Figure 5
Figure 5. Runx1 binds and activates the Cebpa promoter. (A) Gel shift assay was conducted with radiolabeled WT probe from the murine Cebpa promoter containing 2 Runx1 consensus binding sites and nuclear extracts from 293T cells transfected with 6 μg of empty CMV vector (−) or with 3 μg of CMV-CBFβ and 3 μg of CMV-RUNX1c (RX1), in the absence of competitor, with 5- or 25-fold excess unlabeled WT competitor, or with 5- or 25-fold excess M12 competitor mutant in both sites (left panel). Gel shift assay was also conducted after incubating the indicated radiolabeled probes with a 293T cell extract expressing exogenous Runx1 (right panel). Bracket on the left denotes specific Runx1 gel shift complexes. The sequence of the 2 adjacent Runx1 sites and of mutant variants is shown below. (B) 32Dcl3 cells were subjected to ChIP with the use of rabbit anti-Runx1 antiserum or normal rabbit IgG, followed by genomic DNA PCR with the use of oligonucleotides centered at −260 bp (prom.) or −2.5 kb of the Cebpa gene or within the β-actin promoter. Data representative of 4 experiments are shown. Binding was quantified relative to input, and this value was set to 1.0 for ChIP with IgG on the Cebpa promoter. (C) 32Dcl3 cells were transduced with 5 μg of CEBPA-LUC or CEBPA(M1*)–LUC harboring 4-bp changes in the more upstream Runx1 consensus site at −285 bp, together with 0.25 μg of CMV-βGal. Luciferase and β-galactosidase activities were assessed 2 days later. Normalized luciferase activity of each reporter relative CMV-βGal activity is shown, with activity of CEBPA-LUC set to 1.0 in each experiment (mean of SE of 3 determinations, each done in triplicate). (D) 32Dcl3-RUNX1-ER cells proliferating in IL-3 were exposed to 4HT for the indicated times. Total cellular proteins were assessed for C/EBPα, PU.1, or α-tubulin expression by Western blot analysis (left panel), and total cellular RNAs were assessed for Cebpa and Pu.1 mRNA expression, relative to mS16 mRNA (right panel; mean and SE from 3 determinations). (E) 32Dcl3-RUNX1-ER or 32Dcl3-pBabePuro cells were cultured without or with 50 μg/mL cycloheximide for 30 minutes, followed by continued culture with or without 4HT for 6 hours. Total cellular RNAs were then analyzed for Cebpa expression (mean and SE from 3 determinations). (F) 32Dcl3-KRAB-RUNT-ER cells exposed to 4HT for 0, 8, or 24 hours were assessed for C/EBPα and α-tubulin expression by Western blot analysis (left panel) and for Cebpa mRNA expression (right graph; mean and SE from 3 determinations).

Runx1 binds and activates the Cebpa promoter. (A) Gel shift assay was conducted with radiolabeled WT probe from the murine Cebpa promoter containing 2 Runx1 consensus binding sites and nuclear extracts from 293T cells transfected with 6 μg of empty CMV vector (−) or with 3 μg of CMV-CBFβ and 3 μg of CMV-RUNX1c (RX1), in the absence of competitor, with 5- or 25-fold excess unlabeled WT competitor, or with 5- or 25-fold excess M12 competitor mutant in both sites (left panel). Gel shift assay was also conducted after incubating the indicated radiolabeled probes with a 293T cell extract expressing exogenous Runx1 (right panel). Bracket on the left denotes specific Runx1 gel shift complexes. The sequence of the 2 adjacent Runx1 sites and of mutant variants is shown below. (B) 32Dcl3 cells were subjected to ChIP with the use of rabbit anti-Runx1 antiserum or normal rabbit IgG, followed by genomic DNA PCR with the use of oligonucleotides centered at −260 bp (prom.) or −2.5 kb of the Cebpa gene or within the β-actin promoter. Data representative of 4 experiments are shown. Binding was quantified relative to input, and this value was set to 1.0 for ChIP with IgG on the Cebpa promoter. (C) 32Dcl3 cells were transduced with 5 μg of CEBPA-LUC or CEBPA(M1*)–LUC harboring 4-bp changes in the more upstream Runx1 consensus site at −285 bp, together with 0.25 μg of CMV-βGal. Luciferase and β-galactosidase activities were assessed 2 days later. Normalized luciferase activity of each reporter relative CMV-βGal activity is shown, with activity of CEBPA-LUC set to 1.0 in each experiment (mean of SE of 3 determinations, each done in triplicate). (D) 32Dcl3-RUNX1-ER cells proliferating in IL-3 were exposed to 4HT for the indicated times. Total cellular proteins were assessed for C/EBPα, PU.1, or α-tubulin expression by Western blot analysis (left panel), and total cellular RNAs were assessed for Cebpa and Pu.1 mRNA expression, relative to mS16 mRNA (right panel; mean and SE from 3 determinations). (E) 32Dcl3-RUNX1-ER or 32Dcl3-pBabePuro cells were cultured without or with 50 μg/mL cycloheximide for 30 minutes, followed by continued culture with or without 4HT for 6 hours. Total cellular RNAs were then analyzed for Cebpa expression (mean and SE from 3 determinations). (F) 32Dcl3-KRAB-RUNT-ER cells exposed to 4HT for 0, 8, or 24 hours were assessed for C/EBPα and α-tubulin expression by Western blot analysis (left panel) and for Cebpa mRNA expression (right graph; mean and SE from 3 determinations).

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