Fig 1: Tumor-derived ASXL1 mutant interferes with the BAP1-ASXL1-FOXK1/K2 axis to down-regulate tumor suppressor genes. (A) The mRNA expression of tumor suppressor genes in the indicated groups of K562 cells, as determined by RNA-seq analysis. (B–F) The occupancy of FOXK1, FOXK2, ASXL1, EZH2 and SUZ12 at the promoter regions of indicated genes, analyzed by using the Cistrome Data Browser and UCSC genome browser. Moreover, BAP1 and H2AK119Ub enrichment at promoters of these genes were detected by ChIP-qPCR in ASXL1+/N590 knockout K562 cells and FOXK1 and/or FOXK2 knockout K562 pools. IgG was included as negative control for ChIP-qPCR. According to ChIP-Seq retrieved from the GTRD database (https://gtrd.biouml.org/), CMTM7 is not a direct target gene of FOXK1 and FOXK2, although BAP1 is enriched at the promoter region of CMTM7. This gene thus is included as a negative control. Asterisks denote statistical significance with two-tailed Student’s t-test or one-way ANOVA (B–F). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 for the indicated comparison; ns = not significant
Fig 2: LINC00586 epigenetically silenced ASXL1 transcription through LSD1-mediated H3K4me2 demethylation. (A) Relative LINC00586 expression levels in nuclear and cytosolic fractions of HCTl16 and LoVo cells were determined by real-time qPCR. Nuclear controls: U6; Cytosolic controls: GAPDH. (B) RIP assay was performed to determine LINC00586 binding with LSD1. A significant enrichment of endogenous LINC00586 was detected in the anti-LSD1 RIP fraction (relative to nonspecific IgG control) in HCTl16 and LoVo cells. (C) HCTl16 and LoVo cells were subject to ChIP-PCR assays using anti-LSD1, anti-H3K4me2 or nonspecific IgG control for ASXL1 enrichment. (D) ChIP-PCR assays of ASXL1 enrichment in HCTl16 and LoVo cells following LSD1 knockdown and/or LINC00586 overexpression. *p < 0.05 by unpaired t test or by one-way ANOVA with Tukey’s test.
Fig 3: High expression of LINC00586 inhibits the expression of ASXL1 in CRC cells. (A) The mRNA and protein expression of ASXL1 was analyzed by real-time qPCR and immunoblotting analysis in CRC tissues (n = 54) and surrounding non-neoplastic mucosa (n = 54). (B) The mRNA and protein expression of ASXL1 was analyzed by real-time qPCR and immunoblotting analysis in a panel of CRC cell lines (HCTl16, LoVo, HT-29, SW480, and SW620) and FHC. (C,D), The mRNA and protein expression of ASXL1 was analyzed by real-time qPCR (C) and immunoblotting analysis (D) in HCTl16 and LoVo cells upon LINC00586 knockdown or overexpression. *p < 0.05 by unpaired t test or by one-way ANOVA with Tukey’s test [only for (B)].
Fig 4: Tumor-derived ASXL1 mutants promote leukemia cell growth through increased cell cycle progression and decreased apoptosis. (A and B) Cell proliferation of ASXL1+/N590 and ASXL1+/- K562 clones, as well as ASXL1+/N646 and ASXL1+/- Kasumi-1 clones under normoxia or hypoxia (1% O2) condition for the indicated time periods. (C) The ratios of G0/G1-phase and G2/M-phase in ASXL1+/N590 and ASXL1+/- clones under hypoxia (1% O2, 24 h). (D) Cell apoptosis and viability in ASXL1+/N590 and ASXL1+/- clones upon serum starvation. The indicated K562 cells were maintained in RPMI-1640 medium without FBS for 48 h. (E) Apoptosis-related markers in ASXL1+/N590 and ASXL1+/- clones upon serum starvation. Cells were cultured as mentioned above in (D). (F) ASXL1+/N590 and ASXL1+/- K562 clones were treated with 2-MeOE2 (HIF-1a inhibitor) for 24 h, and the cytotoxicity was determined by CCK-8 assay as described in the “MATERIALS AND METHODS” section. (G) Working model. C-terminally truncated ASXL1 mutant is expressed at a much higher protein level than wild-type ASXL1, and loses interaction with transcription factors such as FOXK1 and FOXK2, but still interacts with BAP1. Thus, the mutant ASXL1 protein inhibits the interaction between BAP1 and wild-type ASXL1 in a dominant-negative manner, reduces BAP1 enrichment and increases H2AK119 mono-ubiquitination at the promoters of FOXK1/K2 target genes, and impairs the function of BAP1-ASXL1-FOXK1/K2 axis to regulate target genes and leukemia cell growth. Asterisks denote statistical significance with two-tailed Student’s t-test (A, B, C, D, F). *P < 0.05; ***P < 0.001 for the indicated comparison; ns = not significant
Fig 5: Tumor-derived C-terminally truncated ASXL1 mutants compete with wild-type ASXL1 for binding with BAP1. (A) AML-derived mutations in ASXL1 gene include frameshift mutations (in red), nonsense (in blue), and missense mutations (in green), modified from a previous study by Schnittger et al. NLS, nuclear localization signal; HARE-HTH, HB1, ASXL, restriction endonuclease Helix-Turn-Helix domain; DEUBAD, DEUBiquitinase adaptor domain; PHD, plant homedomain finger. (B) ASXL1 mutation status in K562 and Kasumi-1 cells, and schematic representation of wild-type ASXL1, Y591X and G646Wfs*12 mutations, and the antibody specific to the N-terminal sequence of ASXL1 protein. See also Fig. S1A and S1B. (C) Western blot analysis of the endogenous proteins of full-length and C-terminally truncated mutant ASXL1 in K562 and Kasumi-1 cells. (D) Increased amount of ASXL1N646-Myc were overexpressed in HEK293T cells together with BAP1-Flag, and the interaction between BAP1-Flag and endogenous ASXL1 was determined by Western blot analysis. Relative Flag-BAP1-binding ASXL1 protein levels were quantified. (E) Cells with deletion of mutant ASXL1 allele (referred to as ASXL1+/-) were generated using the CRISPR/Cas9 system in leukemia cells of K562 (left) and Kausmi-1 (right). Co-IP was performed to detect the interaction between BAP1 and ASXL1. (F) Quantification of the protein levels of BAP1-interacting ASXL1 in (E) is shown. (G) Global H2AK119 mono-ubiquitination and H3K27 trimethylation levels in ASXL1+/N590 and ASXL1+/- cells in K562 (left) and ASXL1+/N646 and ASXL1+/- cells in Kausmi-1 (right). Relative H2AK119 mono-ubiquitination was normalized by Histone H2A. Asterisks denote statistical significance with two-tailed Student’s t-test. *P < 0.05 and **P < 0.01 for the indicated comparison
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