Figure 2.
Figure 2. STAT1 and STAT5 are recruited to a 3′ distal enhancer of Cebpb that is required for upregulation of Cebpb in response to IFN-α. (A) EML cells transduced with empty (top lane) or BCR-ABL (bottom lane) expression vectors were subjected to ChIP-seq analysis using anti-STAT5 antibody. Panels show recruitment of STAT5 around the Cebpb locus, as displayed by the Integrative Genomics Viewer. Representative data from 2 independent samples are shown. (B) Enrichment of STAT5 activated by BCR-ABL on the enhancer region of Cebpb gene identified in panel A, as confirmed by ChIP-PCR. Enrichment levels of STAT5 are shown as red (promoter region) and blue (enhancer region) bars. Data are means ± standard error (SE) of 3 independent experiments. (C) Enrichment of H3K27Ac in the presence or absence of BCR-ABL to the enhancer region of Cebpb. Data are means ± SE of 3 independent experiments. (D) Effect of CRISPR/dCas9-mediated targeting of STAT5 binding sites on Cebpb mRNA expression in EML cells. The gRNA sequence was designed to target STAT5 binding sites in the distal region, as shown in supplemental Figure 3B. The Cebpb mRNA level was normalized against the corresponding level of Gapdh mRNA. The normalized Cebpb mRNA level in dCas9 (+) gRNA (−) EML cells transduced with MIG-empty was defined as 1.0. mRNA expression levels are shown as red (MIG-empty) or blue (MIG-BCR-ABL) bars. Data are means ± SD of 3 independent experiments. (E) dCas9-mediated repression of the Cebpb enhancer impaired BCR-ABL–induced myeloid differentiation. Percentage of c-kit− CD11b+ myeloid cells in BCR-ABL–expressing EML cells transduced with dCas9 and a vector control for gRNA (upper left) or with dCas9 and a vector expressing gRNA targeting the STAT5 binding sites (lower left). Data are means ± SD of 2 independent experiments (right). Representative flow cytometric data are shown. (F) Chromatin obtained from EML cells treated with IFN-α was subjected to ChIP-PCR using anti-STAT1 (blue bars; top) and anti-STAT5 (blue bars; bottom) antibodies. Normal immunoglobulin G (red bars) was used as a control for the ChIP experiments. Data are means ± SD of 3 independent experiments. (G) Induction of Cebpb mRNA expression by IFN-α in EML cells was significantly impaired by a combination of dCas9 and gRNA targeting the STAT5 binding sites. Cells were treated with 100 U/mL IFN-α for 3 hours. Cebpb mRNA level was normalized against the corresponding level of Gapdh mRNA. Normalized Cebpb mRNA levels are shown relative to the level in untreated dCas9 (+) gRNA (−) cells, shown as red (untreated) or blue (IFN-α–treated) bars. (H) Involvement of STAT1 and STAT5 in IFN-α–mediated upregulation of Cebpb mRNA. EML cells stably expressing BCR-ABL were retrovirally transduced with the indicated dn mutants of STAT1 and STAT5. On day 3, transduced cells were purified and subjected to quantitative RT-PCR before and after a 3-hour treatment with IFN-α. Expression level of Cebpb mRNA in IFN-α–treated cells is shown relative to the level in nontreated cells. Data are means ± SD of 3 independent experiments. *P < .05; **P < .01.

STAT1 and STAT5 are recruited to a 3′ distal enhancer of Cebpb that is required for upregulation of Cebpb in response to IFN-α. (A) EML cells transduced with empty (top lane) or BCR-ABL (bottom lane) expression vectors were subjected to ChIP-seq analysis using anti-STAT5 antibody. Panels show recruitment of STAT5 around the Cebpb locus, as displayed by the Integrative Genomics Viewer. Representative data from 2 independent samples are shown. (B) Enrichment of STAT5 activated by BCR-ABL on the enhancer region of Cebpb gene identified in panel A, as confirmed by ChIP-PCR. Enrichment levels of STAT5 are shown as red (promoter region) and blue (enhancer region) bars. Data are means ± standard error (SE) of 3 independent experiments. (C) Enrichment of H3K27Ac in the presence or absence of BCR-ABL to the enhancer region of Cebpb. Data are means ± SE of 3 independent experiments. (D) Effect of CRISPR/dCas9-mediated targeting of STAT5 binding sites on Cebpb mRNA expression in EML cells. The gRNA sequence was designed to target STAT5 binding sites in the distal region, as shown in supplemental Figure 3B. The Cebpb mRNA level was normalized against the corresponding level of Gapdh mRNA. The normalized Cebpb mRNA level in dCas9 (+) gRNA (−) EML cells transduced with MIG-empty was defined as 1.0. mRNA expression levels are shown as red (MIG-empty) or blue (MIG-BCR-ABL) bars. Data are means ± SD of 3 independent experiments. (E) dCas9-mediated repression of the Cebpb enhancer impaired BCR-ABL–induced myeloid differentiation. Percentage of c-kit− CD11b+ myeloid cells in BCR-ABL–expressing EML cells transduced with dCas9 and a vector control for gRNA (upper left) or with dCas9 and a vector expressing gRNA targeting the STAT5 binding sites (lower left). Data are means ± SD of 2 independent experiments (right). Representative flow cytometric data are shown. (F) Chromatin obtained from EML cells treated with IFN-α was subjected to ChIP-PCR using anti-STAT1 (blue bars; top) and anti-STAT5 (blue bars; bottom) antibodies. Normal immunoglobulin G (red bars) was used as a control for the ChIP experiments. Data are means ± SD of 3 independent experiments. (G) Induction of Cebpb mRNA expression by IFN-α in EML cells was significantly impaired by a combination of dCas9 and gRNA targeting the STAT5 binding sites. Cells were treated with 100 U/mL IFN-α for 3 hours. Cebpb mRNA level was normalized against the corresponding level of Gapdh mRNA. Normalized Cebpb mRNA levels are shown relative to the level in untreated dCas9 (+) gRNA (−) cells, shown as red (untreated) or blue (IFN-α–treated) bars. (H) Involvement of STAT1 and STAT5 in IFN-α–mediated upregulation of Cebpb mRNA. EML cells stably expressing BCR-ABL were retrovirally transduced with the indicated dn mutants of STAT1 and STAT5. On day 3, transduced cells were purified and subjected to quantitative RT-PCR before and after a 3-hour treatment with IFN-α. Expression level of Cebpb mRNA in IFN-α–treated cells is shown relative to the level in nontreated cells. Data are means ± SD of 3 independent experiments. *P < .05; **P < .01.

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