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. Author manuscript; available in PMC: 2015 Oct 23.
Published in final edited form as: Mol Cell. 2014 Sep 25;56(2):298–310. doi: 10.1016/j.molcel.2014.08.022

Figure 2. BS69 binds H3.3K36me3 in vitro via its PWWP domain.

Figure 2

(A) Schematic diagrams of human BS69 and mutants carrying PWWP deletion and point mutations denoted by red stars. (B) Recombinant GST-BS6950-401 specifically recognizes H3.3K36me3 peptide in the biotinylated histone peptide pull-down assays. (C) Sequential ChIP using H3K36me3 and anti-FLAG antibodies in HeLa cells overexpressing H3.3-FLAG shows co-occupancy of H3K36me3 and H3.3-FLAG at indicated locales of BS69 target genes. (D) GST-BS6950-401 FW point mutant (F293A, W294A) loses the ability to bind H3.3K36me3 in vitro. (E) and (F) MicroScale Thermophoresis analysis determined the Kd of GST-BS6950-401 WT (E) interaction with H3.3K36me3 to be 50 μM. The Kd value of the interaction between the BS69 PWWP point mutant and H3.3K36me3 (F) was determined to be 277 μM. (G) HA-ChIP and q-PCR demonstrated that the ectopically expressed WT BS69 but not the BS69 PWWP deletion mutant binds the BS69 targets (identified through BS69 ChIP). (H) The binding of GST-BS6950-401 to H3.3K36me3 peptide is abolished by phosphorylation at position S31. Data are represented as mean ± SEM from 3 biological replicates (C,G), *P<0.05, **P<0.01. See also Figure S2.