Figure 2.
RUNX1-ETO and its truncated variant, AE9a, found in t(8:21) AML demonstrate dominant-negative effects on ADAR2 transcription. (A) Schematic diagram of the RUNX1 binding sites along the 4 kb region upstream of the TSS of the ADAR2 gene. The numbers below the double line indicate the nucleotide position with respect to the ADAR2 TSS. Black bars indicate 4 putative RUNX1 binding sites (TGTGGT) centered at positions −3558, −3151, −2945, and −1369 base pairs relative to the TSS. Black arrows indicate the locations of primers used for chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (PCR). Notably, the primers were designed to amplify R1, R2, and R3 regions which cover site 1, sites 2 and 3, and site 4, respectively. (B) ChIP-qPCR analysis of RUNX1 or RUNX1-ETO protein binding to the indicated ADAR2 regulatory region (R1, R2, or R3) in Kasumi-1 cells. Anti-RUNX1 or anti-ETO antibodies were used to pull down RUNX1 or RUNX1-ETO, respectively. Immunoglobulin G (IgG) was used as a negative control. Notably, the anti-RUNX1 antibody recognizes the 200 to 300 amino acids of RUNX1 protein and only recognizes RUNX1, whereas the anti-ETO antibody was used for immunoprecipitation to specifically pull down RUNX1-ETO in Kasumi-1 cells, which do not express WT ETO protein. Data were presented as the mean ± standard deviation (SD) of technical triplicates from a representative experiment. (C) Luciferase activities associated with each of the indicated sequences upstream of the TSS of ADAR2. HEK293T cells were transfected with each of the indicated reporter constructs containing fragment A1, A2, A3, or A4. S1, S2, S3, and S4 refer to RUNX binding sites 1, 2, 3, and 4, respectively. Relative luciferase activity represents firefly luciferase activity normalized against the internal control Renilla luciferase, calculated as fold difference against the activity of the pGL3 EV. Data were presented as the mean ± SD of 3 independent experiments (∗∗∗∗P < .0001 using two-tailed Student t test.). (D) Luciferase activities associated with the A1 fragment in HEK293T cells (panel C) cotransfected with either RUNX1 alone or RUNX1 together with RUNX1-ETO or AE9a (RUNX1, RUNX1 + RE, or RUNX1 + AE9a, respectively). Luciferase activity was normalized to the A1 construct coexpressed with EV control and defined as Luciferase activity (fold). Data were presented as the mean ± SD of 3 independent experiments. (E) Similar to panel D, except that the A2 luciferase construct (panel C) was used. (F) Bar chart showing the relative expression of RUNX1, RUNX1-ETO/AE9a, and ADAR2 expression in Kasumi-1 cells, upon short hairpin RNA (shRNA)–mediated knockdown of RUNX1 (shRUNX1-1 and shRUNX1-2) or RUNX1-ETO/AE9a (shRE-1 and shRE-2). The relative expression was calculated as described in supplemental Materials and Methods. Data were presented as mean ± SD of 3 independent experiments (∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 using two-tailed Student t test).

RUNX1-ETO and its truncated variant, AE9a, found in t(8:21) AML demonstrate dominant-negative effects on ADAR2 transcription. (A) Schematic diagram of the RUNX1 binding sites along the 4 kb region upstream of the TSS of the ADAR2 gene. The numbers below the double line indicate the nucleotide position with respect to the ADAR2 TSS. Black bars indicate 4 putative RUNX1 binding sites (TGTGGT) centered at positions −3558, −3151, −2945, and −1369 base pairs relative to the TSS. Black arrows indicate the locations of primers used for chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (PCR). Notably, the primers were designed to amplify R1, R2, and R3 regions which cover site 1, sites 2 and 3, and site 4, respectively. (B) ChIP-qPCR analysis of RUNX1 or RUNX1-ETO protein binding to the indicated ADAR2 regulatory region (R1, R2, or R3) in Kasumi-1 cells. Anti-RUNX1 or anti-ETO antibodies were used to pull down RUNX1 or RUNX1-ETO, respectively. Immunoglobulin G (IgG) was used as a negative control. Notably, the anti-RUNX1 antibody recognizes the 200 to 300 amino acids of RUNX1 protein and only recognizes RUNX1, whereas the anti-ETO antibody was used for immunoprecipitation to specifically pull down RUNX1-ETO in Kasumi-1 cells, which do not express WT ETO protein. Data were presented as the mean ± standard deviation (SD) of technical triplicates from a representative experiment. (C) Luciferase activities associated with each of the indicated sequences upstream of the TSS of ADAR2. HEK293T cells were transfected with each of the indicated reporter constructs containing fragment A1, A2, A3, or A4. S1, S2, S3, and S4 refer to RUNX binding sites 1, 2, 3, and 4, respectively. Relative luciferase activity represents firefly luciferase activity normalized against the internal control Renilla luciferase, calculated as fold difference against the activity of the pGL3 EV. Data were presented as the mean ± SD of 3 independent experiments (∗∗∗∗P < .0001 using two-tailed Student t test.). (D) Luciferase activities associated with the A1 fragment in HEK293T cells (panel C) cotransfected with either RUNX1 alone or RUNX1 together with RUNX1-ETO or AE9a (RUNX1, RUNX1 + RE, or RUNX1 + AE9a, respectively). Luciferase activity was normalized to the A1 construct coexpressed with EV control and defined as Luciferase activity (fold). Data were presented as the mean ± SD of 3 independent experiments. (E) Similar to panel D, except that the A2 luciferase construct (panel C) was used. (F) Bar chart showing the relative expression of RUNX1, RUNX1-ETO/AE9a, and ADAR2 expression in Kasumi-1 cells, upon short hairpin RNA (shRNA)–mediated knockdown of RUNX1 (shRUNX1-1 and shRUNX1-2) or RUNX1-ETO/AE9a (shRE-1 and shRE-2). The relative expression was calculated as described in supplemental Materials and Methods. Data were presented as mean ± SD of 3 independent experiments (∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 using two-tailed Student t test).

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