Fig. 1

FTO is highly expressed in t(8;21) AML and upregulated by AML1-ETO via PU.1. A qPCR analysis of FTO mRNA expression in bone marrow (BM) cells from 26 patients with de novo t(8;21) AML and 7 normal controls (NC). B Comparison of FTO mRNA expression in BM cells from the 26 de novo t(8;21) patients with or without primary refractory AML using qPCR assay. C FTO mRNA expression in different stages of disease, including at diagnosis and achieving first CR, in the BM samples from 6 t(8;21) AML patients with or without c-KIT mutations by qPCR assay. D Comparison of event-free survival of patients with t(8;21) AML (n = 26) using the Kaplan–Meier method grouped by the levels of expression of FTO (high vs. low). E Pearson correlation of the FTO mRNA expression and AML1-ETO in BM cells from 26 patients with de novo t(8;21) AML. F, G Decreased FTO expression after knockdown of AML1-ETO (siAE) in SKNO-1 and Kasumi-1 cells detected by qPCR (F) and western blotting (G). H ChIP-seq of GSE65427 depicting the loci of SPI1 in Kasumi-1 cells. The blue rectangle reveals a colocalization of AML1 (N-terminus) and ETO (C-terminus) targeting peaks, which represents AML1-ETO peaks, on the promoter of SPI1 in Kasumi-1 cells. ChIP-seq peak regions were called using MACS at a p value cut-off of 10e−8 (Kasumi-1_ETO_C-terminus) or 10e−5 (Kasumi-1_AML1_N-terminus). I ChIP-qPCR assays showing the direct binding of AML1 and ETO within 200 bp upstream of the SPI1 promoter in SKNO-1 and Kasumi-1 cells. The location of targeted amplified region (named ‘Target’) and negative control site (NC) are shown in detail in Additional file 1: Fig. S1D. J Pearson correlation of SPI1 with the expression of AML1-ETO in BM cells from 26 patients with de novo t(8;21) AML. K, L qPCR (K) and western blotting (L) showing the increased expression of PU.1 after AML1-ETO knockdown (siAE) in SKNO-1 and Kasumi-1 cells. M Pearson correlation of SPI1 with the expression of FTO in BM cells from 26 patients with de novo t(8;21) AML. N, O Decreased expression of FTO after forced expression of PU.1 in SKNO-1 and Kasumi-1 cells detected by qPCR (N) and western blotting (O). P Schematic diagrams of the PU.1 binding sites along the FTO promoter. Putative PU.1 binding sites were predicted by JASPER and are indicated with red vertical arrows. Blue horizontal lines indicate the amplified regions for ChIP-qPCR in Q. The lower panel indicates 4 FTO promoter fragments containing sites 1, 2, or 3 on the luciferase reporter vector. NC, negative control site. Q ChIP-qPCR assays showing the direct binding of PU.1 within 1 kb upstream of the FTO promoter in SKNO-1 and Kasumi-1 cells. The 5 qPCR amplified regions containing PU.1 putative binding sites predicted by JASPAR (S1–S5) and negative control site (NC) are shown in detail in P. R The effect of increasing amounts (50, 100, and 200 ng) of pcDNA3.0-PU.1-CDS on the relative luciferase activity of pGL3-basic vector containing different lengths of the FTO promoter (P1–P4, see P and Additional file 1: Fig. S1I), as analyzed by dual luciferase reporter assays