP5 and MLL1SET.

Mentions: Given the highly synergistic function of Ash2L and MLL1SET in H3 K4 methylation and the ability for both proteins to interact with substrate SAM, we postulate that Ash2L/RbBP5 and MLL1SET may interact with the same set of H3 and SAM substrates at the catalytic center of the MLL1 complex. To test this, we performed pull-down assays for MLL1SET and Ash2L/RbBP5 with or without the presence of H3 or SAM. As shown in Fig. 6, MLL1SET was not able to stably interact with Ash2L and RbBP5 in the absence of WDR5, consistent with our previous observation [16]. However, in the presence of H3 or methylation product AdoHcy, MLL1SET could be partially recovered by Flag-Ash2L immunoprecipitation (Fig. 6A). The pull-down efficiency for MLL1SET was further enhanced when both H3 and AdoHcy were present (Fig. 6A). AdoHcy was used during the IP because Flag-IP was blocked in the presence of SAM, probably due to methylation of the M2 antibody by MLL1. Co-purification of Ash2L/RbBP5 and MLL1SET in the presence of H3 and AdoHcy suggests that they probably interact with the same molecules. This result implies that Ash2L/RbBP5 probably functions at the catalytic center of the MLL1 core complex or at least in close proximity to it. The H3- and AdoHcy-dependent stabilization was only apparent in the absence of WDR5 (data not shown), which held MLL1 core complex together by simultaneously interacting with MLL1 and RbBP5 [16], [21].


An Ash2L/RbBP5 heterodimer stimulates the MLL1 methyltransferase activity through coordinated substrate interactions with the MLL1 SET domain.

Cao F, Chen Y, Cierpicki T, Liu Y, Basrur V, Lei M, Dou Y - PLoS ONE (2010)

H3 and SAM mediate Ash2L/RbBP5 and MLL1SET interaction.(A) 150 nM recombinant proteins Flag-Ash2L, His-RbBP5 and His-MLL1SET were mixed in the presence of ∼7.5 µM histone H3, ∼10 µM methylation product AdoHcy or both as indicated on top. After Flag-IP, eluates from M2 agarose beads were separated on SDS-PAGE and immunoblotted for MLL1SET, Ash2L and RbBP5 (indicated on right). 25% input was loaded as the control. (B) “Single catalytic center” model for the regulation of the MLL1 core complex. In this model, Ash2L/RbBP5 and MLL1SET interact with the same H3 substrate and substrate SAM and form a shared active center for catalysis. This structure is further stabilized by WDR5, which simultaneously interacts with RbBP5 and MLL1SET.
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pone-0014102-g006: H3 and SAM mediate Ash2L/RbBP5 and MLL1SET interaction.(A) 150 nM recombinant proteins Flag-Ash2L, His-RbBP5 and His-MLL1SET were mixed in the presence of ∼7.5 µM histone H3, ∼10 µM methylation product AdoHcy or both as indicated on top. After Flag-IP, eluates from M2 agarose beads were separated on SDS-PAGE and immunoblotted for MLL1SET, Ash2L and RbBP5 (indicated on right). 25% input was loaded as the control. (B) “Single catalytic center” model for the regulation of the MLL1 core complex. In this model, Ash2L/RbBP5 and MLL1SET interact with the same H3 substrate and substrate SAM and form a shared active center for catalysis. This structure is further stabilized by WDR5, which simultaneously interacts with RbBP5 and MLL1SET.
Mentions: Given the highly synergistic function of Ash2L and MLL1SET in H3 K4 methylation and the ability for both proteins to interact with substrate SAM, we postulate that Ash2L/RbBP5 and MLL1SET may interact with the same set of H3 and SAM substrates at the catalytic center of the MLL1 complex. To test this, we performed pull-down assays for MLL1SET and Ash2L/RbBP5 with or without the presence of H3 or SAM. As shown in Fig. 6, MLL1SET was not able to stably interact with Ash2L and RbBP5 in the absence of WDR5, consistent with our previous observation [16]. However, in the presence of H3 or methylation product AdoHcy, MLL1SET could be partially recovered by Flag-Ash2L immunoprecipitation (Fig. 6A). The pull-down efficiency for MLL1SET was further enhanced when both H3 and AdoHcy were present (Fig. 6A). AdoHcy was used during the IP because Flag-IP was blocked in the presence of SAM, probably due to methylation of the M2 antibody by MLL1. Co-purification of Ash2L/RbBP5 and MLL1SET in the presence of H3 and AdoHcy suggests that they probably interact with the same molecules. This result implies that Ash2L/RbBP5 probably functions at the catalytic center of the MLL1 core complex or at least in close proximity to it. The H3- and AdoHcy-dependent stabilization was only apparent in the absence of WDR5 (data not shown), which held MLL1 core complex together by simultaneously interacting with MLL1 and RbBP5 [16], [21].

Bottom Line: Histone H3 lysine 4 (K4) methylation is a prevalent mark associated with transcription activation and is mainly catalyzed by the MLL/SET1 family histone methyltransferases.Taken together, our results show that the Ash2L/RbBP5 heterodimer plays a critical role in the overall catalysis of MLL1 mediated H3 K4 methylation.The results we describe here provide mechanistic insights for unique regulation of the MLL1 methyltransferase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT
Histone H3 lysine 4 (K4) methylation is a prevalent mark associated with transcription activation and is mainly catalyzed by the MLL/SET1 family histone methyltransferases. A common feature of the mammalian MLL/SET1 complexes is the presence of three core components (RbBP5, Ash2L and WDR5) and a catalytic subunit containing a SET domain. Unlike most other histone lysine methyltransferases, all four proteins are required for efficient H3 K4 methylation. Despite extensive efforts, mechanisms for how three core components regulate MLL/SET1 methyltransferase activity remain elusive. Here we show that a heterodimer of Ash2L and RbBP5 has intrinsic histone methyltransferase activity. This activity requires the highly conserved SPRY domain of Ash2L and a short peptide of RbBP5. We demonstrate that both Ash2L and the MLL1 SET domain are capable of binding to S-adenosyl-L- [methyl-(3)H] methionine in the MLL1 core complex. Mutations in the MLL1 SET domain that fail to support overall H3 K4 methylation also compromise SAM binding by Ash2L. Taken together, our results show that the Ash2L/RbBP5 heterodimer plays a critical role in the overall catalysis of MLL1 mediated H3 K4 methylation. The results we describe here provide mechanistic insights for unique regulation of the MLL1 methyltransferase activity. It suggests that both Ash2L/RbBP5 and the MLL1 SET domain make direct contacts with the substrates and contribute to the formation of a joint catalytic center. Given the shared core configuration among all MLL/SET1 family HMTs, it will be interesting to test whether the mechanism we describe here can be generalized to other MLL/SET1 family members in the future.

Show MeSH