Fig 2: Gene expression analysis of WT and ASXL1-mutated U937 cells by RNA-seq. (A) Linear correlation of fold changes of all genes between MT+/- (MT1-MT3) and MT-/- (MT11-MT13) groups. Pearson R-value, 0.2192; P<0.0001. (B) Venn diagrams indicate the number of differentiated gene sets in the MT+/- and MT-/- groups, compared with the WT group, as determined by Gene Set Enrichment Analysis. Representative commonly skewed gene sets in MT+/- and MT-/- were listed. (C) Venn diagrams demonstrating the significantly upregulated (red) and downregulated (blue) genes (P<0.05; fold change >2; false discovery rate-corrected P<0.20) in the MT+/- or MT-/- groups compared with the WT (WTblk, WT1 and WT2) group. Expression levels and fold changes of significantly upregulated and downregulated genes are shown by (D) heat maps and (E) bar graphs. ASXL1, additional sex combs-like 1; MT, mutated; WT, wild-type; WTblk, wild-type bulk parental.

Fig 3: 6mA Deposition Triggers Proteolysis of the Sensor Proteins ASXL1 and MPND(A) Domain structures of ASXL1, showing the positions of the HARE-HTH and PHD domains and the central proline-rich region (PRR), and the MYSM1 and MPND deubiquitinases, with the percentage amino acid identity and similarity between RAMA domains. JAMM/DUB denotes the deubiquitinase catalytic domain.(B) In vitro DNA pull-down assays. Purified recombinant HARE-HTH and RAMA domains were incubated in the presence of untreated resin or resin coated with duplex DNA containing unmodified adenine (A) or 6mA. After extensive washing, bound protein was resolved by SDS-PAGE and detected by immunoblotting using the indicated antisera (n = 3 experiments).(C) HEK293T cells expressing Flag-ASXL1.591 and either empty vector, Flag-Mettl4, or Flag-Dam were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting with the indicated antisera (upper panels). Dot blot analysis shows relative 6mA levels in genomic DNA recovered from the same samples (n = 3 experiments).(D) Cells expressing Flag-RAMA, Flag-Mettl4, or Flag-Dam were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting (upper panels). Dot blot analysis shows relative 6mA levels in genomic DNA recovered from the same samples (n = 2 experiments).(E) Accumulation of Asxl1 and reduction of H2A-K119Ub and H3K27me3 in Mettl4 KO cells. Whole-cell extracts (upper panels) and purified histones (lower panels) were prepared from spleens isolated from individual WT or Mettl4 KO mice and proteins resolved by SDS-PAGE and immunoblotting (n = 2 experiments).(F) Accumulation of Mpnd in Mettl4 KO cells. Protein extracts were prepared from spleens isolated from individual WT or Mettl4 KO mice and resolved by SDS-PAGE and immunoblotting (n = 2 experiments).See also Figures S4 and S5.

Fig 4: Effects of ASXL1 mutations on the differentiation of PMA-treated U937 cells. Following treatment with various concentrations of PMA, cells were harvested at 96 h. (A) Representative images of Wright-Giemsa-stained WT and ASXL1-mutated U937 cell lines, which attached to chamber slides following 200 ng/ml PMA-induced differentiation. (B) CD11b expression in cells harvested from slide chambers, as determined by flow cytometric analysis. (C) Percentages of CD11b-positive cells in WT and MT U937 cells calculated using flow cytometry data. Data are presented as the means ± standard error of the mean. *P<0.05 vs. the WT group (WTblk, WT1 and WT2), one-way analysis of variance. ASXL1, additional sex combs-like 1; CD11b, cluster of differentiation 11b; PE, phycoerythrin; PMA, phorbol 12-myristate 13-acetate; MT, mutated; WT, wild-type; WTblk, wild-type bulk parental.

Fig 5: Generation of ASXL1-mutated U937 cell lines using the CRISPR/Cas9 system. (A) Schematic diagrams of ASXL1 mRNA, and ASXL1-WT and ASXL1-MT proteins. The target site of gRNA, frameshift mutation and truncated proteins are indicated. (B) Representative electropherograms, as determined by Sanger sequencing, for the evaluation of ASXL1 mutations in exon 8 introduced by CRISPR/Cas9. Inserted nucleotides are underlined in red. (C) ASXL1 mutations in exon 8 of all mutated cell lines with associated changes in ASXL1 protein. All genomic and protein sequence variants are represented using the Human Genome Variation Society nomenclature. WT indicates a WT sequence without mutations. (D) Immunoblot analysis of ASXL1 in WT and MT cell lines. Immunoprecipitated protein samples were subjected to 8% SDS-PAGE and immunoblot analysis. (E) Representative images of Wright-Giemsa-stained WTblk, WT1, MT2 and MT11 U937 cells in logarithmic growth phase. ASXL1, additional sex combs-like 1; CRISPR/Cas9, Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein-9 nuclease; Ex, exon; gRNA, guide RNA; NR, nuclear receptor; PHD, plant homeodomain; MT, mutated; WT, wild-type; WTblk, wild-type bulk parental.