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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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Related in: MedlinePlus

Ash2L/RbBP5 binds to substrate SAM.(A) SAM binding assays for Ash2L/RbBP5 and MLL1SET by UV cross-linking. Left, SAM binding assays for the MLL1 core complex with or without UV cross-linking. Equal molar (∼3 µM) of each MLL1 core component was used. Right, SAM binding by Ash2L and MLL1SET could be chased off by 1000x excess cold SAM (∼0.35 mM) but not by ATP (∼0.35 mM). Black lines indicated expected positions for each protein. 10-fold molar excess of Ash2L/RbBP5 (relative to MLL1SET) was used in this experiment to reduce the exposure time for detecting Ash2L SAM binding. Black lines indicated expected positions for each protein. (B) SAM binding assay for 3 µM equivalent of Ash2L alone or Ash2L/RbBP5 in the presence of increasing amount of MLL1SET. Molar ratios of MLL1SET versus Ash2L/RbBP5 or Ash2L were indicated on top. (C) Image-J quantitation of the results in (B). Y-axis, arbitrary unit for pixel count. (D) Reciprocal SAM binding assays as performed in (B) except ∼3 µM wild type or H3907A MLL1SET was mixed with increasing amount of Ash2L/RbBP5. The molar ratios of Ash2L/RbBP5 versus MLL1SET were indicated on top. (E) Image-J quantitation of the results in (D). Y-axis, arbitrary unit for pixel count. In (B) and (D), Coomassie staining for the same gels was included on bottom. The positions for Ash2L and RbBP5 were indicated on left. *, non-specific protein.
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pone-0014102-g003: Ash2L/RbBP5 binds to substrate SAM.(A) SAM binding assays for Ash2L/RbBP5 and MLL1SET by UV cross-linking. Left, SAM binding assays for the MLL1 core complex with or without UV cross-linking. Equal molar (∼3 µM) of each MLL1 core component was used. Right, SAM binding by Ash2L and MLL1SET could be chased off by 1000x excess cold SAM (∼0.35 mM) but not by ATP (∼0.35 mM). Black lines indicated expected positions for each protein. 10-fold molar excess of Ash2L/RbBP5 (relative to MLL1SET) was used in this experiment to reduce the exposure time for detecting Ash2L SAM binding. Black lines indicated expected positions for each protein. (B) SAM binding assay for 3 µM equivalent of Ash2L alone or Ash2L/RbBP5 in the presence of increasing amount of MLL1SET. Molar ratios of MLL1SET versus Ash2L/RbBP5 or Ash2L were indicated on top. (C) Image-J quantitation of the results in (B). Y-axis, arbitrary unit for pixel count. (D) Reciprocal SAM binding assays as performed in (B) except ∼3 µM wild type or H3907A MLL1SET was mixed with increasing amount of Ash2L/RbBP5. The molar ratios of Ash2L/RbBP5 versus MLL1SET were indicated on top. (E) Image-J quantitation of the results in (D). Y-axis, arbitrary unit for pixel count. In (B) and (D), Coomassie staining for the same gels was included on bottom. The positions for Ash2L and RbBP5 were indicated on left. *, non-specific protein.

Mentions: Given the intrinsic activity of the Ash2L/RbBP5 heterodimer, we next examined whether it bound to substrate SAM, an essential step in catalysis. We found that Ash2L was capable of binding to SAM in the presence of RbBP5 and MLL1SET after UV treatment (Fig. 3A). In contrast, no SAM binding by RbBP5 was detected in the same reaction (Fig. 3A). SAM binding by Ash2L was UV-dependent and could be competed off by excess amount of unlabeled SAM (1000 fold, ∼0.35 mM) (Fig. 3A). As a control, excess amount of ATP (1000 fold, ∼0.35 mM) could not compete off the SAM binding by Ash2L or MLL1SET. Interestingly, stable Ash2L SAM binding required both RbBP5 and MLL1SET. As shown in Fig. 3B, SAM-binding by Ash2L was severely comprised when MLL1SET and RbBP5 were either sub-stoichiometric or absent. Quantitation of Ash2L SAM binding in Fig. 3B was included in Fig. 3C. In contrast to Ash2L, MLL1SET-SAM interaction was stable and was not affected by Ash2L/RbBP5 (Fig. 3D). Adding excess amount of Ash2L/RbBP5 to wild type MLL1SET did not enhance SAM binding by wild type MLL1SET. However, Ash2L/RbBP5 was able to partially rescue the SAM binding deficiency of the MLL1SET mutant H3907A, which was deficient in SAM binding by itself (Fig. 3D) [3]. Quantitation of Ash2L SAM binding in Fig. 3D was included in Fig. 3E. These results suggest that two proteins in the MLL1 core complex (i.e. Ash2L and MLL1SET) are capable of SAM binding and their interactions with SAM can potentially be influenced by each other.


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)

Ash2L/RbBP5 binds to substrate SAM.(A) SAM binding assays for Ash2L/RbBP5 and MLL1SET by UV cross-linking. Left, SAM binding assays for the MLL1 core complex with or without UV cross-linking. Equal molar (∼3 µM) of each MLL1 core component was used. Right, SAM binding by Ash2L and MLL1SET could be chased off by 1000x excess cold SAM (∼0.35 mM) but not by ATP (∼0.35 mM). Black lines indicated expected positions for each protein. 10-fold molar excess of Ash2L/RbBP5 (relative to MLL1SET) was used in this experiment to reduce the exposure time for detecting Ash2L SAM binding. Black lines indicated expected positions for each protein. (B) SAM binding assay for 3 µM equivalent of Ash2L alone or Ash2L/RbBP5 in the presence of increasing amount of MLL1SET. Molar ratios of MLL1SET versus Ash2L/RbBP5 or Ash2L were indicated on top. (C) Image-J quantitation of the results in (B). Y-axis, arbitrary unit for pixel count. (D) Reciprocal SAM binding assays as performed in (B) except ∼3 µM wild type or H3907A MLL1SET was mixed with increasing amount of Ash2L/RbBP5. The molar ratios of Ash2L/RbBP5 versus MLL1SET were indicated on top. (E) Image-J quantitation of the results in (D). Y-axis, arbitrary unit for pixel count. In (B) and (D), Coomassie staining for the same gels was included on bottom. The positions for Ash2L and RbBP5 were indicated on left. *, non-specific protein.
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pone-0014102-g003: Ash2L/RbBP5 binds to substrate SAM.(A) SAM binding assays for Ash2L/RbBP5 and MLL1SET by UV cross-linking. Left, SAM binding assays for the MLL1 core complex with or without UV cross-linking. Equal molar (∼3 µM) of each MLL1 core component was used. Right, SAM binding by Ash2L and MLL1SET could be chased off by 1000x excess cold SAM (∼0.35 mM) but not by ATP (∼0.35 mM). Black lines indicated expected positions for each protein. 10-fold molar excess of Ash2L/RbBP5 (relative to MLL1SET) was used in this experiment to reduce the exposure time for detecting Ash2L SAM binding. Black lines indicated expected positions for each protein. (B) SAM binding assay for 3 µM equivalent of Ash2L alone or Ash2L/RbBP5 in the presence of increasing amount of MLL1SET. Molar ratios of MLL1SET versus Ash2L/RbBP5 or Ash2L were indicated on top. (C) Image-J quantitation of the results in (B). Y-axis, arbitrary unit for pixel count. (D) Reciprocal SAM binding assays as performed in (B) except ∼3 µM wild type or H3907A MLL1SET was mixed with increasing amount of Ash2L/RbBP5. The molar ratios of Ash2L/RbBP5 versus MLL1SET were indicated on top. (E) Image-J quantitation of the results in (D). Y-axis, arbitrary unit for pixel count. In (B) and (D), Coomassie staining for the same gels was included on bottom. The positions for Ash2L and RbBP5 were indicated on left. *, non-specific protein.
Mentions: Given the intrinsic activity of the Ash2L/RbBP5 heterodimer, we next examined whether it bound to substrate SAM, an essential step in catalysis. We found that Ash2L was capable of binding to SAM in the presence of RbBP5 and MLL1SET after UV treatment (Fig. 3A). In contrast, no SAM binding by RbBP5 was detected in the same reaction (Fig. 3A). SAM binding by Ash2L was UV-dependent and could be competed off by excess amount of unlabeled SAM (1000 fold, ∼0.35 mM) (Fig. 3A). As a control, excess amount of ATP (1000 fold, ∼0.35 mM) could not compete off the SAM binding by Ash2L or MLL1SET. Interestingly, stable Ash2L SAM binding required both RbBP5 and MLL1SET. As shown in Fig. 3B, SAM-binding by Ash2L was severely comprised when MLL1SET and RbBP5 were either sub-stoichiometric or absent. Quantitation of Ash2L SAM binding in Fig. 3B was included in Fig. 3C. In contrast to Ash2L, MLL1SET-SAM interaction was stable and was not affected by Ash2L/RbBP5 (Fig. 3D). Adding excess amount of Ash2L/RbBP5 to wild type MLL1SET did not enhance SAM binding by wild type MLL1SET. However, Ash2L/RbBP5 was able to partially rescue the SAM binding deficiency of the MLL1SET mutant H3907A, which was deficient in SAM binding by itself (Fig. 3D) [3]. Quantitation of Ash2L SAM binding in Fig. 3D was included in Fig. 3E. These results suggest that two proteins in the MLL1 core complex (i.e. Ash2L and MLL1SET) are capable of SAM binding and their interactions with SAM can potentially be influenced by each other.

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
Related in: MedlinePlus