doi: 10.1038/leu.2017.194.
Epub 2017 Jun 20.
The molecular pathogenesis of the NUP98-HOXA9 fusion protein in acute myeloid leukemia
Affiliations
- PMID: 28630438
- PMCID: PMC5596207
- DOI: 10.1038/leu.2017.194
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The molecular pathogenesis of the NUP98-HOXA9 fusion protein in acute myeloid leukemia
Leukemia.
2017 Sep.
No abstract available
Conflict of interest statement
The authors declare no conflict of interest.
Figures
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.
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.
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