Fig 1: AML1-ETO epigenetically activa SETDB2 expression.Notes: (A) Schematic diagrams of the CpG islands along the SETDB2 promoter. Numbers indicate the nucleotides relative to SETDB2 (+1 nt). Vertical lines indicate CpG dinucleotides. Lower panels: A series of constructs and their mutants. (B) Luciferase reporter activities of human 293T cells transiently co-transfected for 48 h with luciferase reporter constructs containing the wild-type sequence of SETDB2 promoter or its mutant counterparts, together with AML1-ETO or mock cDNA. (C) After ChIP using the indicated antibodies or IgG, qRT–PCR was performed to evaluate the specificity of protein binding in the region containing the predicted AML1-binding site. (D) After Kasumi-1 cells were treated with C646 (30 nM) for 24 h, qRT–PCR and Western blot were performed to quantify SETDB2 levels. Expression values are shown as mean ± SEM. *P<0.05.
Fig 2: EP300 stimulates CREB-dependent GPX4 transcription. (A) STRING analysis revealed CREB interacted with methyltransferase and acyltransferase (confidence=0.980). (B) Co-immunoprecipitation experiments performed in A549 cells using indicated antibodies, and further analysis of CREB, EP300 and SETDB2 expression by IB. (C) Schematic diagram shows the domains in CREB and EP300 protein. (D and E) Co-immunoprecipitation experiments performed using anti-HA or anti-CREB in A549 and H1299 cells with indicated CREB or EP300 plasmids overexpressed, and further analysis of Myc and HA levels by IB. (F and G) The enrichments of H3K27Ac, EP300 and CREB at −2 k, CREB motif or 2 k regions of GPX4 promoter were calculated as the percentage of input chromosomal DNA via ChIP using the corresponding antibodies in (F) A549 and (G) H1299 cells with CREB or EP300 overexpressed or knocked down. Anti-IgG was used as the parallel control. (H-K) The enrichments of H3K27Ac, EP300 and CREB at −2 k, CREB motif or 2 k regions of GPX4 promoter were calculated as the percentage of input chromosomal DNA via ChIP using the corresponding antibodies in (H and J) A549 and H1299 (I and K) cells with WT or mutant (H and I) CREB or (J and K) EP300 overexpressed. Anti-IgG was used as the parallel control. (L and M) Cell viability and MDA, respectively, were measured in A549 and H1299 cells treated with erastin (10 µM) with or without ectopically expressed CREB with or without EP300 knockdown. The data are presented as the mean ± SD from three biological replicates (including IB). **P<0.01 indicates statistical significance. Data from F-M were analyzed using a one-way ANOVA followed by Bonferroni's post hoc test. EP300, E1A binding protein P300; CREB, cAMP response element-binding protein; GPX4, glutathione peroxidase 4; SETDB2, SET domain bifurcated histone lysine methyltransferase 2; IB, immunoblotting; ChIP, chromatin immunoprecipitation; WT, wild-type; MDA, MDA, malondialdehyde; sh, short hairpin; Ctrl, control.
Fig 3: Impact of shRNA-mediated knockdown of SETDB2 on AML1-ETO -positive AML cell lines.Notes: (A) After SKNO-1 and Kasumi-1 cells were infected with shRNA lentivirus for 48 h, qRT–PCR and Western blot were performed to quantify SETDB2 levels. (B) Effect of SETDB2 knockdown compared to scramble control on colony formation. (C) Effect of SETDB2 knockdown compared to scramble control on proliferation. Expression values are shown as mean ± SEM. *P<0.05.
Fig 4: SETDB2 knockdown increases sensitivity to epigenetic inhibitors.Notes: Dose-response curves are shown for control or SETDB2 shRNA-treated SKNO-1 and Kasumi-1 cells cultured in increasing concentrations of JQ1 (A) and C646 (B). IC50 for JQ1 in combination with control or SETDB2 shRNA is 197.2 versus 63.36 nM (for SKNO-1 cells) and 84.9 versus 29.78 nM (for Kasumi-1 cells), respectively. IC50 for C646 in combination with control or SETDB2 shRNA is 916.6 versus 244.7 nM (for SKNO-1 cells) and 510.5 versus 154.7 nM (for Kasumi-1 cells), respectively.
Fig 5: The expression of SETDB2 in AML.Notes: (A) Quantification of SETDB2 expression in patients with AML1-ETO-positive AML and normal BM subpopulations by qRT-PCR. (B) Quantification of SETDB2 expression in AML patients with AE, PML-RARa fusions, or Inv(16), and normal human BM CD34+ cells by qRT-PCR. (C) Quantification of SETDB2 expression in AML cell lines by qRT-PCR. (D) Quantification of SETDB2 expression in patients with AML1-ETO -positive AML or AML1-ETO -negative AML by qRT-PCR. (E) Sequential analyses of SETDB2 mRNA levels in mononuclear cells isolated from bone marrow samples of three individual patients with AML1-ETO -positive AML at different stages of disease, including newly diagnosed, remission and relapse. Expression values are shown as mean ± SEM. *P<0.05.
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