Figure 6
Figure 6. Runx1 binds and activates transcription via a 450-bp conserved 37 kb Cebpa enhancer. (A) Comparison of the murine and human Cebpa genomic loci identifies 8 regions of homology upstream of the single Cebpa exon (top). Alignment of a 453-bp region from 37 kb of the murine Cebpa locus (M) with a related region from 41 kb in the human CEBPA locus (H) is also shown. The 4 conserved Runx1 sites (R1-R4) are indicated in bold (bottom). (B) Double-stranded DNA probes containing sites R1-R4, or mutant variants in which the core Runx1 consensus 5′-ACCACA was mutated to 5′-TGCACA, were radiolabeled and subjected to gel shift analysis with the use of nuclear extracts from 293T cells transduced with empty CMV vector (−) or with CMV-RUNX1c and CMV-CBFβ (RX1, left panels). Radiolabeled sites R1-R4 were also subjected to gel shift analysis alone or in the presence of 5- or 25-fold excess unlabeled WT or mutant oligonucleotides (right panels). (C) 32Dcl3 cells were subjected to ChIP with the use of 2 μg of rabbit anti-Runx1 antiserum or normal rabbit IgG, followed by genomic DNA PCR with the use of oligonucleotides centered between R1 and R2 or surrounding R3 and R4, at −2.5 kb of the Cebpa promoter, or within the β-actin promoter. Binding was quantified relative to input, and this value was set to 1.0 for ChIP with IgG on the R1-R2 region of the Cebpa enhancer (panel 1). NIH 3T3 were subjected to ChIP with the use of 2 μg of Runx1 antiserum, followed by enhancer or promoter PCR (panel 2). 32Dcl3 cells were also subjected to ChIP with 0.5 μg of H3K4me1 or 1 μg of p300 antisera (panels 3 and 4). Data are mean and SE of 3 determinations. (D) 32Dcl3 cells were transduced with 5 μg of luciferase reporters containing the murine Cebpa promoter alone (Prom), the promoter with the conserved 37 kb region positioned upstream (Enh+Prom), or the later construct harboring either mutation of the promoter Runx1 site 1 (Enh+mProm) or mutation of sites R1-R4 in the enhancer region (mEnh+Prom), 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 to CMV-βGal activity is shown, with activity of CEBPA(Prom)–LUC set to 1.0 in each experiment (mean of SE of 3 determinations, each done in triplicate).

Runx1 binds and activates transcription via a 450-bp conserved 37 kb Cebpa enhancer. (A) Comparison of the murine and human Cebpa genomic loci identifies 8 regions of homology upstream of the single Cebpa exon (top). Alignment of a 453-bp region from 37 kb of the murine Cebpa locus (M) with a related region from 41 kb in the human CEBPA locus (H) is also shown. The 4 conserved Runx1 sites (R1-R4) are indicated in bold (bottom). (B) Double-stranded DNA probes containing sites R1-R4, or mutant variants in which the core Runx1 consensus 5′-ACCACA was mutated to 5′-TGCACA, were radiolabeled and subjected to gel shift analysis with the use of nuclear extracts from 293T cells transduced with empty CMV vector (−) or with CMV-RUNX1c and CMV-CBFβ (RX1, left panels). Radiolabeled sites R1-R4 were also subjected to gel shift analysis alone or in the presence of 5- or 25-fold excess unlabeled WT or mutant oligonucleotides (right panels). (C) 32Dcl3 cells were subjected to ChIP with the use of 2 μg of rabbit anti-Runx1 antiserum or normal rabbit IgG, followed by genomic DNA PCR with the use of oligonucleotides centered between R1 and R2 or surrounding R3 and R4, at −2.5 kb of the Cebpa promoter, or within the β-actin promoter. Binding was quantified relative to input, and this value was set to 1.0 for ChIP with IgG on the R1-R2 region of the Cebpa enhancer (panel 1). NIH 3T3 were subjected to ChIP with the use of 2 μg of Runx1 antiserum, followed by enhancer or promoter PCR (panel 2). 32Dcl3 cells were also subjected to ChIP with 0.5 μg of H3K4me1 or 1 μg of p300 antisera (panels 3 and 4). Data are mean and SE of 3 determinations. (D) 32Dcl3 cells were transduced with 5 μg of luciferase reporters containing the murine Cebpa promoter alone (Prom), the promoter with the conserved 37 kb region positioned upstream (Enh+Prom), or the later construct harboring either mutation of the promoter Runx1 site 1 (Enh+mProm) or mutation of sites R1-R4 in the enhancer region (mEnh+Prom), 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 to CMV-βGal activity is shown, with activity of CEBPA(Prom)–LUC set to 1.0 in each experiment (mean of SE of 3 determinations, each done in triplicate).

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