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Essential role of lncRNA binding for WDR5 maintenance of active chromatin and embryonic stem cell pluripotency.

Yang YW, Flynn RA, Chen Y, Qu K, Wan B, Wang KC, Lei M, Chang HY - Elife (2014)

Bottom Line: Here we identify a RNA binding pocket on WDR5, and discover a WDR5 mutant (F266A) that selectively abrogates RNA binding without affecting MLL complex assembly or catalytic activity.We identify a family of ESC messenger and lncRNAs that interact with wild type WDR5 but not F266A mutant, including several lncRNAs known to be important for ESC gene expression.These results suggest that specific RNAs are integral inputs into the WDR5-MLL complex for maintenance of the active chromatin state and embryonic stem cell fates.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, United States.

ABSTRACT
The WDR5 subunit of the MLL complex enforces active chromatin and can bind RNA; the relationship between these two activities is unclear. Here we identify a RNA binding pocket on WDR5, and discover a WDR5 mutant (F266A) that selectively abrogates RNA binding without affecting MLL complex assembly or catalytic activity. Complementation in ESCs shows that WDR5 F266A mutant is unable to accumulate on chromatin, and is defective in gene activation, maintenance of histone H3 lysine 4 trimethylation, and ESC self renewal. We identify a family of ESC messenger and lncRNAs that interact with wild type WDR5 but not F266A mutant, including several lncRNAs known to be important for ESC gene expression. These results suggest that specific RNAs are integral inputs into the WDR5-MLL complex for maintenance of the active chromatin state and embryonic stem cell fates. DOI: http://dx.doi.org/10.7554/eLife.02046.001.

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WDR5 F266A lncRNA binding mutant does not affect MLL complex formation or catalytic function, but shows decreased ability to activate target genes in 293T cells.(A) WDR5 binding affinity to RbBP5, MLL1, or H3 peptides by isothermal calorimetry. ND, not detectable. (B) WDR5 F266A does not have decreased binding to RbBP5, Ash2L and MLL1SET domain proteins in GST protein pull down assays. (C) WDR5 F266A does not affect histone methylase activity of the MLL1 complex. (D) GAL4-WDR5 F266 is defective in activating luciferase expression, as seen in luciferase titration tests.DOI:http://dx.doi.org/10.7554/eLife.02046.005
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fig2: WDR5 F266A lncRNA binding mutant does not affect MLL complex formation or catalytic function, but shows decreased ability to activate target genes in 293T cells.(A) WDR5 binding affinity to RbBP5, MLL1, or H3 peptides by isothermal calorimetry. ND, not detectable. (B) WDR5 F266A does not have decreased binding to RbBP5, Ash2L and MLL1SET domain proteins in GST protein pull down assays. (C) WDR5 F266A does not affect histone methylase activity of the MLL1 complex. (D) GAL4-WDR5 F266 is defective in activating luciferase expression, as seen in luciferase titration tests.DOI:http://dx.doi.org/10.7554/eLife.02046.005

Mentions: To confirm the lncRNA selectivity of the F266A mutation, we examined its effects on MLL complex structure and catalytic activity in vitro. As seen by isothermal calorimetry, the F266A mutation minimally affects the affinity of WDR5 for RbBP5, MLL1, or H3 peptides, in contrast to the Y228A mutation (RbBP5 binding defective) or D107A mutation (MLL1 and H3 binding defective) (Figure 2A). This was further confirmed using GST protein pull down assays, in which GST-WDR5 F266A displays no deficits in binding RbBP5, Ash2L, and MLL1 purified proteins (Figure 2B). Finally, in contrast to the MLL1/H3 or RbBP5 binding mutants, the F266A mutation did not significantly decrease in vitro MLL1 complex methylase activity compared to wild type (Figure 2C). Thus, the F266A mutation appears to singularly affect lncRNA binding, without altering MLL complex structure or catalytic activity.10.7554/eLife.02046.005Figure 2.WDR5 F266A lncRNA binding mutant does not affect MLL complex formation or catalytic function, but shows decreased ability to activate target genes in 293T cells.


Essential role of lncRNA binding for WDR5 maintenance of active chromatin and embryonic stem cell pluripotency.

Yang YW, Flynn RA, Chen Y, Qu K, Wan B, Wang KC, Lei M, Chang HY - Elife (2014)

WDR5 F266A lncRNA binding mutant does not affect MLL complex formation or catalytic function, but shows decreased ability to activate target genes in 293T cells.(A) WDR5 binding affinity to RbBP5, MLL1, or H3 peptides by isothermal calorimetry. ND, not detectable. (B) WDR5 F266A does not have decreased binding to RbBP5, Ash2L and MLL1SET domain proteins in GST protein pull down assays. (C) WDR5 F266A does not affect histone methylase activity of the MLL1 complex. (D) GAL4-WDR5 F266 is defective in activating luciferase expression, as seen in luciferase titration tests.DOI:http://dx.doi.org/10.7554/eLife.02046.005
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: WDR5 F266A lncRNA binding mutant does not affect MLL complex formation or catalytic function, but shows decreased ability to activate target genes in 293T cells.(A) WDR5 binding affinity to RbBP5, MLL1, or H3 peptides by isothermal calorimetry. ND, not detectable. (B) WDR5 F266A does not have decreased binding to RbBP5, Ash2L and MLL1SET domain proteins in GST protein pull down assays. (C) WDR5 F266A does not affect histone methylase activity of the MLL1 complex. (D) GAL4-WDR5 F266 is defective in activating luciferase expression, as seen in luciferase titration tests.DOI:http://dx.doi.org/10.7554/eLife.02046.005
Mentions: To confirm the lncRNA selectivity of the F266A mutation, we examined its effects on MLL complex structure and catalytic activity in vitro. As seen by isothermal calorimetry, the F266A mutation minimally affects the affinity of WDR5 for RbBP5, MLL1, or H3 peptides, in contrast to the Y228A mutation (RbBP5 binding defective) or D107A mutation (MLL1 and H3 binding defective) (Figure 2A). This was further confirmed using GST protein pull down assays, in which GST-WDR5 F266A displays no deficits in binding RbBP5, Ash2L, and MLL1 purified proteins (Figure 2B). Finally, in contrast to the MLL1/H3 or RbBP5 binding mutants, the F266A mutation did not significantly decrease in vitro MLL1 complex methylase activity compared to wild type (Figure 2C). Thus, the F266A mutation appears to singularly affect lncRNA binding, without altering MLL complex structure or catalytic activity.10.7554/eLife.02046.005Figure 2.WDR5 F266A lncRNA binding mutant does not affect MLL complex formation or catalytic function, but shows decreased ability to activate target genes in 293T cells.

Bottom Line: Here we identify a RNA binding pocket on WDR5, and discover a WDR5 mutant (F266A) that selectively abrogates RNA binding without affecting MLL complex assembly or catalytic activity.We identify a family of ESC messenger and lncRNAs that interact with wild type WDR5 but not F266A mutant, including several lncRNAs known to be important for ESC gene expression.These results suggest that specific RNAs are integral inputs into the WDR5-MLL complex for maintenance of the active chromatin state and embryonic stem cell fates.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, United States.

ABSTRACT
The WDR5 subunit of the MLL complex enforces active chromatin and can bind RNA; the relationship between these two activities is unclear. Here we identify a RNA binding pocket on WDR5, and discover a WDR5 mutant (F266A) that selectively abrogates RNA binding without affecting MLL complex assembly or catalytic activity. Complementation in ESCs shows that WDR5 F266A mutant is unable to accumulate on chromatin, and is defective in gene activation, maintenance of histone H3 lysine 4 trimethylation, and ESC self renewal. We identify a family of ESC messenger and lncRNAs that interact with wild type WDR5 but not F266A mutant, including several lncRNAs known to be important for ESC gene expression. These results suggest that specific RNAs are integral inputs into the WDR5-MLL complex for maintenance of the active chromatin state and embryonic stem cell fates. DOI: http://dx.doi.org/10.7554/eLife.02046.001.

Show MeSH
Related in: MedlinePlus