Lee SY et al. (2012), The role of heterodimeric AP-1 protein comprise...

XB-ART-45707

J Biol Chem 2012 Sep 07;28737:31342-8. doi: 10.1074/jbc.M112.387266.

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The role of heterodimeric AP-1 protein comprised of JunD and c-Fos proteins in hematopoiesis.

Lee SY , Yoon J , Lee MH , Jung SK , Kim DJ , Bode AM , Kim J , Dong Z .

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Activator protein-1 (AP-1) regulates a wide range of cellular processes including proliferation, differentiation, and apoptosis. As a transcription factor, AP-1 is commonly found as a heterodimer comprised of c-Jun and c-Fos proteins. However, other heterodimers may also be formed. The function of these dimers, specifically the heterodimeric AP-1 comprised of JunD and c-Fos (AP-1(JunD/c-Fos)), has not been elucidated. Here, we identified a function of AP-1(JunD/c-Fos) in Xenopus hematopoiesis. A gain-of-function study performed by overexpressing junD and c-fos and a loss-of-function study using morpholino junD demonstrate a critical role for AP-1(JunD/c-Fos) in hematopoiesis during Xenopus embryogenesis. Additionally, we confirmed that JunD of AP-1(JunD/c-Fos) is required for BMP-4-induced hematopoiesis. We also demonstrated that BMP-4 regulated JunD activity at the transcriptional regulation and post-translational modification levels. Collectively, our findings identify AP-1(JunD/c-Fos) as a novel hematopoietic transcription factor and the requirement of AP-1(JunD/c-Fos) in BMP-4-induced hematopoiesis during Xenopus hematopoiesis.

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???displayArticle.pmcLink??? PMC3438963
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Species referenced: Xenopus laevis
Genes referenced: actl6a fos jun jund jund.2 klf17 klf4 lmo2 tal1 ventx1.2
???displayArticle.antibodies??? FLAG Ab1 HA Ab3 Jun Ab1
???displayArticle.morpholinos??? jund MO1 jund MO2 jun MO1 jun MO2

Phenotypes: Xla Wt + fos + jund (fig.2.c) [+]

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	FIGURE 1. AP-1JunD/c-Fos is required for hematopoiesis. A and B, co-expression of junD and c-fos induces hematopoietic makers (SCL, GATA1A, Neptune, and LMO2) and globin. Animal caps, explanted from embryos injected with the indicated concentration of mRNAs encoding junD or/and c-fos, were incubated until stages 18-20 (A) or 24-28 (B) and used for qRT-PCR analysis. C, co-expression of junD and c-fos enhances the promoter activities of the SCL but not the mutant SCL-mAP1. Embryos injected with the SCL- or SCL-mAP1-luciferase reporter gene alone or together with 2 ng of junD and c-fos were incubated until stages 18-20. Luciferase activity was measured. Values are shown as means Â± S.D. from at least three independent experiments. RLU, relative luciferase activity. D, MO junD (20 ng) specifically knocks down the translation of the overexpressed C-terminal HA-tagged XJunD protein at stage 18. Actin served as a specificity control. E, mouse junD rescues SCL and globin, which are repressed by MO junD expression without changing the dorsal mesoderm marker, actin. qRT-PCR analysis of whole embryos expressing MO junD (20 ng) alone or in combination with 1 ng of mouse junD at stages 20-24. F, illustration of the scheme of the experiment. One blastomere of two-cell embryos was injected with mRNA encoding Î²-galactosidase together with MO junD (20 ng) or mouse junD (1 ng) (mjunD) as illustrated. G and H, embryos were stained for Î²-galactosidase (Î²-gal) activity at stage 30 (blue stain) followed by in situ hybridization analysis of globin expression (purple stain). G, the expression of globin is repressed in the MO junD-injected side. H, mouse junD mRNA (mjunD) rescues globin expression that is repressed by expression of MO junD. EF1Î±, loading control; w.e., whole embryo was used as a positive control for PCR; âˆ’RT, control reaction without reverse transcriptase; con, animal cap samples obtained from non-injected embryos. , p value < 0.01; *, p value < 0.001. IB, immunoblot.
	FIGURE 2. AP-1JunD/c-Fos converts dorsal-fated tissue into ventral-fated tissue (ventral blood island). A and D, illustration of the scheme of the experiment. GFP mRNA (200 pg, B) only (B and F) or GFP together with AP-1 (junD and c-fos, 500 pg) mRNA (C and E) were injected into dorsal animal blastomeres (D1) or ventral animal blastomeres (V1) of eight-cell stage embryos and then cultured until stages 28ï¿½30. Embryos were fixed, and GFP expression was observed by green fluorescent microscopy. B and C, dorsally expressed GFP (B, upper panel) is partially transferred into the ventral blood island region (C, three arrows in upper panel). E and F, embryos injected with either GFP alone (F, upper panel) or together with AP-1JunD/c-Fos (E, upper panel) show a similar expression pattern of GFP, which is expressed at the ventroposterior epidermis. The ventral blood island is indicated by in situ hybridization of globin (Bï¿½F, lower panel). The number (n) of phenotypes for each group is presented.
	FIGURE 3. AP-1JunD/c-Fos is required for hematopoiesis induced by BMP-4. A and B, animal caps, explanted from embryos injected with the indicated mRNAs were incubated until stage 20-24 and used for qRT-PCR analysis (A) or benzidine staining (B). A, AP-1JunD/c-Fos and BMP-4 synergistically induce hematopoietic markers and globin. B, blood formation stained by benzidine is synergistically enhanced by AP-1JunD/c-Fos and BMP-4. C, the activity of the (AP-1)4-luciferase reporter gene is synergistically activated by co-injection of AP-1JunD/c-Fos and BMP-4. An (AP-1)4-luciferase assay using animal cap explants derived from embryos injected with the (AP-1)4-luciferase reporter gene alone or in combination with the indicated mRNA was performed. Luciferase activity was measured at stage 18. Values are averages from at least three independent experiments. RLU, relative luciferase activity. D, animal caps, explanted from embryos injected with the indicated mRNAs or MO junD (20 ng), were incubated until stage 20ï¿½24 and used for qRT-PCR analysis. MO JunD selectively blocks BMP-4-induced expression of globin, LMO2, and SCL. Injection of mouse junD mRNA rescues the BMP-4 induction of globin, LMO2, and SCL as well as the activity of the (AP-1)4-luciferase reporter gene in MO junD-injected animal caps (E). EF1Î±, loading control; w.e., whole embryo was used as a positive control for PCR; âˆ’rt, control reaction without reverse transcriptase. , p value < 0.01; *, p value < 0.001.
	FIGURE 4. Transcriptional and post-translational modification of AP-1JunD/c-Fos by BMP-4 is required for hematopoiesis. A, BMP-4 activates the transcription of junD, but not c-fos. Xvent1 (ventral marker) was used as a positive control for BMP-4. Animal caps derived from embryos injected with the indicated mRNA were excised and cultured until stage 13 and used for qRT-PCR analysis. con, animal cap samples obtained from non-injected embryos. B, BMP-4 enhances phosphorylation of XJunD at serine 67; in contrast, dominant-negative BMP receptor inhibits the phosphorylation of XJunD. Embryos injected with wild-type HA-junD (W) or mutant HA-junD (M2) alone or in combination with 1 ng of BMP-4 or dominant-negative BMP receptor mRNA were used for Western blotting. Phosphorylation of JunD was analyzed by Western blot using anti-phospho-Jun (Î±-73). Western blotting with anti-HA shows that equal amounts of expressed JunD were loaded. C, band density was measured using the NIH ImageJ program. Dï¿½F, animal caps, explanted from embryos injected with the indicated mRNAs or in combination with the (AP-1)4-luciferase reporter gene or SCL-luciferase reporter gene, were used for qRT-PCR analysis (D) and the luciferase assay (E and F). The concentration of each mRNA injected into embryos was 1 ng. D, AP-1M2JunD/c-Fos shows a lower induction of hematopoietic markers and globin, compared with AP-1JunD/c-Fos. E and F, the AP-1- and SCL-luciferase activities are consistent with D. Data are shown as means Â± S.D. of values from at least three independent experiments. RLU, relative luciferase activity. , p value < 0.01; *, p value < 0.001.

References [+] :

Ameyar, A role for AP-1 in apoptosis: the case for and against. 2003, Pubmed

	FIGURE 1. AP-1JunD/c-Fos is required for hematopoiesis. A and B, co-expression of junD and c-fos induces hematopoietic makers (SCL, GATA1A, Neptune, and LMO2) and globin. Animal caps, explanted from embryos injected with the indicated concentration of mRNAs encoding junD or/and c-fos, were incubated until stages 18-20 (A) or 24-28 (B) and used for qRT-PCR analysis. C, co-expression of junD and c-fos enhances the promoter activities of the SCL but not the mutant SCL-mAP1. Embryos injected with the SCL- or SCL-mAP1-luciferase reporter gene alone or together with 2 ng of junD and c-fos were incubated until stages 18-20. Luciferase activity was measured. Values are shown as means Â± S.D. from at least three independent experiments. RLU, relative luciferase activity. D, MO junD (20 ng) specifically knocks down the translation of the overexpressed C-terminal HA-tagged XJunD protein at stage 18. Actin served as a specificity control. E, mouse junD rescues SCL and globin, which are repressed by MO junD expression without changing the dorsal mesoderm marker, actin. qRT-PCR analysis of whole embryos expressing MO junD (20 ng) alone or in combination with 1 ng of mouse junD at stages 20-24. F, illustration of the scheme of the experiment. One blastomere of two-cell embryos was injected with mRNA encoding Î²-galactosidase together with MO junD (20 ng) or mouse junD (1 ng) (mjunD) as illustrated. G and H, embryos were stained for Î²-galactosidase (Î²-gal) activity at stage 30 (blue stain) followed by in situ hybridization analysis of globin expression (purple stain). G, the expression of globin is repressed in the MO junD-injected side. H, mouse junD mRNA (mjunD) rescues globin expression that is repressed by expression of MO junD. EF1Î±, loading control; w.e., whole embryo was used as a positive control for PCR; âˆ’RT, control reaction without reverse transcriptase; con, animal cap samples obtained from non-injected embryos. , p value < 0.01; *, p value < 0.001. IB, immunoblot.
	FIGURE 2. AP-1JunD/c-Fos converts dorsal-fated tissue into ventral-fated tissue (ventral blood island). A and D, illustration of the scheme of the experiment. GFP mRNA (200 pg, B) only (B and F) or GFP together with AP-1 (junD and c-fos, 500 pg) mRNA (C and E) were injected into dorsal animal blastomeres (D1) or ventral animal blastomeres (V1) of eight-cell stage embryos and then cultured until stages 28ï¿½30. Embryos were fixed, and GFP expression was observed by green fluorescent microscopy. B and C, dorsally expressed GFP (B, upper panel) is partially transferred into the ventral blood island region (C, three arrows in upper panel). E and F, embryos injected with either GFP alone (F, upper panel) or together with AP-1JunD/c-Fos (E, upper panel) show a similar expression pattern of GFP, which is expressed at the ventroposterior epidermis. The ventral blood island is indicated by in situ hybridization of globin (Bï¿½F, lower panel). The number (n) of phenotypes for each group is presented.
	FIGURE 3. AP-1JunD/c-Fos is required for hematopoiesis induced by BMP-4. A and B, animal caps, explanted from embryos injected with the indicated mRNAs were incubated until stage 20-24 and used for qRT-PCR analysis (A) or benzidine staining (B). A, AP-1JunD/c-Fos and BMP-4 synergistically induce hematopoietic markers and globin. B, blood formation stained by benzidine is synergistically enhanced by AP-1JunD/c-Fos and BMP-4. C, the activity of the (AP-1)4-luciferase reporter gene is synergistically activated by co-injection of AP-1JunD/c-Fos and BMP-4. An (AP-1)4-luciferase assay using animal cap explants derived from embryos injected with the (AP-1)4-luciferase reporter gene alone or in combination with the indicated mRNA was performed. Luciferase activity was measured at stage 18. Values are averages from at least three independent experiments. RLU, relative luciferase activity. D, animal caps, explanted from embryos injected with the indicated mRNAs or MO junD (20 ng), were incubated until stage 20ï¿½24 and used for qRT-PCR analysis. MO JunD selectively blocks BMP-4-induced expression of globin, LMO2, and SCL. Injection of mouse junD mRNA rescues the BMP-4 induction of globin, LMO2, and SCL as well as the activity of the (AP-1)4-luciferase reporter gene in MO junD-injected animal caps (E). EF1Î±, loading control; w.e., whole embryo was used as a positive control for PCR; âˆ’rt, control reaction without reverse transcriptase. , p value < 0.01; *, p value < 0.001.
	FIGURE 4. Transcriptional and post-translational modification of AP-1JunD/c-Fos by BMP-4 is required for hematopoiesis. A, BMP-4 activates the transcription of junD, but not c-fos. Xvent1 (ventral marker) was used as a positive control for BMP-4. Animal caps derived from embryos injected with the indicated mRNA were excised and cultured until stage 13 and used for qRT-PCR analysis. con, animal cap samples obtained from non-injected embryos. B, BMP-4 enhances phosphorylation of XJunD at serine 67; in contrast, dominant-negative BMP receptor inhibits the phosphorylation of XJunD. Embryos injected with wild-type HA-junD (W) or mutant HA-junD (M2) alone or in combination with 1 ng of BMP-4 or dominant-negative BMP receptor mRNA were used for Western blotting. Phosphorylation of JunD was analyzed by Western blot using anti-phospho-Jun (Î±-73). Western blotting with anti-HA shows that equal amounts of expressed JunD were loaded. C, band density was measured using the NIH ImageJ program. Dï¿½F, animal caps, explanted from embryos injected with the indicated mRNAs or in combination with the (AP-1)4-luciferase reporter gene or SCL-luciferase reporter gene, were used for qRT-PCR analysis (D) and the luciferase assay (E and F). The concentration of each mRNA injected into embryos was 1 ng. D, AP-1M2JunD/c-Fos shows a lower induction of hematopoietic markers and globin, compared with AP-1JunD/c-Fos. E and F, the AP-1- and SCL-luciferase activities are consistent with D. Data are shown as means Â± S.D. of values from at least three independent experiments. RLU, relative luciferase activity. , p value < 0.01; *, p value < 0.001.