The NHR1 domain of A-E interacts with p300, which potentiates its transcriptional activating properties. (A) The NHR1 domain is required for the interaction of A-E with p300. An antibody to Flag was used for immunoprecipitation (IP); antibodies to p300 or Flag were used for Western blotting (WB). (B) AE9a also interacts with p300. An antibody to hemagglutinin (HA) was used for immunoprecipitation; antibodies to p300 or HA were used for Western blotting. (C) Knockdown of p300 decreased the expression of A-E–activated target genes. Kasumi-1 cells were transduced with shRNAs against p300 or a control shRNA. Quantitative polymerase chain reaction (qPCR) was performed to quantify the level of target gene expression (± SD; n = 3). (D) A-E and p300 colocalize on some A-E up-regulated genes, as shown by ChIP-seq assays. Representative examples of A-E and p300 co-occupancy are shown as custom tracks in the UCSC genome browser. The locations of the Runx1 consensus binding sites are shown by green lines on the x axis. DNA binding was also analyzed by qPCR amplification of the regions indicated by the red and green arrows on the x axis (± SD; n = 3).

Inhibition of p300 or lysine acetylation blocks leukemogenesis by inhibiting cell growth of A-E–activated target genes. (A) Lys-CoA-Tat (50 μM) inhibited the growth of primary t(8;21)⁺ leukemia cells and Kasumi-1 cells (± SD; n = 3) but not HL-60 cells or normal human CD34⁺ HSPCs. (B) Lys-CoA-Tat (50 μM) blocks A-E Lys⁴³ and histone H3 acetylation in Kasumi-1 cells, as detected by antibodies to acetyl A-E Lys⁴³ or to histone H3. (C) The binding of TAF_II250 and TAF7 to A-E peptides that contain acetylated or unacetylated Lys²⁴ or Lys⁴³ was studied using a peptide pull-down assay. Antibodies to TAF_II250 or TAF7 were used for Western blotting. (D) A hypothetical model showing how p300, and acetylation of A-E by p300, might cooperate to induce leukemia.

The enhanced self-renewal capacity and transcriptional activation induced by A-E in human HSPCs requires the NHR1 domain. (A) The NHR1 domain (amino acids 245 to 436) is required for A-E to increase CAFC formation by human CD34⁺ cells transduced with the indicated retroviral vector. The number of cobblestone areas at week 5 is shown (± SD; n = 3). (B) Deletion of NHR1 affects the ability of A-E to promote self-renewal in liquid culture. CD34 expression was examined weekly in MIGR1-, A-E–, and A-E ΔNHR1–transduced human CD34⁺ cells (± SD; n = 3). (C) M-CSFR, GM-CSF, and IL-3 promoter activity was examined in cells transfected with MIGR1, A-E, or A-E ΔNHR1 (± SD; n = 3). All values were standardized to the level of Renilla luciferase activity.

The acetylation of A-E Lys⁴³ by p300 is required for A-E–induced leukemogenesis. (A) Acetylation of Flag-tagged A-E by p300. Antibodies to Flag or to acetyl lysine were used for immunoprecipitation or Western blotting. (B) The ability of A-E to bind p300 is required for its lysine acetylation. Antibodies to Flag or to acetyl A-E Lys²⁴ or Lys⁴³ were used for immunoprecipitation or Western blotting. (C) Lethally irradiated recipient mice were injected with HSPCs transduced with AE9a or AE9a ΔNHR1. The number of GFP⁺ c-kit⁺ cells in the peripheral blood is shown 15 weeks after transplant. (D) Survival of mice receiving AE9a-transduced or AE9a mutant–transduced HSPCs (P < 0.0001, n = 15 per group). (E) Acetylation of A-E Lys⁴³ was detected in blast cells from t(8;21)⁺ leukemia patients. Antibodies to ETO, acetyl A-E Lys⁴³, or AML1 were used for immunoprecipitation or Western blotting (29).