Essential role of lncRNA binding for WDR5 maintenance of active chromatin and embryonic stem cell pluripotency
An orchestra of chromatin readers , writers , and erasers act on diverse covalent histone modifications to establish particular cell fates ( Rando and Chang , 2009 ) . By maintaining such histone modifications through cell divisions , the cell state can then be epigenetically transferred from generation to generation , eventually establishing tissues and complex organisms . Histones are modified by large protein complexes with multiple coenzymes that modulate and regulate catalytic activity , thus allowing fine regulation of histone marks . Histone H3 lysine 4 trimethylation ( H3K4me3 ) is a mark of transcriptionally active chromatin , generated through the catalytic activity of the MLL family of proteins . The MLL family of H3K4 methylases are evolutionarily conserved ( known as COMPASS in yeast and trithorax in Drosophila ) ( Smith et al . , 2011 ) , and MLL translocations are an important cause of human leukemias ( Meyer et al . , 2009 ) . Members of the MLL family encode single SET domain-containing H3K4 methylases , and are associated with WDR5 , Ash2L , RbBP5 , and additional proteins to regulate activity . Of the various cofactors that regulate MLL activity , WDR5 is a particularly important multifunctional adaptor protein that can discriminate posttranslational modifications on histone tails , as well as bind to the MLL complex to regulate gene activation ( Wysocka et al . , 2005; Migliori et al . , 2012 ) . WDR5 is particularly important for mammalian embryonic stem cell ( ESC ) self renewal and maintenance of active chromatin for pluripotency genes , and WDR5 is required for efficient generation of induced pluripotent stem cells from differentiated somatic cells ( Ang et al . , 2011; Li et al . , 2012 ) . WDR5 has recently been shown to bind individual long noncoding RNAs ( lncRNAs ) ( Wang et al . , 2011; Gomez et al . , 2013 ) . LncRNAs are capped , spliced , polyadenylated RNA transcripts ranging from several hundred to kilobases in length ( Derrien et al . , 2012; Rinn and Chang , 2012 ) . Specific lncRNAs bind repressive or activating chromatin modification complexes , and localize these activities to specific gene loci ( reviewed by Wang and Chang ( 2011 ) ) . For example , the lncRNA XIST binds the Polycomb Repressive Complex 2 ( PRC2 ) to cause histone H3 lysine 27 trimethylation and silence the X chromosome for dosage compensation in females ( Morey and Avner , 2011 ) . As another example , the lncRNA HOTAIR acts as a molecular scaffold , binding both PRC2 and the H3K4 demethylase LSD1 complex to silence hundreds of loci throughout the genome ( Rinn et al . , 2007; Gupta et al . , 2010; Tsai et al . , 2010; Chu et al . , 2011 ) . Additional lncRNAs can bind to messenger RNAs to control their decay via interaction with the Staufen 1 protein ( Gong and Maquat , 2011; Kretz et al . , 2013 ) . In contrast , several lncRNAs bind to WDR5 to facilitate H3K4me3 and epigenetic activation . HOTTIP is an enhancer-like lncRNA of the human HOXA locus that coordinates expression of HOXA9 to HOXA13 , which are important for distal identity ( Wang et al . , 2011 ) . HOTTIP RNA directly binds to the WDR5 protein to recruit the MLL H3K4 methylase complex to maintain H3K4me3 . In addition , recent research has shown that the NeST lncRNA also binds WDR5 to upregulate IFN-γ expression through H3K4me3 ( Gomez et al . , 2013 ) , suggesting the existence of multiple different enhancing lncRNAs that function via WDR5 interactions . Many enhancers and promoters often produce lncRNAs , and some of these lncRNAs have enhancer-like functions in the activation of nearby genes by binding the Mediator complex ( Kim et al . , 2010; Orom et al . , 2010; Hung et al . , 2011; Rada-Iglesias et al . , 2011; Lai et al . , 2013 ) . Currently , it is believed that lncRNAs function by guiding localization of chromatin modification machinery as simple scaffolds . However , like coenzymes that modulate protein activity , it is possible that lncRNAs may affect protein function more directly . To examine the roles of RNA binding on WDR5 functionality , here we identify a RNA binding site on WDR5 , and characterized its function in vitro and in vivo . Inability to bind RNAs does not affect MLL complex formation or catalytic activity in vitro . However , ability to bind RNAs is crucial for WDR5 function in cells . We find that WDR5 binds over a thousand endogenous RNAs and that RNA binding is essential for WDR5 maintenance of ESC pluripotency . Inability to bind RNAs greatly reduced duration of WDR5 protein and its occupancy on chromatin , impaired global H3K4me3 levels , and reduced expression of genes required for embryonic stem cell state . Overall , these results suggest an unexpected role for RNA binding in regulating the lifespan of epigenetic fates .
To determine the lncRNA binding site , we performed alanine scanning mutagenesis of WDR5 , guided by its crystal structure ( Trievel and Shilatifard , 2009; Odho et al . , 2010; Avdic et al . , 2011 ) ( Figure 1A , B ) . WDR5 is a barrel-shaped protein with several charged clefts on its surface , several of which are known to mediate protein–protein interactions with MLL , histone H3 tail , or RbBP5 ( Figure 1A , Figure 1—figure supplement 1A , B ) . We generated 19 WDR5 point mutants , expressed them in Escherichia coli as GST-fusion proteins , and purified them to homogeneity ( Figure 1—figure supplement 1C ) . Four out of 19 mutants significantly reduced the ability to retrieve HOTTIP lncRNA in vitro: Y228A , L240A , K250A , and F266A . These WDR5 mutations defined a cleft between blades 5 and 6 , partially encompassing the same surface previously described to bind RbBP5 amino acids 371–381 ( Odho et al . , 2010; Avdic et al . , 2011 ) . Thus , a focal binding site defines the interaction between WDR5 and HOTTIP . To confirm that HOTTIP and RbBP5 bind to the same or overlapping sites on WDR5 , we pre-incubated wild type GST-WDR5 with an excess of RbBP5 peptide ( amino acids 371–381 ) or control H3K4me3 peptide ( amino acids 1–20 ) , and then assayed for HOTTIP binding ( Figure 1C ) . Whereas addition of H3K4me3 peptide had no effect , pre-incubation with RbBP5 peptide prevented HOTTIP binding to WDR5 , thus confirming the shared binding cleft . 10 . 7554/eLife . 02046 . 003Figure 1 . HOTTIP lncRNA binding surface overlaps with the RbBP5 binding surface on WDR5 . ( A ) Crystal structure of WDR5 ( PDB:3P4F , visualized with PyMol ) reveal that mutations abrogating HOTTIP RNA binding align along a cleft between blades 5 and 6 , opposite the H3K4/MLL binding site . This binding surface overlaps the site for RbBP5 . Top: schematic of tested and HOTTIP binding mutations . Bottom: magnification of HOTTIP binding cleft . Yellow: MLL peptide . Blue: RbBP5 peptide . ( B ) qRT-PCR results of indicated GST-WDR5 mutants tested by in vitro assay for binding to HOTTIP RNA or control Histone 1H2BG RNA . ( C ) RbBP5 peptide competition assay indicates that RbBP5 amino acids 371–381 can fully inhibit HOTTIP binding to WDR5 . Wild type data same as in Figure 1B . ( D ) Average fold pulldown of full length HOTTIP by cell-based native RNA immunoprecipitation of select WDR5 mutants . Top: qRT-PCR results of RNA immunoprecipitation of FLAG-WDR5 mutants . All values are normalized to input , then to FLAG pulldown of wild type and positive control D107A . Negative RNA controls ( U1 , HOTAIR ) and reaction without reverse transcription ( -RT ) show minimal enrichment ( Figure 1—figure supplement 1 , data not shown ) . Bottom: representative western blots of immunoprecipitations . DOI: http://dx . doi . org/10 . 7554/eLife . 02046 . 00310 . 7554/eLife . 02046 . 004Figure 1—figure supplement 1 . lncRNA binding surface of WDR5 and WDR5 RIP-qRT-PCR standards . ( A–B ) Electrostatic surface contouring of the WDR5 crystal structure ( PDB:3P4F , visualized with PyMol ) reveal that mutations abrogating HOTTIP RNA binding de_ne an electropositively charged region . Orange: WDR5 residues important for HOTTIP binding . Cyan: MLL peptide . Grey: RbBP5 peptide . ( C ) Silver stain of post-assay glutathione-bound GST-WDR5 mutants indicates no protein degradation or loading bias occurred during in vitro binding assay . ( D ) Average levels of input HOTTIP RNA for cell-based native RNA immunoprecipitation assays of WDR5 mutants by qRT-PCR , relative to GAPDH . ( E ) Average fold pulldown of HOTAIR or U1 control RNAs by cell-based native RNA immunoprecipitation of WDR5 , demonstrating no enrichment compared with IgG control . DOI: http://dx . doi . org/10 . 7554/eLife . 02046 . 004 To verify the lncRNA binding site in living cells , we conducted in vivo RNA immunoprecipitation ( RIP ) experiments with select WDR5 mutants in 293T cells ( Figure 1D ) . Whereas the D107A and R181A mutations caused little effect on RbBP5 binding compared with wild type ( ∼95% ) , the K250 mutation reduced RbBP5 binding ( ∼62 . 5% ) as previously described ( Odho et al . , 2010; Avdic et al . , 2011 ) . Furthermore , the F266A mutation actually increased RbBP5 binding ( ∼120% ) , suggesting that loss of binding to HOTTIP increases the ability to bind RbBP5 . Consistent with the direct in vitro binding assay , both K250A and F266A mutations fully abrogated WDR5 binding to HOTTIP in vivo . In contrast , mutations at D107A and R181A showed minimal effects on WDR5-HOTTIP interactions . K250A and F266A did not affect the accumulation of HOTTIP lncRNA ( Figure 1—figure supplement 1D ) , and both negative controls U1 and HOTAIR did not enrich binding to WDR5 ( Figure 1—figure supplement 1E ) . Taken together , the in vitro and in vivo analyses demonstrate that HOTTIP RNA binds WDR5 through a specific binding pocket . To pinpoint the functional consequences of a selective lncRNA-binding mutation of WDR5 , we further analyzed WDR5 F266A . In contrast to the other HOTTIP binding mutations , WDR5 F266A is defective in lncRNA binding in vitro and in vivo , but without any defects in binding MLL complex subunits RbBP5 or MLL1 in immunoprecipitation experiments ( Figure 1D ) . We reasoned that the F266A mutation offered an experimental strategy to isolate the requirement of lncRNA binding for WDR5 function . 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 . ( 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 To test the effects of the F266A mutation on gene activation in cells , we transfected increasing amounts of plasmids encoding GAL4-WDR5 WT or GAL4-WDR5 F266A to activate luciferase reporter gene expression ( Figure 2D ) , as previously described ( Wysocka et al . , 2005 ) . Surprisingly , WDR5 F266A demonstrated a severe defect in luciferase gene activation , requiring >100-fold transfected plasmid compared with wild type to achieve similar reporter gene activity . Thus , even though the WDR5 F266A mutation can bind the full MLL complex and does not affect catalytic activity , the inability to bind lncRNAs strongly compromises WDR5 function in activating gene expression by reporter gene assay . 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 . ( 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 GFP+ cells were sorted using consistent fluorescence parameters , cultured for 4 days in the presence ( +dox ) and absence ( −dox ) of doxycycline , and then analyzed by western blot and qRT-PCR . As expected , both cell lines demonstrated similar knockdown of endogenous mouse WDR5 ( Figure 3B ) , with equal transcription of exogenous human WDR5 RNA ( Figure 3C ) . Unexpectedly , more human WDR5 WT protein was present when compared with human WDR5 F266A ( Figure 3D ) . In WDR5 WT ESCs , promoter shutoff of WDR5 resulted in ∼50% reduction of H3K4me3 ( Lanes 1 and 2 ) , as previously reported ( Ang et al . , 2011 ) . Interestingly , ESCs harboring WDR5 F266A displayed a similar global 50% reduction of H3K4me3 ( Lane 3 ) , although WDR5 F266A protein was expressed at lower levels . In addition , promoter shutoff of WDR5 F266A caused a drastic >80% reduction of H3K4me3 levels ( Lanes 3 and 4 ) . Thus , WDR5 F266A exhibits a striking inability to maintain global H3K4me3 levels . We more closely examined the subcellular localization of WDR5 WT and WDR5 F266A , and found that WDR5 F266A was depleted in both nuclear ( Figure 3E ) and chromatin fractions ( Figure 3F ) . The alteration in cellular distribution of WDR5 F266A can be explained by differential stability in nuclear vs cytoplasmic pools , or alternatively , by a role of F266 in directly controlling nuclear localization . We believe that the former is the correct explanation based on the fact that fusion of F266A to a strong nuclear localization signal ( GAL4 DNA binding domain ) did not rescue its function ( Figure 2D ) . To further assess WDR5 protein stability directly , we conducted pulse chase experiments using doxycycline withdrawal to shutoff FLAG-hWDR5 transcription , and followed the fate of pre-existing WDR5 WT or F266A protein in the nucleus and cytoplasm ( Figure 3G ) . We found that in the cytoplasm , both WDR5 WT and F266A have equally short half-lives of less than 12 hr . In contrast , nuclear WDR5 WT persists for up to 24 hr , but WDR5 F266A is turned over at least 12 hr sooner ( Figure 3G ) . Thus , ability to bind lncRNAs appears to be essential for WDR5 protein stability , suggesting a RNA-mediated post-translational regulation of WDR5 . 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 ) . We performed RIPiT-seq with tandem immunopurified FLAG-HA-tagged WDR5 from formaldehyde crosslinked ESCs . To afford high stringency to the data , we searched for RNAs that were enriched in the WDR5 WT but not in the mock infected or WDR5 F266A datasets . WDR5 WT , but not F266A or control , is associated with 1434 RNAs , comprised of mRNAs , lncRNAs , pri-miRNAs , and snoRNAs ( Figure 4A , B ) . Thus , a family of ESC RNAs bind WDR5 through the same surface as HOTTIP enhancer-like RNA that act in other cell types , dramatically expanding the known RNA targets of WDR5 . WDR5 binds to 23 previously identified ESC lncRNAs ( Guttman et al . , 2011; Ulitsky et al . , 2011; Ng et al . , 2012; Sheik Mohamed et al . , 2010 ) . Importantly , six lncRNA partners of WDR5 have been shown to be required for ESC pluripotency or differentiation when individually depleted by shRNAs ( Figure 4B , lncRNAs with ESC phenotype in orange ) ( Guttman et al . , 2011 ) . Prior protein interaction surveys with repressive chromatin modification complexes had failed to identify partners for these lncRNAs except lincRNA-1592 ( Guttman et al . , 2011 ) , and their interaction with WDR5-MLL provides an explanation for their functions in ESC biology . 10 . 7554/eLife . 02046 . 007Figure 4 . WDR5 binds to a family of ESC RNAs . ( A ) WDR5 RIPiT-seq retrieves diverse class of RNAs . ( B ) 1434 RNAs bind to wild type WDR5 but not F266A mutant or vector control . Each column is a RIPiT-seq experiment; each row is a transcript . Red indicates enrichment over input and WDR5 F266A mutant . Known ESC lncRNAs are listed; those with known functions in ESC pluripotency or differentiation are highlighted in orange . Two lincRNA loci that are both bound by WDR5 on chromatin and generate lincRNAs that bind WDR5 are highlighted in orange bold font . ( C ) Overlap of genes retrieved by WDR5 RIPiT-seq vs ChIPseq . ( D ) Metagene analysis of WDR5 ChIP-seq signal in the indicated classes of genes . DOI: http://dx . doi . org/10 . 7554/eLife . 02046 . 00710 . 7554/eLife . 02046 . 008Figure 4—figure supplement 1 . 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 Previous reports of RNA-WDR5 interaction suggest the possibility for both cis-acting tethering and trans-acting mechanisms ( Wang et al . , 2011; Gomez et al . , 2013 ) . To investigate whether and how these mechanisms are utilized genome-wide , we compared RNA interactors of WDR5 identified by RIPiT-seq vs chromatin occupancy sites of WDR5 identified by ChIP-seq . Ang et al . ( 2011 ) reported 9303 genes that are bound by WDR5 and other trithorax group proteins in ESCs with high confidence , and 590 of these loci produced RNAs that physically interacts with WDR5 ( Figure 4C ) . Restricting the analysis to the top quintile of WDR5-bound promoters still showed very limited overlap ( 100 out of 1849 promoters produce RNAs that also bind WDR5 ) . Plotting average WDR5 ChIP-seq signal at ChIP-seq only or genes with both ChIP-seq and RIPiT-seq signal revealed that their WDR5 binding profiles are indistinguishable , highlighting the value of the RIPiT data set to reveal RNA involvement ( Figure 4D ) . In contrast , the class of genes with RIPiT signal only had substantially lower WDR5 chromatin occupancy at their promoters ( Figure 4D ) ; one or more of these RNAs may be decoys that remove WDR5 from cognate loci . The RNAs that both bind to WDR5 and have strong WDR5 ChIP-seq signal at their promoters are candidates for additional cis regulators of WDR5 recruitment . Two such lncRNAs , lincRNA-1552 and lincRNA-1592 , were shown to previously impact ESC biology , and lincRNA-1552 is a key node in the pluripotency gene regulatory network . Depletion of lincRNA-1552 led to mis-expression of ∼100 mRNAs including Nanog and Pou5f1 , and the promoter of linc-1552 is bound by Pou5f1 , Klf4 , Nanog , Zfx , n-Myc , and c-Myc ( Guttman et al . , 2011 ) , suggesting a positive feedback loop has been described for other ESC lncRNAs ( Sheik Mohamed et al . , 2010 ) . Further , the expression of linc-1552 is repressed by retinoic acid-induced ESC differentiation ( Guttman et al . , 2011 ) . This example highlights of how WDR5 can interact with a lincRNA important to ESC core transcription factor circuitry to control ESC pluripotency . Additionally , a previously reported screen in ESCs identified 901 protein-coding genes important for ESC pluripotency , and 23 of the corresponding mRNAs were enriched in WDR5 RIPiT-seq , including Myc and Bmp4 mRNAs ( Ivanova et al . , 2006 ) ( Supplementary file 1 ) . Our results also imply many ESC promoters can recruit WDR5 by mechanisms independent of RNA , consistent with other reports ( Ang et al . , 2011; Jiang et al . , 2011 ) . The discovery of many RNAs that bind WDR5 but not at its cognate genomic loci suggests that some WDR5-binding RNAs do not bind WDR5 as nascent transcripts , but only interact with WDR5 at a later stage of maturation and possibly for action in trans . Thus , WDR5 binds over 1400 endogenous RNAs in ESCs with potential for both cis and trans chromatin regulation . Loss of RNA binding also impacts the ability of WDR5 to promote ESC self-renewal . ( Figure 5A , B ) . In WDR5 WT ESCs , promoter shut-off caused a ∼50% reduction in the number of alkaline phosphatase-positive colonies , a specific indicator of ESC state . Notably , F266A WDR5 ESCs grown in the presence of doxycycline displayed a similar ∼50% reduction in colony number . By morphology , both WDR5 WT ESCs–dox and WDR5 F266A ESCs +dox formed similar small , partially differentiated colonies . Doxycycline withdrawal in WDR5 F266A ESCs caused a >90% reduction in number of colonies , with the majority of cells forming clusters of fully differentiated alkaline phosphatase-negative cells . 10 . 7554/eLife . 02046 . 009Figure 5 . WDR5 F266A mutation causes defects in ESC self renewal and increases differentiation . ( A and B ) Alkaline phosphatase staining and morphology of ESC colonies after 6 days growth in conditions specified . WDR5 F266A demonstrates reduced alkaline phosphatase positive colonies with increased differentiation compared with wild type . ( A ) Representative wells and colony morphology . Scale bar represents 200 μm . ( B ) Quantitation of colonies per well . ( C ) Microarray data reveal that both WDR5 F266A +dox and WDR5 WT −dox displayed loss of self renewal genes . WDR5 F266A +dox further demonstrates increased expression of ectodermal and mesodermal markers . Mixl1 is a mesendoderm marker indicative of potential for both endoderm and mesoderm . ( D and E ) qRT-PCR validation of ( D ) reduced expression of ESC pluripotency genes and ( E ) ectopic expression of differentiation genes with either WDR5 F266A expression or shutoff of WDR5 WT . qRT-PCR values were first normalized to the housekeeping gene beta-actin , then normalized to WT . Mean +/− SD are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 02046 . 009 WDR5 directly binds to the chromatin of key genes required for in ESC self renewal ( Ang et al . , 2011 ) . Genome-wide expression profiling further confirmed that WDR5 F266A was unable to sustain the gene expression program associated with ESC self-renewal and pluripotency , and instead allowed ectopic expression of genes indicative of ectodermal and mesodermal lineages ( Figure 5C ) . Validation of microarray results by qRT-PCR further demonstrated that ESCs expressing WDR5 F266A had significantly reduced mRNA levels of pluripotency regulators Pou5f1 , Nanog , Sox2 , and Esrrb , but upregulation of differentiation markers Cdx2 , Fgf5 , and Nestin ( Figure 5D , E ) .
Guided by the crystal structure and comprehensive mutagenesis , we identified the lncRNA binding cleft in WDR5 . WDR5 is a multifunctional adaptor protein that can interact with multiple subunits of the MLL complex , such as MLL itself and RbBP5 , as well as with regulators and substrates of the complex , such as HOTTIP and histone H3 arginine 2 symmetric demethylation ( H3R2me2s ) ( Migliori et al . , 2012 ) . The MLL and H3R2me2s share the same binding pocket , and we found that HOTTIP and RbBP5 share a distinct binding pocket . While Pou5f1 has been reported to bind WDR5 , we found no difference in the WDR5 F266A lncRNA binding mutant for its interaction with Pou5f1 ( data not shown ) . Nonetheless , these multitudes of interactions suggest that molecular arrangements in this complex may be dynamic and intricate . By strategic structure-guided mutagenesis , we identified WDR5 F266A as a mutant that has a specific defect in binding the lncRNA HOTTIP , but does not impact interactions with MLL1 , RbBP5 , or Ash2L . More importantly , the F266A mutation does not diminish MLL1 complex catalytic activity in vitro . Thus , although HOTTIP and RbBP5 share an overlapping binding pocket , the specific residues required for binding appear fundamentally different . This result is not surprising , given the electrostatic and three dimensional structure differences between peptides vs RNAs . The WDR5 F266A mutant is largely deficient in gene activation in cells , suggesting that direct lncRNA-protein interactions are important for WDR5 function in vivo . We replaced endogenous WDR5 in mouse ESCs with a RNA binding mutant , WDR5 F266A , and found that the RNA binding interface is critical for the accumulation of nuclear and chromatin WDR5 . These results suggest that in addition to their roles in recruiting protein complexes to specific genomic loci , lncRNA binding may also regulate the longevity of their interacting protein complexes ( Figure 6 ) . When able to bind RNAs , wild type WDR5 displays a long nuclear and chromatin half-life , thus allowing formation of the MLL1 complex for H3K4me3 and gene activation of ESC pluripotency genes . However , when unable to bind RNAs , WDR5 mutant is rapidly turned over , causing loss of H3K4me3 and reduced expression of pluripotency genes . Thus , RNA binding may directly or indirectly regulate protein turnover for epigenetic regulation . It is possible that WDR5 protein that is not properly complexed or localized on chromatin are degraded by default , or WDR5 degradation may be a regulated mechanism ( Nakagawa and Xiong , 2011 ) . In our study , ability to bind RNAs extends WDR5 protein half-life by an additional 12 hr to approximately 24 hr , which starts to approach the time scale of an entire cell division cycle ( especially in ESCs ) . With the extended protein longevity , lncRNAs may then regulate protein occupancy on chromatin during cellular maintenance , as well as aid in transmission of epigenetic regulators across cell generations . In this manner , lncRNAs may play crucial roles in regulating the multigenerational transmission of epigenetic proteins required for maintenance of cell state . 10 . 7554/eLife . 02046 . 010Figure 6 . Model for RNA-mediated switch of protein turnover to activate target genes . Expressed lncRNAs bind the WDR5 protein to increase protein stabilization on chromatin , which facilitates MLL complex assembly and methyltransferase activity for target gene activation . Without lncRNA binding , WDR5 fails to associate effectively with chromatin and is rapidly degraded . DOI: http://dx . doi . org/10 . 7554/eLife . 02046 . 010 Given the dynamic regulation of nucleosome/chromatin mark turnover , protein stability also greatly affects the epigenetic landscape within the cell . Nucleosomes are turned over in a dynamic and regulated fashion at specific genomic loci ( Dion et al . , 2007; Mito et al . , 2007; Deal et al . , 2010 ) , and histone tails can be proteolyzed during differentiation to erase prior histone modifications ( Duncan et al . , 2008 ) . In our example , the loss of stabilized WDR5 leads to an inability to maintain global H3K4me3 level , loss of expression of pluripotency regulators , and loss of ESC self renewal . Interestingly , the impact of F266A mutation on H3K4me3 level is modest in steady state conditions , but becomes quite striking with transcriptional shutoff of WDR5 . We interpret these results to indicate that WDR5 stability is important for the robustness of the active chromatin state , exemplified by H3K4me3 . In the absence of stabilized WDR5 , H3K4me3 becomes much more sensitive to fluctuations in the transcriptional levels of WDR5 and likely other subunits of the MLL complex . Thus , these results reveal a new facet for RNA regulation in the connection between protein half-lives and the temporal transmission of epigenetic information via chromatin . RIPiT-seq reveals nearly 1500 cellular RNAs in ESCs that are associated with WDR5 complex in a manner dependent on the WDR5 RNA binding pocket . These results support and substantially broaden the scope of RNAs that may impact WDR5’s function , including many lncRNAs and mRNAs that collectively may equal the copy number of WDR5 protein . The WDR5 F266A lncRNA-binding deficient mutant provides an ideal reagent to act as nonbinding control in the systematic genome-wide identification of functional WDR5-binding RNAs . Our data find that some of the recent discovered lncRNAs important for ESC self-renewal and pluripotency are associated with WDR5 ( Guttman et al . , 2011; Chakraborty et al . , 2012 ) , including five lncRNAs with ESC phenotypes that do not bind repressive chromatin complexes . However , there are numerous additional RNAs that have not been characterized with respect to cell fate control or chromatin function , and thus suggest additional layers of regulation at the level of lncRNAs . Comparative analysis of genomic locations of WDR5 occupancy and WDR5 RNA binding suggest configurations that are compatible with both cis or trans regulation . Studies of other chromatin modification complexes have shown that a single complex ( e . g . , PRC2 ) can interact with thousands of RNAs , each of which can regulate a subset of genes targeted by the complex ( Khalil et al . , 2009; Zhao et al . , 2010 ) . Additionally , promiscuous binding of RNAs may allow PRC2 and other silencing factors to survey the genome and distinguish transcribed vs non-transcribed regions ( Davidovich et al . , 2013; Di Ruscio et al . , 2013; Kaneko et al . , 2013 ) . A similar model likely applies to MLL and other gene activating complexes as well . The MLL complex possesses multiple components with RNA-binding capabilities . While WDR5 binds HOTTIP , NeST , and many other RNAs as shown here ( Wang et al . , 2011; Gomez et al . , 2013 ) , MLL1 itself also has domains that bind RNAs in specific and nonspecific fashions ( Krajewski et al . , 2005; Bertani et al . , 2011 ) . Cyp33 , an allosteric regulator of MLL , also contains a RNA recognition motif , and its regulation of MLL can be controlled in an RNA-dependent manner ( Hom et al . , 2010; Wang et al . , 2010 ) . The presence of multiple RNA interaction domains suggests intimate and potentially multiple roles for RNAs in the control of H3K4me3 and programming active chromatin .
HEK293T/17 cells were obtained from the American Type Culture Collection ( ATCC ) . The HEK293T 5XUAS-Luciferase cell line has been previously described ( Wysocka et al . , 2005 ) . V6 . 5 mouse embryonic stem cells were grown on 0 . 2% gelatinized plates in Knockout DMEM supplemented with 15% FBS , 1% Glutamax ( 35050; Invitrogen , Carlsbad , CA ) , 1% nonessential amino acids , 1% Pen/Strep , 0 . 2% β-mercaptoethanol , LIF ( ESGRO1107; 1:10000; Millipore , Billerica , MA ) , and 2 µg/ml doxycycline ( D9891; Sigma , St . Louis , MO ) . To maintain consistent levels of doxycycline , media was changed every 2 days . Antibodies used were: anti-FLAG-M2 ( F1804; Sigma , St . Louis , MO ) , anti-β actin ( ab8227; Abcam , Cambridge , MA ) , anti-RbBP5 ( A300-109A; Bethyl , Montgomery , TX ) , anti-H3K4me3 ( ab8580; Abcam , Cambridge , MA ) , anti-β tubulin ( ab6046; Abcam , Cambridge , MA ) , anti-H3 ( ab1791; Abcam , Cambridge , MA ) , anti-WDR5 ( 07-706; Millipore , Billerica , MA ) , and anti-HA ( MMS-101P; Covance , Princeton , NJ ) . All immunoprecipitations were conducted using anti-FLAG-M2 ( A2220; Sigma , St . Louis , MO ) or mouse IgG ( A0919; Sigma , St . Louis , MO ) agarose beads . Unless noted , all expression vectors were cloned into pcDNA3 or pcDNA3 . 1 + backbones . Hottip ( F:CCTAAAGCCACGCTTCTTTG , R:TGCAGGCTGGAGATCCTACT ) Hist1h2bg ( F:GGCATCATGAATTCCTTCGT , R:GCTTGTTGTAGTGGGCCAGA ) Hotair ( F:GGTAGAAAAAGCAACCACGAAGC , R:ACATAAACCTCTGTCTGTGAGTGCC ) Flag-Wdr5 ( F:GACTACAAAGACGATGACGACAA , R:TCCCAGCTTGTGACCAGATA ) Gapdh ( F:AGGTGGAGGAGTGGGTGTCGCTGTT , R:CCGGGAAACTGTGGCGTGATGG ) Rnu1 ( F:ATACTTACCTGGCAGGGGAG , R:CAGGGGGAAAGCGCGAACGCA ) Actb ( F:GCTGTATTCCCCTCCATCGTG , R:CACGGTTGGCCTTAGGGTTCAG ) Esrrb ( F:GGGTAGAGCCCACTTGTTCA , R:AGGTAGCCTGGGTTTTTGCT ) Cdx2 ( F:CCTGCGACAAGGGCTTGTTTAG , R:TCCCGACTTCCCTTCACCATAC ) Fgf5 ( F:AAAGTCAATGGCTCCCACGAA , R:GGCACTTGCATGGAGTTTTCC ) Gata6 ( F:ACAGCCCACTTCTGTGTTCCC , R:GTGGGTTGGTCACGTGGTACAG ) Hand1 ( F:GCGTCAGTACCCTGATGCCTTC , R:AAAGAGGAGGTAAGAGGACGGAAG ) Nanog ( F:TGGTCCCCACAGTTTGCCTAGTTC , R:CAGGTCTTCAGAGGAAGGGCGA ) Nestin ( F: AGGCGCTGGAACAGAGATT , R: TTCCAGGATCTGAGCGATCT ) Pou5f1 ( F:GTGGAGGAAGCCGACAACAATGA , R:CAAGCTGATTGGCGATGTGAG ) Sox2 ( F:CAGGAGAACCCCAAGATGCACAA , R:AATCCGGGTGCTCCTTCATGTG ) GST pulldown and competition assays were performed as previously described , with modifications ( Wang et al . , 2011 ) . GST-tagged WDR5 wild type , mutants , and GST control proteins were expressed and purified from E . coli as previously described ( Smith and Johnson , 1988 ) . GST proteins were bound to glutathione sepharose 4B ( Amersham/GE Healthcare , Pittsburgh , PA ) , blocked with 0 . 2 mg/ml BSA ( Ambion , Carlsbad , CA ) in 2X binding buffer ( 40 mM HEPES pH 7 . 6 , 200 mM KCl ) at 4°C for 1 hr . For peptide competition assays , indicated peptides ( Elim Biopharmaceuticals ) were also added . 0 . 04 mg/ml heparin ( H3149; 1:1; Sigma , St . Louis , MO ) was added to further block nonspecific RNA binding . Protein-bound beads were then incubated with folded T7-transcribed HOTTIP or histone 1H2BG mRNA for 45 min ( T7; Promega , Madison , WI ) . Beads were washed twice with PB200 ( 20 mM HEPES pH 7 . 6 , 200 mM KCl , 0 . 05% NP-40 ) and once with DEPC-treated water to remove detergents . After resuspension in water , beads were directly used in qRT-PCR reactions to determine RNA levels . To confirm lack of degradation , bead-bound proteins were analyzed by Silver Stain Plus ( Bio-Rad , Hercules , CA ) . Native cell-based RNA immunoprecipitation was performed as previously described with modifications ( Dignam et al . , 1983 ) . 293T cells were transfected with pcDNA3 . 1+FLAG-WDR5 and RNA expression plasmid . After 48–72 hr , cells were harvested by scraping in cold PBS , spun down , and pellets were then snap frozen in liquid nitrogen and stored at −80°C . Cells were resuspended in Buffer A ( 10 mM HEPES pH 7 . 5 , 1 . 5 mM MgCl2 , 10 mM KCl , 0 . 5 mM DTT , 1 . 0 mM PMSF , supplemented with RNaseOUT [1:100; Invitrogen , Carlsbad , CA] ) , and cell membranes were lysed with 0 . 25% NP-40 . After centrifugation , resulting nuclei were further lysed in Buffer C ( 20 mM HEPES pH 7 . 5 , 10% glycerol , 0 . 42M KCl , 4 mM MgCl2 , 0 . 5 mM DTT , 1 . 0 mM PMSF supplemented with RNaseOUT [1:100] ) . Resulting whole cell lysates were immunoprecipitatied with mouse anti-FLAG-M2 or control mouse IgG agarose beads , and washed four times with wash buffer ( 50 mM Tris–HCl pH 7 . 9 , 10% glycerol , 100 mM KCl , 5 mM MgCl2 , 10 mM β-mercaptoethanol , 0 . 1% NP-40 ) . To confirm immunoprecipitation , FLAG peptide-eluted protein-RNA complexes were analyzed by western blot . Coimmunoprecipitated RNA was extracted using Trizol LS ( Life Technologies , Carlsbad , CA ) and Qiagen RNeasy columns , treated with TURBO DNAfree , and then analyzed by qRT-PCR ( Brilliant II Sybr Green ) . Resulting values were defined as fraction of input , then normalized to the positive control sample . RIPiT-seq was performed large as described previously ( Singh et al . , 2013 ) with the following modifications . ESCs were fixed for 15 min with 1% formaldehyde in 1xPBS at 25°C . The reaction was stopped by added 1/10th the volume of 1 . 25M Glycine . For a typical RIPiT-seq experiment chromatin was prepared from ∼1 × 107 cells by thawing previously crosslinked ESC pellets and resuspending in 880 μl of Nuclear Lysis Buffer ( 50 mM Tris–HCl pH 7 , 10 mM EDTA , 1% SDS ) supplemented with fresh Protease Inhibitor Cocktail ( Roche , Indianapolis , IN ) , 10 mM PMSF , and RNaseOUT ( 1:100 ) . Chromatin was sheared in a Covaris E220 ultrasonicator to a size of 200–500 bp . The first IP was performed with 50 μl of FLAG-M2 agarose slurry ( Sigma , St . Louis , MO ) for 4 hr at 4°C on rotation . Samples were washed one time 1 ml in series at 4°C for 5 min each with the following buffers: High Stringency Buffer ( 15 mM Tris–HCl pH 7 , 5 mM EDTA pH 8 , 2 . 5 mM EGTA pH 8 , 1% Triton-X100 , 1% Sodium Deoxycholate , 120 mM NaCl , 25 mM KCl ) , High Salt Buffer ( 15 mM Tris–HCl pH 7 , 5 mM EDTA pH 8 , 2 . 5 mM EGTA pH 8 , 1% Triton-X100 , 1% Sodium Deoxycholate , 1M NaCl ) and NT2 Buffer ( 50 mM Tris–HCl pH 7 , 150 mM NaCl , 1 mM MgCl2 , 0 . 0005% NP-40 ) . Captured FH-WDR5 complexes were then eluted from the FLAG-M2 agarose by incubating each sample in 500 μl of FLAG Elution Buffer ( 50 mM Tris–HCl pH 7 . 5 , 250 mM NaCl , 0 . 5% NP-40 , 0 . 1% Sodium Deoxycholate ) and 0 . 5 μg/ml FLAG Peptide ( PAN Facility , Stanford University ) for 25 min at 4°C on rotation . The FLAG elution was repeated once , achieving a total elution volume of 1 ml . FH-WDR5 complexes were then captured by adding 10 μl HA-agarose slurry ( Pierce ) to each sample for 1 hr at 4°C on rotation . The samples were then washed as previously done on the FLAG-M2 beads . After washing each sample was resuspended in 100 μl of ProteinaseK Buffer ( 10 mM Tris–HCl pH 7 , 100 mM NaCl , 1 mM EDTA pH 8 , 0 . 5% SDS ) and 5 μl of ProteinaseK ( Ambion , Carlsbad , CA ) for 45 min at 50°C in a Thermomixer ( Eppendorf , Hauppauge , NY ) at 1000 rpm . After ProteinaseK treatment 500 μl of Trizol LS ( Qiagen , Venlo , Limburg ) was added to each sample , vortexed for 10 s , and incubated at 25°C for 5 min . Total RNA was recovered from each sample with the miRNeasy kit ( Qiagen , Venlo , Limburg ) following the manufacturer’s instructions , treated with TURBO-DNAfree ( Ambion , Carlsbad , CA ) , and repurified with the miRNeasy kit . The Ovation v2 Kit ( NuGEN , San Carlos , CA ) was used according to the manufactures instructions to produce dsDNA from the purified RNA enriched by the RIPiT . DNA isolated from the Ovation v2 Kit was then used in the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina Kit ( NEB , Ipswich , MA ) following the manufactures instructions to produce barcoded deep sequencing libraries . Library PCR material was quality checked and quantified on an Agilent BioAnalyzer 2100 and samples were sequenced with a run type of 1 × 50 bp on an Illumina HiSeq 2500 machine . Sequencing reads were mapped to the mouse genome ( mm9 assembly ) using TopHat ( version 1 . 1 . 3 ) ( Trapnell et al . , 2009 ) . Each sample generated 13 . 6 to 51 . 1 million mapped sequences , recorded in BAM/SAM format . A non-redundant mm9 transcriptome was assembled from UCSC RefSeq genes , UCSC genes , and predictions from ( Ulitsky et al . , 2011 ) and ( Guttman et al . , 2011 ) . Gene expression in the form of log2d RPKM was calculated using a self-developed script . An enrichment score of each gene in each sample was defined as the fold change of log2d RPKM between the pull down sample vs its corresponding input . A gene was defined enriched in wide-type WDR5 pull down if the minimum enrichment score of this gene in both wide-type replicates is greater than 0 and greater than the maximum of that in empty vector and mutated samples . There were 1434 enriched genes , whose enriched score were clustered using Cluster and plotted in Treeview . WDR5 ChIPseq peaks were downloaded from Ang et al . ( 2011 ) , and genes whose promoter region ( 2 kb upstream and 1 kb downstream of its TSS ) overlaps with any WDR5 ChIPseq peak were defined as WDR5 bounded genes . Overlay of WDR5 bounded genes with WDR5 RIPseq enriched genes was shown and p value of the significance was estimated using Fisher’s Exact test . RIPiT data are available at Gene Expression Omnibus under the accession number GSE53035 . The Kd values of the WDR5-RbBP5330–381 , WDR5-MLL13762–3802 , WDR5-H31–10 were determined by a ITC200 calorimeter ( MicroCal ) in buffer 25 mM Tris , 150 mM NaCl , pH 8 . 0 . The latter components were used as titrants . The concentration of macromolecule solution is 60–100 µM and the concentration of titrant is 600–1000 µM . ITC data were analyzed and fit using Origin 7 ( OriginLab ) . 40 µg GST-fused WDR5 ( WT or mutants ) mixed with MLL1SET , Ash2L , RbBP5 were incubated with 10 µl glutathione-Sepharose 4B beads for 2 hr at 4°C . After extensive wash , the bound protein were eluted in 50 mM Tris–HCl , pH 8 . 0 , 300 mM NaCl , 15 mM reduced glutathione . The input samples and eluted samples were loaded on 12% SDS-PAGE and Coomassie stained . Reactions were carried out at room temperature for 1 hr in the presence of [3H]-SAM ( S-adenosyl-L- [methyl-3H] methionine ) as previously described ( Cao et al . , 2010 ) . 0 . 25 mM unmodified H3K4 peptides ( 10 mer:ARTKQTARKS ) was used as substrate . 0 . 5 µM of WDR5 , RbBP5 , Ash2L and MLL1SET proteins were used . Each assay was performed in triplicate . pCMX-GAL4-WDR5 and pRL Renilla Luciferase plasmid were transfected into HEK293T 5XUAS-Luciferase cells using Lipofectamine 2000 . 40–48 hr after transfection , cells were harvested and analyzed using the Dual-Luciferase Reporter Assay System ( Promega , Madison , WI ) . Luciferase readings were normalized to Renilla readings . Lentiviral constructs were generated that that constitutively knock down mouse WDR5 while allowing expression of a doxycycline-inducible human WDR5 . The rescue vector pLKO . tre ( Ang et al . , 2011 ) was modified by replacing the shRNA-immune mouse WDR5-cDNA with either wild type or F266A mutated human WDR5 . The resulting pLKO vectors and rtTA ( from Marius Wernig , Stanford University ) constructs were cotransfected with packaging plasmids ( pLKO: second generation pCMV-dR8 . 2 dvpr and pCMV-VSVG| rTta: third generation pMDLg/pRRE , pCMV-VSVG , pRSV-Rev ) into 293T cells using Lipofectamine 2000 ( Invitrogen , Carlsbad , CA ) . After 16 hr , media was changed . Viruses were harvested after 48 and 72 hr , pooled , and then clarified by centrifugation . Viruses were concentrated using Lenti-X concentrator , incubated with 8 µg/ml polybrene ( Sigma H9268 , St . Louis , MO ) and then used to infect V6 . 5 ESCs . GFP+ cells were sorted ( Stanford Shared FACS Facility ) and then cultured as described above . For RNA analysis , 5000 GFP+ cells were plated in media with or without doxycycline , and then harvested after 6 days . For qRT-PCR analysis , resulting values were normalized to control β actin levels , and then normalized to WT+dox . Lentiviral constructs were generated that contained either no cDNA , N-terminally FLAG-HA tagged WT WDR5 ( human ) , or N-terminally FLAG-HA tagged WDR5 F266A ( human ) mutant . The F266A mutant construct has a 20 amino acid N-terminal truncation , which does not affect in vivo function or MLL complex formation . The parent vector was N103 ( kind gift from Dr Jerry Crabtree Lab ) and contains a puromycin resistance cassette . The resulting modified N103 vectors were reverse transfected with packaging plasmids MD2G and PSPAX2 ( kind gift from Dr Jerry Crabtree Lab ) into 293T cells using FuGENE HD ( Promega , Madison , WI ) according to the manufactures protocol . After 16 hr , media was changed . Viruses were harvested after 48 and 72 hr , pooled , and then clarified by centrifugation . Viruses were concentrated using Lenti-X concentrator , incubated with 8 µg/ml polybrene ( H9268; Sigma , St . Louis , MO ) and then used to infect V6 . 5 ESCs . Two days after infection clones with successful integration were selected by added 1 µg/ml Puromycin to ESC media . ESC colonies were screened for expression of the FH-WDR5 constructs and only clones with near endogenous levels of the FH-WDR5 or FH-WDR5-F266A proteins were selected . After isolation and expansion of clonal populations of ESCs with near endogenous levels of FH-tagged wild type or F266A mutant WDR5 nuclei were isolated as described above . Nuclei were lysed in 300 μl of FPLC Buffer ( 50 mM Tris–HCl pH 7 . 5 , 250 mM NaCl , 10% glycerol , and 0 . 1% NP-40 ) . The lysates were loaded onto a HiPrep Sephacryl S-500 HR Gel Filtration Column ( GE Healthcare Life Sciences , Pittsburgh , PA ) using a Pharmacia Biotech FPLC pump system . Fractions ( 330 μl per fraction ) were collected on ice and then subjected to SDS-PAGE and western blot analysis . ESCs were fractionated as previously described ( Tee et al . , 2010 ) , with modifications . To isolate nuclei and cytoplasm , Pellets were lysed in nuclei isolation buffer ( NIB: 10 mM Tris pH 7 . 5 , 60 mM KCl , 15 mM NaCl , 1 . 5 mM MgCl2 , 1 mM CaCl2 , 250 mM Sucrose , 10% glycerol , and 0 . 1% NP-40 ) . Lysates were centrifuged at low speed to separate cytoplasm ( supernatant ) from nuclei ( pellet ) . Cells were washed once with NIB without NP-40 , resuspended in NIB without NP-40 , and treated with Turbo DNase ( Ambion , Carlsbad , CA ) . EDTA was added to a final concentration of 10 mM to neutralize the DNase . After centrifugation , the resulting washed pellet was resuspended in NIB , and NaCl was added to a final concentration of 500 mM to lyse the nuclei . For chromatin isolation , cells were lysed with cytoskeleton buffer ( CSK: 10 mM PIPES KOH pH 7 , 100 mM NaCl , 300 mM sucrose , 3 mM MgCl2 , 0 . 5% Triton X-100 ) , centrifuged , and the resulting pellet was washed twice more with CSK buffer . The washed pellet was resuspended in CSK buffer , and treated with Turbo DNase to release chromatin-bound proteins . For colony forming assays , FLAG-tagged WDR5 rescue v6 . 5 ESCs were counted by flow cytometer , as well as hemocytometer , and 250 cells were plated onto 12 well plates with full media with or without doxycycline . After 6 days , cells were fixed with 1:1 methanol:acetone , then stained using Vector Blue ( Vector Laboratories , Burlingame , CA ) per the manufacturer’s instructions . cDNA was synthesized , labeled , and hybridized to Affymetrix Mouse 430 2 . 0 arrays in biologic duplicates ( Stanford Protein and Nucleic Acid Facility ) . Arrays were normalized by robust multi-array average ( RMA ) using justRMA package in R , and probes that had an expression value greater than 50 in at least one sample were defined as expressed probes . For each expressed probe , its expressions were log2ed , and the gene expression was defined as the average expression of all the expressed probes that attached to this gene . Differential expression was performed using significance analysis of microarrays ( SAM ) 3 . 0 ( Tusher et al . , 2001 ) with a false discovery rate less than 5% , an average fold change ≥2 in any compared groups . Heatmaps show mean-centered gene expression , based on previously described expression patterns ( Ang et al . , 2011 ) . Microarray data are available at Gene Expression Omnibus under the accession number GSE36513 .
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 .
If all the DNA contained within a single human cell were stretched out it would be about three meters long . To fit this length of DNA into the nucleus of the cell , it is packaged into a compact structure called chromatin . If a cell wants to express one of the genes in the DNA in order to produce a protein , it must unpack part of the chromatin to give an enzyme called RNA polymerase access to the DNA to produce messenger RNA . However , other enzymes—often working with other enzymes , proteins and molecules called cofactors—can modify the structure of the chromatin in a way that leads to changes in the expression of nearby genes . The protein WDR5 binds to enzymes and helps to modify chromatin so that nearby genes can be ‘switched on’ . However , WDR5 also binds to RNA molecules that are not involved in the expression of genes as proteins . These long non-coding RNA molecules ( lncRNAs ) are thought to act as scaffolds that guide the WDR5-enzyme complex to specific stretches of DNA . However , it is not clear if lncRNAs might be performing roles that affect the function of these enzyme complexes directly—as has been observed for other cofactors . Yang , Flynn et al . have now investigated the role of lncRNA molecules further , and identified a pocket on the structure of WDR5 where these molecules bind . A mutation in this pocket that blocks binding to lncRNA did not stop purified WDR5 protein from forming active enzyme complexes when tested in the lab . However , it seems that WDR5 needs to bind to the lncRNAs to function properly inside living cells . WDR5 binds to hundreds of lncRNAs within a cell , and the mutation that blocks this binding reduced both the amount of time that WDR5 protein survives in a cell and the amount of time it spends bound to chromatin . As such , this mutation seems to reduce the ability of the WDR5-enzyme complex to activate chromatin and ‘switch on’ genes . WDR5 is also known to be important in mammalian embryonic stem cells—cells that have the potential to become all the different types of cells found in the body . Yang , Flynn et al . uncovered that the mutations in WDR5 that abolish lncRNA binding also affect the expression of genes that help mouse stem cells to maintain this potential . Since different cell types have distinct patterns of active chromatin , the next challenge will be to understand whether different lncRNAs bind to WDR5 to switch on unique set of genes in each cell type .