Fig. 6: An H3.3K36me3-binding PWWP module is essential for chromatin association of ZM, as well as ZM-enforced proto-oncogene activation and oncogenic transformation.

a Scheme of the used ZM mutations within its ZMYND11 segment. b Proliferation of murine HSPCs stably transduced with ZM, WT or mutant (insert, immunoblot of ZM; n = 3 biological replicates per group, with data presented as mean ± SD). c Wright-Giemsa staining of HSPCs ten days post-transduction of ZM, WT or mutant (scale bar = 10 μm). d RT-qPCR of the indicated ZM direct target genes in HSPCs stably transduced with ZM, WT or the indicated mutant. qPCR signals from three independent experiments were normalized to those of 18S RNA and then to WT and presented as mean ± SD. e Pulldown using H3.3K36me3-containing peptide and nuclear extract of 293T cells stably expressing 3XHA-3XFlag-tagged ZM, either WT or the indicated mutant. f, g Immunoblot for 3XHA-3XFlag-tagged ZM in soluble nucleoplasmic (Sol) and chromatin-bound (Chr) fractions, prepared from 293T cells with stable expression of WT or mutant ZM (f) or those WT ZM stable expression cells with SETD2 knock down (siSETD2; g) or mock treatment (siControl). SETD2 knockdown led to global reduction of H3K36me3. Tubulin and H3 serve as fractionation controls. h ChIP-qPCR examining ZM occupancy to the indicated gene in 293T stable expression cells. ChIP signals from three independent experiments were normalized to those of input and then to WT and presented as mean ± SD. i Average signal profiles and clustered heatmaps displaying 3XHA-3XFlag-tagged ZM∆M, ZM∆Z, and ZM CUT&RUN signals (detected with HA antibody) over all genes in 293T cells. j IGV views of ZM∆M, ZM∆Z, and ZM CUT&RUN signals (detected with Flag or HA antibody) at the indicated gene in 293T cells.