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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 mutation decreases protein stability and localization to chromatin.(A) Schematic of lentiviral vectors, modified from (Ang et al., 2011). (B) Western blot demonstrating successful mouse WDR5 knockdown. (C) qRT-PCR results demonstrating equal RNA expression of human WDR5 WT and WDR5 F266A. (D) Western blot of WDR5 WT and WDR5 F266A protein expression, also with 4 days after doxycycline removal. (E) WDR5 F266A is defective in nuclear accumulation, compared with WDR5 WT. (F) WDR5 F266A reduces chromatin association, as seen in chromatin isolation experiments. (G) WDR5 F266A mutation decreases protein stability in the nucleus after doxycycline withdrawal. Both WDR5 WT and WDR5 F266A are similarly unstable in the cytoplasmic fraction.DOI:http://dx.doi.org/10.7554/eLife.02046.006
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fig3: WDR5 F266A mutation decreases protein stability and localization to chromatin.(A) Schematic of lentiviral vectors, modified from (Ang et al., 2011). (B) Western blot demonstrating successful mouse WDR5 knockdown. (C) qRT-PCR results demonstrating equal RNA expression of human WDR5 WT and WDR5 F266A. (D) Western blot of WDR5 WT and WDR5 F266A protein expression, also with 4 days after doxycycline removal. (E) WDR5 F266A is defective in nuclear accumulation, compared with WDR5 WT. (F) WDR5 F266A reduces chromatin association, as seen in chromatin isolation experiments. (G) WDR5 F266A mutation decreases protein stability in the nucleus after doxycycline withdrawal. Both WDR5 WT and WDR5 F266A are similarly unstable in the cytoplasmic fraction.DOI:http://dx.doi.org/10.7554/eLife.02046.006

Mentions: WDR5 has been recently shown to be required to maintain H3K4 trimethylation (H3K4me3) in mouse embryonic stem cell (ESC) genes for pluripotency and self renewal (Ang et al., 2011). To test whether WDR5 requires lncRNA binding for physiologic activity, we created ‘rescue’ complementation ESC lines that replace endogenous mouse WDR5 with either wild type human WDR5 or F266A mutant WDR5 (Figure 3A). ESCs were infected by lentiviruses containing two tandem gene expression cassettes (Ang et al., 2011). The first cassette constitutively expresses a highly efficient shRNA to repress endogenous mouse WDR5. WDR5 deficiency is rescued by the second cassette, in which human WDR5 wild type (WT) or human WDR5 F266A linked to GFP is expressed under control of a doxycycline (dox)-inducible promoter. In the presence of dox, the ability of WDR5 mutant to support ESC self renewal can be compared; upon dox withdrawal, the half-life of the mutant protein and its regulatory impact are further revealed.10.7554/eLife.02046.006Figure 3.WDR5 F266A mutation decreases protein stability and localization to chromatin.


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 mutation decreases protein stability and localization to chromatin.(A) Schematic of lentiviral vectors, modified from (Ang et al., 2011). (B) Western blot demonstrating successful mouse WDR5 knockdown. (C) qRT-PCR results demonstrating equal RNA expression of human WDR5 WT and WDR5 F266A. (D) Western blot of WDR5 WT and WDR5 F266A protein expression, also with 4 days after doxycycline removal. (E) WDR5 F266A is defective in nuclear accumulation, compared with WDR5 WT. (F) WDR5 F266A reduces chromatin association, as seen in chromatin isolation experiments. (G) WDR5 F266A mutation decreases protein stability in the nucleus after doxycycline withdrawal. Both WDR5 WT and WDR5 F266A are similarly unstable in the cytoplasmic fraction.DOI:http://dx.doi.org/10.7554/eLife.02046.006
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921674&req=5

fig3: WDR5 F266A mutation decreases protein stability and localization to chromatin.(A) Schematic of lentiviral vectors, modified from (Ang et al., 2011). (B) Western blot demonstrating successful mouse WDR5 knockdown. (C) qRT-PCR results demonstrating equal RNA expression of human WDR5 WT and WDR5 F266A. (D) Western blot of WDR5 WT and WDR5 F266A protein expression, also with 4 days after doxycycline removal. (E) WDR5 F266A is defective in nuclear accumulation, compared with WDR5 WT. (F) WDR5 F266A reduces chromatin association, as seen in chromatin isolation experiments. (G) WDR5 F266A mutation decreases protein stability in the nucleus after doxycycline withdrawal. Both WDR5 WT and WDR5 F266A are similarly unstable in the cytoplasmic fraction.DOI:http://dx.doi.org/10.7554/eLife.02046.006
Mentions: WDR5 has been recently shown to be required to maintain H3K4 trimethylation (H3K4me3) in mouse embryonic stem cell (ESC) genes for pluripotency and self renewal (Ang et al., 2011). To test whether WDR5 requires lncRNA binding for physiologic activity, we created ‘rescue’ complementation ESC lines that replace endogenous mouse WDR5 with either wild type human WDR5 or F266A mutant WDR5 (Figure 3A). ESCs were infected by lentiviruses containing two tandem gene expression cassettes (Ang et al., 2011). The first cassette constitutively expresses a highly efficient shRNA to repress endogenous mouse WDR5. WDR5 deficiency is rescued by the second cassette, in which human WDR5 wild type (WT) or human WDR5 F266A linked to GFP is expressed under control of a doxycycline (dox)-inducible promoter. In the presence of dox, the ability of WDR5 mutant to support ESC self renewal can be compared; upon dox withdrawal, the half-life of the mutant protein and its regulatory impact are further revealed.10.7554/eLife.02046.006Figure 3.WDR5 F266A mutation decreases protein stability and localization to chromatin.

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