Figure 7
Figure 7. Bmi1 is a direct downstream target of C/ebpαDN. (A-C) External features of the LDL-EGFP+/−;Cre+/− transgenic embryos without injection (A, uninj), and the LDL- EGFP-C/ebpα+/−;Cre+/− transgenic embryos injected with either unrelated 5-mismatch (B) or bmi1-specifcic morpholino oligonucleotides (C). The circulation congestion is readily detected in the C/ebpαDN-expressing embryos injected with control morpholino (B, arrowheads) but largely rescued by bmi1 knockdown (C, arrowheads). Boxes with dashed lines indicate the regions amplified and shown at the bottom. (D-F) WISH analyses of αE1-globin in the indicated transgenic embryos at 80 hpf. The red arrowheads indicate that the increased number of erythrocytes with expression of αE1-globin transcripts can be corrected to normal level in the LDL-EGFP-C/ebpα+/−;Cre+/− embryos injected with the bmi1-specifcic morpholino oligonucleotide (F). (G) Statistics of circulation congestion in the transgenic embryos shown in panels A to C. Normal, Moderate, and Severe indicate the extent of circulation congestion at the CHT region. (H) Conservation of the zebrafish bmi1 promoter region 5.5 kb upstream of the TSS with murine Bmi1 promoter region by rVista software. Diamonds and corresponding dashed lines with Arabic numerals represent the positions of 11 predicted C/EBP binding motifs. Horizontal lines with Roman numerals I to XI indicate the locations of primers used in the E-ChIP analyses. (I) The nucleotide acid sequence of the zebrafish bmi1 promoter region between −5203 and −5023 bp, corresponding to the conserved region indicated by the red diamond in panel H. Red dots indicate the nucleotide acid identities compared with murine bmi1 promoter region between −11 780 and −11 612 bp upstream of TSS. The predicated C/EBP binding motif is indicated by a box. (J-K) E-ChIP analyses of chromatins extracted from the LDL-EGFP+/−;Cre+/− and LDL-EGFP-C/ebpα+/−;Cre+/− embryos at 22 hpf. PCR reactions were performed using the primers located to the indicated promoter region (I-XI) in panel H. The EGFP-tagged C/ebpα specifically interacts with the C/EBP binding motifs II and I. The results were repeated 3 times with separate clutches of embryos and batches of chromatin preparations.

Bmi1 is a direct downstream target of C/ebpαDN. (A-C) External features of the LDL-EGFP+/−;Cre+/− transgenic embryos without injection (A, uninj), and the LDL- EGFP-C/ebpα+/−;Cre+/− transgenic embryos injected with either unrelated 5-mismatch (B) or bmi1-specifcic morpholino oligonucleotides (C). The circulation congestion is readily detected in the C/ebpαDN-expressing embryos injected with control morpholino (B, arrowheads) but largely rescued by bmi1 knockdown (C, arrowheads). Boxes with dashed lines indicate the regions amplified and shown at the bottom. (D-F) WISH analyses of αE1-globin in the indicated transgenic embryos at 80 hpf. The red arrowheads indicate that the increased number of erythrocytes with expression of αE1-globin transcripts can be corrected to normal level in the LDL-EGFP-C/ebpα+/−;Cre+/− embryos injected with the bmi1-specifcic morpholino oligonucleotide (F). (G) Statistics of circulation congestion in the transgenic embryos shown in panels A to C. Normal, Moderate, and Severe indicate the extent of circulation congestion at the CHT region. (H) Conservation of the zebrafish bmi1 promoter region 5.5 kb upstream of the TSS with murine Bmi1 promoter region by rVista software. Diamonds and corresponding dashed lines with Arabic numerals represent the positions of 11 predicted C/EBP binding motifs. Horizontal lines with Roman numerals I to XI indicate the locations of primers used in the E-ChIP analyses. (I) The nucleotide acid sequence of the zebrafish bmi1 promoter region between −5203 and −5023 bp, corresponding to the conserved region indicated by the red diamond in panel H. Red dots indicate the nucleotide acid identities compared with murine bmi1 promoter region between −11 780 and −11 612 bp upstream of TSS. The predicated C/EBP binding motif is indicated by a box. (J-K) E-ChIP analyses of chromatins extracted from the LDL-EGFP+/−;Cre+/− and LDL-EGFP-C/ebpα+/−;Cre+/− embryos at 22 hpf. PCR reactions were performed using the primers located to the indicated promoter region (I-XI) in panel H. The EGFP-tagged C/ebpα specifically interacts with the C/EBP binding motifs II and I. The results were repeated 3 times with separate clutches of embryos and batches of chromatin preparations.

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