Extended Data Figure 4 ∣. WDR5 associates with the APC/C and TBP on distinct surfaces.

a, The WIN (WDR5 interaction motif)-binding site on WDR5 is critical for APC/C engagement, while the WBM (WDR5 binding motif)-binding surface is dispensable. A secondary binding surface (4A) is also important for WDR5’s association with the APC/C. The WBM-binding surface on WDR5 is critical for TFIID association, while the WIN-binding site is dispensable. HEK 293T cells were transfected with the indicated ^FLAGWDR5 variants and cells were synchronized in mitosis. ^FLAGWDR5 was affinity purified and bound proteins were determined by Western blotting. This experiment was performed five independent times with similar results.

b, Reciprocal IPs show that the APC/C binds WDR5 through its WIN-binding site. Endogenous APC/C was purified from 293T cells expressing the indicated ^FLAGWDR5 variants, and bound proteins were determined by SDS-PAGE and Western blotting. This experiment was performed three independent times with similar results.

c, Heatmap of bait-normalized total spectral counts identified from ^FLAGWDR5-purified mass spectrometry experiments. HeLa cells were transfected with ^FLAGWDR5 for 24 h prior to mitotic synchronization.

d, The WDR5 inhibitor MM-102 impairs WDR5’s association with the APC/C. Mitotic HeLa S3 cells were released into MM-102 for 2 h prior to IP experiments. Under these conditions, MM-102 did not prevent the association of WDR5 with MLL and RBBP5. This experiment was performed two independent times with similar results.

e, Expression of wild type WDR5 but not WDR5^ΔWIN rescues the pluripotency defect caused by WDR5 depletion in H1 hESCs. H1 hESCs virally expressing siRNA-resistant WDR5 variants (WDR5 versus WDR5^ΔWIN) were depleted of endogenous WDR5 (W) or treatment with control siRNA (C). Expression of OCT4 and NANOG was determined by Western blotting. This experiment was performed once.