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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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Characterization of FLAG-HA ES cell lines.(A) Western blot analysis of individually isolated ESC clones. antiWDR5 blotting reveals the total expression level of WDR5 in mock, FH-WDR5 wild type, and FH-WDR5 F266A mutant ESCs. The FH tag adds ∼8 kDa to WDR5, resulting in the observed shift, where as the F266A sees no shift because the construct is N-terminally shorter. Orange boxes are draw around clones that were isolated and used in the RIPiT experiments due to their near endogenous expression level. (B) FPLC analysis of the selected wild type and F266A FH-WDR5 clones. Each fraction was subjected to western blot analysis for HA (tagged protein), WDR5, Ash2L, and RbBP5. Orange boxes appear around the endogenous large molecular weight MLL complex, where the majority of the tagged FH-WDR5 constructs elute.DOI:http://dx.doi.org/10.7554/eLife.02046.008
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fig4s1: Characterization of FLAG-HA ES cell lines.(A) Western blot analysis of individually isolated ESC clones. antiWDR5 blotting reveals the total expression level of WDR5 in mock, FH-WDR5 wild type, and FH-WDR5 F266A mutant ESCs. The FH tag adds ∼8 kDa to WDR5, resulting in the observed shift, where as the F266A sees no shift because the construct is N-terminally shorter. Orange boxes are draw around clones that were isolated and used in the RIPiT experiments due to their near endogenous expression level. (B) FPLC analysis of the selected wild type and F266A FH-WDR5 clones. Each fraction was subjected to western blot analysis for HA (tagged protein), WDR5, Ash2L, and RbBP5. Orange boxes appear around the endogenous large molecular weight MLL complex, where the majority of the tagged FH-WDR5 constructs elute.DOI:http://dx.doi.org/10.7554/eLife.02046.008

Mentions: Our in vitro and in vivo data characterizing the WDR5 F266A mutant suggest that RNA binding is an important aspect of WDR5’s cellular function, however currently only two RNAs have been identified as WDR5 partners. To close this gap, we identified WDR5–bound RNAs in the ESC transcriptome. We tested UV-crosslinking with PAR-CLIP, but found that WDR5-RNA interactions have poor inherent UV crosslinking ability. We also found that standard RNA immunoprecipitation (RIP) with FLAG epitope gave substantial background that hampered data interpretation. We then turned to RNA:protein immunoprecipitation in tandem (RIPiT), a method designed to identify RNA targets of RBP complexes with poor UV linking capacity (Singh et al., 2013). Specifically, we fused tandem FLAG and hemagglutinin (HA) tags to wild type WDR5 or the F266A mutant. We established ESC lines expressing FLAG-HA tagged version of WT and F266A WDR5 at near endogenous expression levels, and FPLC analysis confirmed that both tandem-tagged WDR5 proteins quantitatively formed equivalent MLL-WDR5 protein complexes with endogenous subunits (Figure 4—figure supplement 1A,B).


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)

Characterization of FLAG-HA ES cell lines.(A) Western blot analysis of individually isolated ESC clones. antiWDR5 blotting reveals the total expression level of WDR5 in mock, FH-WDR5 wild type, and FH-WDR5 F266A mutant ESCs. The FH tag adds ∼8 kDa to WDR5, resulting in the observed shift, where as the F266A sees no shift because the construct is N-terminally shorter. Orange boxes are draw around clones that were isolated and used in the RIPiT experiments due to their near endogenous expression level. (B) FPLC analysis of the selected wild type and F266A FH-WDR5 clones. Each fraction was subjected to western blot analysis for HA (tagged protein), WDR5, Ash2L, and RbBP5. Orange boxes appear around the endogenous large molecular weight MLL complex, where the majority of the tagged FH-WDR5 constructs elute.DOI:http://dx.doi.org/10.7554/eLife.02046.008
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4s1: Characterization of FLAG-HA ES cell lines.(A) Western blot analysis of individually isolated ESC clones. antiWDR5 blotting reveals the total expression level of WDR5 in mock, FH-WDR5 wild type, and FH-WDR5 F266A mutant ESCs. The FH tag adds ∼8 kDa to WDR5, resulting in the observed shift, where as the F266A sees no shift because the construct is N-terminally shorter. Orange boxes are draw around clones that were isolated and used in the RIPiT experiments due to their near endogenous expression level. (B) FPLC analysis of the selected wild type and F266A FH-WDR5 clones. Each fraction was subjected to western blot analysis for HA (tagged protein), WDR5, Ash2L, and RbBP5. Orange boxes appear around the endogenous large molecular weight MLL complex, where the majority of the tagged FH-WDR5 constructs elute.DOI:http://dx.doi.org/10.7554/eLife.02046.008
Mentions: Our in vitro and in vivo data characterizing the WDR5 F266A mutant suggest that RNA binding is an important aspect of WDR5’s cellular function, however currently only two RNAs have been identified as WDR5 partners. To close this gap, we identified WDR5–bound RNAs in the ESC transcriptome. We tested UV-crosslinking with PAR-CLIP, but found that WDR5-RNA interactions have poor inherent UV crosslinking ability. We also found that standard RNA immunoprecipitation (RIP) with FLAG epitope gave substantial background that hampered data interpretation. We then turned to RNA:protein immunoprecipitation in tandem (RIPiT), a method designed to identify RNA targets of RBP complexes with poor UV linking capacity (Singh et al., 2013). Specifically, we fused tandem FLAG and hemagglutinin (HA) tags to wild type WDR5 or the F266A mutant. We established ESC lines expressing FLAG-HA tagged version of WT and F266A WDR5 at near endogenous expression levels, and FPLC analysis confirmed that both tandem-tagged WDR5 proteins quantitatively formed equivalent MLL-WDR5 protein complexes with endogenous subunits (Figure 4—figure supplement 1A,B).

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