Figure 2
Figure 2. lyz is a direct downstream target of c-Myb. (A) Schematic diagram of the 2.5-kb lyz promoter region. The transcription initiation site is designated as 0. Seven putative c-Myb consensus sites are identified within −0.5∼0 kb proximal region using promo 3.0 online software and their positions (marked by stars) relative to the transcription initiation site are shown. (B) ChIP shows that c-Myb binds to the −0.5-kb proximal promoter region of the lyz promoter. Lysates from the embryos injected with the Myc-tagged c-myb mRNA were precipitated with anti-Myc (lanes 1-3) and anti-Dsred (lane 4, negative control) antibodies. The precipitates were then subjected to semiquantitative PCR analysis of the enrichment of the −0.5∼0-kb proximal region (lanes 1 and 4), the −2.5- ∼ −2-kb distal region (lane 2), and the actin-coding region (lane 3). Lanes 5-7 are input DNA control. (C) Gel shift image shows that 7 FAM-labeled probes containing putative MBSs were incubated with purified His-c-Myb N-terminal (amino acids 33-202) of the zebrafish c-Myb protein, encompassing the highly conserved DNA-binding domain. Excess unlabeled probes compete with the FAM-labeled probes. Equal amounts of proteins were used, specifically bound to the FAM-labeled probes. (D-I) Transient GFP reporter assay. Embryos were injected at the 1-cell stage with pTol-lyzPromoter-GFP reporter constructs driven by an intact 2.4-kb lyz promoter: pTol-lyz:GFP (D) or lyz promoter with subsets of the mutated c-MBSs (mMBS): pTol-lyzmMBS(1-2):GFP (E), pTol-lyzmMBS(3-4):GFP (F), pTol-lyzmMBS(5-7):GFP (G), and pTol-lyzmMBS(1-7):GFP (H). The promoter activity was then measured by calculating the numbers of GFP+ cells at 2 dpf. Representative images (D-H) and GFP+ quantifications (I) of 2-dpf injected embryos (ANOVA, Dunnett T3, n ≥ 50). Scale bars represent 200 μm. (J) Luciferase reporter assay. Bars showed the relative luciferase activity of lyz promoter with different subsets of MBS mutation compared with unmutated control. The statistical significance was calculated using 1-way ANOVA followed by Dunnett T3 correction. An asterisk indicates a statistical difference (*P < .05, **P < .01, ***P < .001, ****P < .0001, triplicated).

lyz is a direct downstream target of c-Myb. (A) Schematic diagram of the 2.5-kb lyz promoter region. The transcription initiation site is designated as 0. Seven putative c-Myb consensus sites are identified within −0.5∼0 kb proximal region using promo 3.0 online software and their positions (marked by stars) relative to the transcription initiation site are shown. (B) ChIP shows that c-Myb binds to the −0.5-kb proximal promoter region of the lyz promoter. Lysates from the embryos injected with the Myc-tagged c-myb mRNA were precipitated with anti-Myc (lanes 1-3) and anti-Dsred (lane 4, negative control) antibodies. The precipitates were then subjected to semiquantitative PCR analysis of the enrichment of the −0.5∼0-kb proximal region (lanes 1 and 4), the −2.5- ∼ −2-kb distal region (lane 2), and the actin-coding region (lane 3). Lanes 5-7 are input DNA control. (C) Gel shift image shows that 7 FAM-labeled probes containing putative MBSs were incubated with purified His-c-Myb N-terminal (amino acids 33-202) of the zebrafish c-Myb protein, encompassing the highly conserved DNA-binding domain. Excess unlabeled probes compete with the FAM-labeled probes. Equal amounts of proteins were used, specifically bound to the FAM-labeled probes. (D-I) Transient GFP reporter assay. Embryos were injected at the 1-cell stage with pTol-lyzPromoter-GFP reporter constructs driven by an intact 2.4-kb lyz promoter: pTol-lyz:GFP (D) or lyz promoter with subsets of the mutated c-MBSs (mMBS): pTol-lyzmMBS(1-2):GFP (E), pTol-lyzmMBS(3-4):GFP (F), pTol-lyzmMBS(5-7):GFP (G), and pTol-lyzmMBS(1-7):GFP (H). The promoter activity was then measured by calculating the numbers of GFP+ cells at 2 dpf. Representative images (D-H) and GFP+ quantifications (I) of 2-dpf injected embryos (ANOVA, Dunnett T3, n ≥ 50). Scale bars represent 200 μm. (J) Luciferase reporter assay. Bars showed the relative luciferase activity of lyz promoter with different subsets of MBS mutation compared with unmutated control. The statistical significance was calculated using 1-way ANOVA followed by Dunnett T3 correction. An asterisk indicates a statistical difference (*P < .05, **P < .01, ***P < .001, ****P < .0001, triplicated).

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