Figure 2
From: The molecular pathogenesis of the NUP98-HOXA9 fusion protein in acute myeloid leukemia
NUP98-HOXA9 has an activator-repressor role in transcriptional regulation driven by p300 and HDAC1 interactions. (a) We applied gene set enrichment analysis (GSEA) to test for enrichment of NHA9 ChIP-seq target gene set among differentially expressed genes using expression array data from hHP-NHA9 cellular model (left panel) and five NHA9 primary samples (right panel). Genes were ranked based on the limma-moderated t statistic. After Kolmogorov–Smirnoff testing, those gene sets with FDR <0.25, a well-established cutoff for the identification of biologically relevant gene sets, were considered enriched (b) Analysis of NHA9 and p300/HDAC1 interactions by co-immunoprecipitation. HEK293FT cells were transfected with pMSCV-NUP98-HOXA9 or pMSCV-empty vectors. Forty-eight hours post-transfection, the immunoprecitpitation was performed using anti-p300 and anti-HDAC1 antibodies and the proteins were analyze by immunoblotting using anti-FLAG antibody. Endogenous GAPDH protein levels were used as a loading control. (c, d) qChIP fold enrichment of p300 and HDAC1 in the regulatory regions of four upregulated (c) and four downregulated (d) target genes of NHA9. The average of three experiments showed the binding, along with the fusion protein, of p300 and HDAC1 to the regulatory regions of the overexpressed and downregulated NHA9 target genes, respectively. (e) Analysis of the hHP-NHA9 response to HDAC inhibitors. Cells were exposed for 72 h to serial dilutions of panobinostat (LBH589) followed by the addition of WST-1 to assess cell viability. The average normalized optical density (OD) values are shown compared to vehicle. 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.
