American Society of Hematology

Figure 7

$Figure 7. Hoxa9 association with enhanceosomes is associated with coactivator recruitment and histone acetylation. (A) Coimmunoprecipitations performed on Hoxa9/Meis1 transformed cells show that Meis1, Stat5, C/ebpα, and Creb1 immunoprecipitate with Hoxa9. Nuclease-treated extracts from BM cells stably transduced with either TAPTAG-Hoxa9/Meis1 (HM1) or HA-Hoxa9/FLAG-Meis1 (HM2) were immunoprecipitated (IP) with anti-HA Affinity Matrix (Roche Applied Science) or anti-FLAG M2 Agarose (Sigma-Aldrich). Proteins were separated by SDS-PAGE and detected by Western blot (WB) analysis. Meis1, Stat5, C/ebpα, and Creb1 coelute with Hoxa9, whereas Hoxa9, C/ebpα, and Creb1, but not Stat5, coimmunoprecipitate with Meis1. (B) Hoxa9 association is closely correlated with association of other enhanceosome components and histone acetylation at representative sites in Cd34, Flt3, and Dnajc10 loci. ChIP experiments were performed with anti-HA Meis1, C/ebpα, Stat5, P300, and histone H3 pan acetyl Abs on a HA epitope-tagged Hoxa9-ER/Meis1-transformed myeloblastic cell line (HM4) as described in “Experimental procedures.” Green bars represent the PCR signal as a percent of input for ChIP on cells cultured for 96 hours in the presence of 4-OHT, and yellow bars represent ratio for cells cultured for 96 hours in the absence of 4-OHT with the exception of C/ebpα, which was cultured for 168 hours. Two control regions not determined to be bound by ChIP-Seq are shown. Additional data are shown in supplemental Figure 6. (C) Model for Hoxa9 regulation of enhanceosome activity. Hoxa9 interacts with enhanceosomes containing as a result of homeodomain domain–DNA interactions, association with Meis1 (Pbx proteins are not shown but may further enhance binding) and direct or indirect interactions with C/ebpα, Creb1 and Stat5a/b. Binding of Hoxa9 promotes P300/CBP recruitment through the Meis1 C terminal domain and potentially other interactions with enhanceosome-associated TFs. A variety of oncogenic alterations, including MLL fusion proteins, CDX2 or CDX4 overexpression, NPMc or NUP98 fusion proteins enforce high level Hoxa9 expression, making enhanceosome coactivator activity refractory to physiologic differentiation signals. The resulting persistent expression of proliferative target genes such as Flt3, Sox4, Lmo2 and Myb leads to leukemic transformation.$

Hoxa9 association with enhanceosomes is associated with coactivator recruitment and histone acetylation. (A) Coimmunoprecipitations performed on Hoxa9/Meis1 transformed cells show that Meis1, Stat5, C/ebpα, and Creb1 immunoprecipitate with Hoxa9. Nuclease-treated extracts from BM cells stably transduced with either TAPTAG-Hoxa9/Meis1 (HM1) or HA-Hoxa9/FLAG-Meis1 (HM2) were immunoprecipitated (IP) with anti-HA Affinity Matrix (Roche Applied Science) or anti-FLAG M2 Agarose (Sigma-Aldrich). Proteins were separated by SDS-PAGE and detected by Western blot (WB) analysis. Meis1, Stat5, C/ebpα, and Creb1 coelute with Hoxa9, whereas Hoxa9, C/ebpα, and Creb1, but not Stat5, coimmunoprecipitate with Meis1. (B) Hoxa9 association is closely correlated with association of other enhanceosome components and histone acetylation at representative sites in Cd34, Flt3, and Dnajc10 loci. ChIP experiments were performed with anti-HA Meis1, C/ebpα, Stat5, P300, and histone H3 pan acetyl Abs on a HA epitope-tagged Hoxa9-ER/Meis1-transformed myeloblastic cell line (HM4) as described in “Experimental procedures.” Green bars represent the PCR signal as a percent of input for ChIP on cells cultured for 96 hours in the presence of 4-OHT, and yellow bars represent ratio for cells cultured for 96 hours in the absence of 4-OHT with the exception of C/ebpα, which was cultured for 168 hours. Two control regions not determined to be bound by ChIP-Seq are shown. Additional data are shown in supplemental Figure 6. (C) Model for Hoxa9 regulation of enhanceosome activity. Hoxa9 interacts with enhanceosomes containing as a result of homeodomain domain–DNA interactions, association with Meis1 (Pbx proteins are not shown but may further enhance binding) and direct or indirect interactions with C/ebpα, Creb1 and Stat5a/b. Binding of Hoxa9 promotes P300/CBP recruitment through the Meis1 C terminal domain and potentially other interactions with enhanceosome-associated TFs. A variety of oncogenic alterations, including MLL fusion proteins, CDX2 or CDX4 overexpression, NPMc or NUP98 fusion proteins enforce high level Hoxa9 expression, making enhanceosome coactivator activity refractory to physiologic differentiation signals. The resulting persistent expression of proliferative target genes such as Flt3, Sox4, Lmo2 and Myb leads to leukemic transformation.

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