Page 112 - 2019_04-Haematologica-web

R. Polak et al.
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Figure 2. ETV6-RUNX1 and ETV6-RUNX1 target genes enhance Vps34 promoter activity. (A) The UCSC genome browser (GRCh37/hg19) was used to analyze the transcription factors that bind to the Vps34 promoter. Several transcription factors known to play an important role in regulation of hematopoiesis were found to be interacting with this promoter region. Using publically available ChIP-seq data, the interaction of the transcription factors RUNX1, GATA1, GATA2, EGR1 and HEY1 with the Vps34 promoter is shown. (B) Umbilical cord blood-derived CD34+ cells were retrovirally transduced to ectopically express the ETV6-RUNX1 fusion protein. Gene expression analysis was performed 20 and 40 hours (hr) after transduction. For flow chart; see left panel. The gene expression levels of GATA1, GATA2, EGR1 and HEY1 are shown. P-values represent the differences between ETV6-RUNX1 positive (green bars) compared to ETV6-RUNX1-negative (white bars) CB-CD34+ cells (FDR-adjusted). (C) (Top) Schematic representation of the -1383 to +58 region of the Vps34 promoter cloned upstream of the Gaussia luciferase gene. Bar graphs: HEK293T cells were co-transfected with a Vps34 promoter construct, RUNX1 and/or CBFβ (left), increasing concentrations of GATA1 and GATA2 (middle) or HEY1 and EGR1 (right) expression constructs. Luciferase activity was determined 48 hr after transfection. eGFP expression was quantified in each sample by flow cyto- metric analysis and used to normalize luciferase activity. Data were depicted as fold induction compared to empty vector controls. Error bars represent Standard Error of Mean. *P≤0.05, **P≤0.01, ***P≤0.001. See also Online Supplementary Figure S2A-C.
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haematologica | 2019; 104(4)