Fig 1: UBXN8 inhibits leukemic growth and reprograms myeloid differentiation in vivo.a Schematic of the experimental design of the xenograft mouse model. b Images of two mice and their excised tumors from the UBXN8 and control groups at the end of the experiment. c Tumor volumes of the two groups (Control: n = 5, UBXN8: n = 5) were measured at the indicated days during the experiment. d The weights of the tumors from the two groups (Control: n = 5, UBXN8: n = 5) were measured at the end of the experiment. e Ectopic expression of UBXN8 significantly increased the expression of CD11b in vivo as examined by immunohistochemical analysis (upper panel: scale bar 100 μm, 10X; lower panel: scale bar 50 μm, 40X). f Schematic model for the epigenetic silencing of the UBXN8 gene by RUNX1-RUNX1T1. In myeloid precursor cells, the occupancy of the RUNX1-binding site (GCATGCGGGTT) on the UBXN8 promoter region by RUNX1 is associated with a chromatin status permissive for transcription. The RUNX1-RUNX1T1 oncoprotein targets this binding site, where it aberrantly recruits DNMTs and methylates CpGs. This induces chromatin packaging that is nonpermissive for UBXN8 gene transcription, which leads to the inhibition of the differentiation of RUNX1-RUNX1T1+ myeloid precursors and consequently promotes myeloid proliferation. Black and white circles indicate methylated and unmethylated CpG dinucleotides, respectively. The numbers indicate the position of the nucleotides relative to the TSS of UBXN8 ( + 1).
Fig 2: The RUNX1-RUNX1T1 fusion protein acts on the RUNX1-binding site in the UBXN8 promoter region and triggers the epigenetic silencing of UBXN8.a Schematic of the RUNX1-binding sites (nt –1370 to –1360, nt –921 to –911) along the UBXN8 gene promoter region. The numbers refer to the nucleotide positions relative to the 5′ end of the TSS of UBXN8 ( + 1). Arrows indicate the location of the primers used in the ChIP assay. b Human 293T cells were transiently cotransfected with luciferase reporter vectors pGL3-LUC containing the sequence of the regulatory regions (shown in Fig. 4a) and increasing amounts of pcDNA3.0 vectors with or without the RUNX1-RUNX1T1-binding sites. Dual-luciferase reporter assays were performed to detect the transcriptional activity of RUNX1-RUNX1T1 on the UBXN8 promoter region. c, d Chromatin was immunoprecipitated using the indicated antibodies or IgG. qRT-PCR analysis was performed to amplify the region in UBXN8 containing the predicted RUNX1-binding site with oligo 1 primers and the distal region lacking the RUNX1 site with oligo 2 primers (shown in Fig. 4a). Amplification of GAPDH was used as a control. Error bars indicate the SD from three different assays (**P < 0.01, ***P < 0.001).
Fig 3: The UBXN8 promoter region is highly methylated in RUNX1-RUNX1T1+ cell lines.a Methylation level at the UBXN8 gene promoter region (–1500 to –1 bp, TSS up 1.5 kb) as analyzed by MCC-Seq was obviously higher in SKNO-1 cells than in SKNO-siAE cells. b Schematic representation of the distribution of the CpG dinucleotides along the UBXN8 promoter region (–1500 to –1 bp, TSS up 1.5 kb). The horizontal line (F-R) indicates the location of the primers used in the BSP assays. c A genomic BSP assay was performed to profile the methylation pattern of the CpG dinucleotides located along the RUNX1-binding site. Black circles and empty circles denote methylated and unmethylated CpG dinucleotides, respectively. d The mRNA and protein expression of UBXN8 was determined in cells treated with 1.0 µM DAC. Error bars indicate the SD from three different assays.
Fig 4: UBXN8 expression is selectively downregulated in RUNX1-RUNX1T1+ AML cell lines.a UBXN8 expression was downregulated in RUNX1-RUNX1T1+ cell lines (U-937AE, SKNO-1 and Kasumi-1 cell lines) compared to monocytes isolated from the two sets of normal bone marrow (NBM) and RUNX1-RUNX1T1- cell lines (U-937, SKNO-siAE, HL-60, THP-1 and MV4-11). Error bars indicate the standard deviation (SD) from three different assays. b UBXN8 protein expression in AML cell lines was measured by Western blotting. c Expression of UBXN8 in monocytes isolated from NBM and different AML subtypes from the BloodSpot database was analyzed. UBXN8 expression was significantly downregulated in t(8;21) AML compared with normal bone marrow monocytes and other AML subtypes. d UBXN8 mRNA expression was plotted against that of RUNX1-RUNX1T1 in t(8;21) AML (n = 60) (GSE13159). The mRNA expression of UBXN8 versus RUNX1-RUNX1T1 showed a negative relationship. (*P < 0.05, ***P < 0.001).
Fig 5: Altered DNA methylation status in RUNX1-RUNX1T1+/− cell lines.The DNA methylation status in SKNO-1 and SKNO-siAE cells was changed in a large number of genes after treatment with 5-aza-2 deoxycytidine. In addition, the UBXN8 gene was one of genes with an altered DNA methylation status.
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