Fig 2: WDR5 Regulates p53-Dependent Gene Regulation during ESC Specification(A) Metaprofiles for ATAC-seq chromatin accessibility in closing (left panel) and opening (right panel) peaks in day 2 Wdr5KO EBs partially rescued by p53 deletion.(B) Boxplots for downregulated (left panel) and upregulated (right panel) genes from RNA-seq show that transcriptional defects in day 2 Wdr5KO EBs (versus WT) are partially rescued by p53 deletion. For downregulated genes, WT versus KO-A or KO-B: p = 2.2e–16. KO-A versus KO-A derived DKO-A: p = 0.009. KO-B versus KO-B derived DKO-B: p = 2.2e–16. For upregulated genes, WT versus KO-A or KO-B: p = 2.2e–16. KO-A versus KO-A derived DKO-A: p = 2.2e–16. KO-B versus KO-B derived DKO-B: p = 2.2e–16.(C) qRT-PCR validation of partially rescued genes E2f3 and Cdkn1a in day 2 or day 3 Wdr5KO EBs by p53 deletion. Data represent mean ± SD (n ≥ 3). **p ≤ 0.01 and ***p ≤ 0.001.(D) Metaprofiles displaying H3K4Me3 peaks centered at WDR5-bound loci (WT) were reduced in day 2 Wdr5KO EBs, but were partially reversed (rescued) by p53 deletion (DKO EBs).

Fig 3: WDR5 Induces Rx+ Neuroectoderm Differentiation via Its RbBP5 Interaction Surface(A and B) WDR5 regulates NE proliferation and differentiation in a dose-dependent manner. WT and WDR5Dox;Wdr5KO ESCs were maintained in the Dox-containing ESC media. ESCs were resuspended in SFEBq differentiation (EB day 0) in different concentrations of Dox. Cell proliferation at EB day 4 (A) and Rx-GFP positive NE at EB day 5 (B) were determined.(C and D) Proliferation and NE differentiation in WDR5Dox; Wdr5KO ESCs stably transfected with FLAG-tagged WT or different WDR5 mutants. Upon differentiation, Dox was removed (no Dox) or added back 12 h later (2.0 ug/ml, T12h). Cell proliferation was determined at EB day 4 and relative cell proliferation was normalized to the respective group with Dox as 1.0 (D). Percentage of Rx-GFP+ NE cells at EB day 5 was determined by flow cytometry (E).(E) Effects of WDR5-RbBP5 interaction mutants with or without Dox-induced WT WDR5 rescue on NE differentiation were determined by Rax mRNA qRT-PCR (EB day 6).(F) WT, but not WDR5-RbBP5 interaction mutant WDR5Q289E, retained capacity to induce Rax-GFP+ NE in Wdr5KO EBs. Representative day 6 EBs were recorded under microscope using bright field or fluorescence channel. Scale bars: 50 μm.(G) Expression of Dox-inducible WDR5-RbBP5 interaction mutants was not able to induce Rx-GFP+ NE differentiation. WDR5Dex; Wdr5KO ESCs (Δ26), maintained in dexamethasone (Dex) containing ES media, which enabled Dex-inducible WDR5 expression to maintain ESC self-renewal, were stably transfected with Dox-inducible forms of various HA-tagged WDR5 mutants. Upon differentiation, Dex was removed and Dox was added 12 h later (2.0 ug/ml, T12h). Percentage of Rx-GFP+ NE cells at EB day 7 was determined by flow cytometry.EV: empty vector backbone control. Data in (A)–(F), (G), (H), and (I) represent mean ± SD (n ≥ 3). **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.001.See also Figures S1 and S2.

Fig 4: Setbp1- and Setbp1(D/N)-induced AML cells are sensitive to WDR5 inhibition(A) Real-time RT-PCR analysis of indicated SETBP1/MLL1 co-bound targets in Setbp1-induced AML cells at 72 h after treatment with OICR-9429 at indicated concentration or control DMSO. Data are represented as mean ± SD (n = 3).(B) Real-time RT-PCR analysis of same genes in Setbp1(D/N)-induced AML cells at 72 h after treatment with OICR-9429 or control DMSO. Data are represented as mean ± SD (n = 3).(C) Relative colony formation by Setbp1-induced AML cells upon treatment with WDR5 inhibitor OICR-9429 at indicated concentration or control DMSO. Data are represented as mean ± SD (n = 3).(D) Relative colony formation by Setbp1(D/N)-induced AML cells upon treatment with WDR5 inhibitor OICR-9429 at indicated concentration or control DMSO. Data are represented as mean ± SD (n = 3).(E) Left panel, real-time RT-PCR analysis of relative indicated SETBP1/MLL1 co-bound targets in Setbp1-induced AML cells after transduction with Wdr5-specific shRNAs (Wdr5-sh1 and -sh2) or a non-targeting control shRNA (NC-sh). Data are represented as mean ± SD (n = 3). Right panel, western blotting analysis of the same transduced cells using indicated antibodies.(F) Same analyses as in (E) for Setbp1(D/N)-induced AML cells.

Fig 5: LANA interacts with MLL1 complex components. (A–D) Reciprocal co-immunopreciptiations (IPs) of LANA and (A) WDR5, (B) MLL1, (C) RbBP5 or (D) ASH2L from whole cell lysates after expression in 293T cells. WDR5, RbBP5, ASH2L, or MLL1 were immunoprecipitated with anti-Flag, and LANA with anti-LANA antibody, or IgG control used. (E) GST or GST-WDR5 precipitation of in vitro translated full length T7 epitope tagged LANA. Ponceau S stained GST and GST-WDR5 are shown. (F) IP of endogenous LANA from KSHV infected BCBL1 cell nuclear lysate followed by detection of MLL1 or WDR5. (A–E) Molecular weight (kDa) indicated at right.