Fig. 6: Binding of ASH1L_PHD to H3K4me3 inhibits enzymatic activity of ASH1L.

a Overlay of ¹H,¹⁵N HSQC spectra of ASH1L_BD in the absence (black) or presence of the indicated molar ratios of ASH1L_SET. b Overlay of ¹H,¹⁵N HSQC spectra of ASH1L_PHD in the absence (black) or presence (blue) of ASH1L_SET. c Liquid histone methyltransferase (HMT) assays of GST-ASH1L_{SET-BD-PHD-BAH} on recombinant nucleosomes carrying H3K4me3 or unmodified NCP. GST-ASH1L_{SET-BD-PHD-BAH} was pre-incubated with substrate and MRG15 (or BSA) prior addition of ³H S-adenosyl methionine. GST, control. Experiment shown was performed in duplicate and was repeated at least three times with different preps of recombinant GST-ASH1L_{SET-BD-PHD-BAH} with similar results. d Western blot analysis of in vitro KMT assays of recombinant GST-ASH1L_SET-BD-PHD on unmodified and H3K4me3-nucleosomes. e Liquid HMT assays of recombinant GST-ASH1L_SET-BD-PHD on H3K4me3-nucleosomes in the absence (BSA) or presence of MRG15. Experiment shown was performed in duplicate and was repeated at least three times with different preps of recombinant proteins with similar results. f Heatmap of gene expression profiles in human lung adenocarcinoma A549 cells transfected with control (shNT) or shASH1L. Blue and red colors indicate down and up regulation of genes, respectively. g IPA analysis of the downregulated 541 genes in ASH1L knockdown cells. The top five hits of each category are listed. Statistical test used was one-sided. h Cell proliferation assays of the control (shControl) and ASH1L knockdown A549 cells. Live cells were counted over a 6-day time course. Error bars represent mean from three replicates ± SEM. i Kaplan-Meier analysis of human lung adenocarcinoma patients with low or high ASH1L gene expression levels. j Overlay of the structures of the H3K4me2-bound ASH1L_BD-PHD and ASH1L_PHD-BAH. k A model of the functional cooperation between ASH1L domains in the context of the nucleosome. DNA and histone tails are shown as black and gray curves, respectively. Histone H3 modifications are indicated by colored circles and labeled. Source data are provided with this paper.