Figure 1
From: The molecular pathogenesis of the NUP98-HOXA9 fusion protein in acute myeloid leukemia
NUP98-HOXA9 binds to enhancers of genes related to leukemogenesis (a) Venn diagrams of NHA9, HOXA9 and NUP98 target genes identified by ChIP-seq experiments on HEK293FT human models and located within +5/−5 kb of an annotated Transcrption Start Site (TSS). Significant ChIP-seq peaks were established at FDR⩽5%. (b) H3K4me1 qChIP fold enrichment in the selected NHA9 target regions using anti-H3K4me1 antibody. The MEIS1 promoter region was used as a negative control. The average of three experiments is shown. Error bars represent s.e.m. (c) NHA9 qChIP fold enrichment on the eight selected NHA9 target enhancer regions using anti-FLAG antibody in the NHA9-expressing hHP cellular model. The average of three experiments is shown. Error bars represent s.e.m. (d) Luciferase assay was performed to analyze the role of NHA9 in regulating the expression of HOXA9, PBX3 and MEIS1. The luciferase constructs containing the enhancer region (using pGL3-Promoter vector, Promega Biotech Ibérica S.L) of HOXA9, PBX3 and MEIS1 were co-transfected into HEK293FT cells with the expression vector pMSCV-NHA9, together with Renilla vector for the purpose of normalization. Luciferase activity was determined 48 h after reporter plasmid transfection in all cases. A significant increase in luciferase activity induced by NHA9 expression was observed in each case, confirming a direct increase of MEIS1, HOXA9 and PBX3 expression through NHA9 interaction with their corresponding enhancer regions. Data are presented as the mean value from two separate experiments with n=3 for each experiment. Error bars represent s.e.m. (e) Expression analysis by qRT-PCR of MEIS1, HOXA9 and PBX3 in the NHA9-expressing hHP cellular model. The expression of the endogenous human housekeeping gene GAPDH was used to normalize the data, which are expressed as the mean of 2−ΔCt values obtained for each sample after normalization based on the hHP-empty vector model. (f) Analysis of the hHP-NHA9 response to HXR9 and CXR9 (control) peptides. hHP-NHA9 cells were plated in 96-well plates in triplicate and exposed to 13 μM of HXR9/CXR9. Cell viability was assessed at different time points. Average normalized optical density (OD) values of three independent experiments are shown. Statistical significance for relative enrichment and proliferation was determined at P<0.05 (*), P<0.01 (**) and P<0.001 (***), using a t-test with Bonferroni correction. N.S corresponds to non-significant comparisons. Error bars represent s.e.m.
