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The Drosophila Transcription Factors Tinman and Pannier Activate and Collaborate with Myocyte Enhancer Factor-2 to Promote Heart Cell Fate.

Lovato TL, Sensibaugh CA, Swingle KL, Martinez MM, Cripps RM - PLoS ONE (2015)

Bottom Line: We found that mesodermal over-expression of Tinman and Pannier resulted in approximately 20% of embryos with ectopic Hand and Sulphonylurea receptor (Sur) expression.By adding MEF2 alongside Tinman and Pannier, a dramatic expansion in the expression of Hand and Sur was observed in almost all embryos analyzed.Two additional cardiac markers were also expanded in their expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of New Mexico, Albuquerque, NM 87131-1091, United States of America.

ABSTRACT
Expression of the MADS domain transcription factor Myocyte Enhancer Factor 2 (MEF2) is regulated by numerous and overlapping enhancers which tightly control its transcription in the mesoderm. To understand how Mef2 expression is controlled in the heart, we identified a late stage Mef2 cardiac enhancer that is active in all heart cells beginning at stage 14 of embryonic development. This enhancer is regulated by the NK-homeodomain transcription factor Tinman, and the GATA transcription factor Pannier through both direct and indirect interactions with the enhancer. Since Tinman, Pannier and MEF2 are evolutionarily conserved from Drosophila to vertebrates, and since their vertebrate homologs can convert mouse fibroblast cells to cardiomyocytes in different activator cocktails, we tested whether over-expression of these three factors in vivo could ectopically activate known cardiac marker genes. We found that mesodermal over-expression of Tinman and Pannier resulted in approximately 20% of embryos with ectopic Hand and Sulphonylurea receptor (Sur) expression. By adding MEF2 alongside Tinman and Pannier, a dramatic expansion in the expression of Hand and Sur was observed in almost all embryos analyzed. Two additional cardiac markers were also expanded in their expression. Our results demonstrate the ability to initiate ectopic cardiac fate in vivo by the combination of only three members of the conserved Drosophila cardiac transcription network, and provide an opportunity for this genetic model system to be used to dissect the mechanisms of cardiac specification.

No MeSH data available.


Related in: MedlinePlus

Tinman and Pannier activate the enhancer in vitro and Tinman is capable of binding to its consensus sequence within the enhancer.(A)Activation of the -2775/-2432 Mef2-lacZ in S2 cells by Tin, Pnr or Tin combined with Pnr. Tin activated the reporter moderately, while activation by Pnr was not significantly above negative controls. When combined, activation of the reporter was increased significantly above that achieved by Tin alone. (B)Electrophoretic mobility shift assay to determine if Tin and Pnr could bind to consensus sites. Free probe had a high mobility when combined with unprogrammed lysate (Un). A complex of probe plus protein was formed in the presence of Tin lysate, which was competed by 300X excess of nonradioactive wild-type sequence (wt comp) but not by 300X excess of nonradioactive mutant sequence (mut comp).
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pone.0132965.g002: Tinman and Pannier activate the enhancer in vitro and Tinman is capable of binding to its consensus sequence within the enhancer.(A)Activation of the -2775/-2432 Mef2-lacZ in S2 cells by Tin, Pnr or Tin combined with Pnr. Tin activated the reporter moderately, while activation by Pnr was not significantly above negative controls. When combined, activation of the reporter was increased significantly above that achieved by Tin alone. (B)Electrophoretic mobility shift assay to determine if Tin and Pnr could bind to consensus sites. Free probe had a high mobility when combined with unprogrammed lysate (Un). A complex of probe plus protein was formed in the presence of Tin lysate, which was competed by 300X excess of nonradioactive wild-type sequence (wt comp) but not by 300X excess of nonradioactive mutant sequence (mut comp).

Mentions: To test the ability of candidate factors to regulate the Mef2 enhancer, we transfected Drosophila S2 cells with the enhancer fused to a lacZ reporter gene, along with plasmids containing the cDNAs of either tin, or pnr, or both factors. After incubation for 48hr, cell lysates were prepared and reporter activity was determined using a quantitative ßGal assay. There was moderate but significant activation of the Mef2-lacZ construct by Tin, while Pnr on its own was unable to significantly activate the enhancer. When Tin and Pnr were combined, activation was more than additive, suggesting that the two factors might work synergistically to activate Mef2 in the heart (Fig 2A).


The Drosophila Transcription Factors Tinman and Pannier Activate and Collaborate with Myocyte Enhancer Factor-2 to Promote Heart Cell Fate.

Lovato TL, Sensibaugh CA, Swingle KL, Martinez MM, Cripps RM - PLoS ONE (2015)

Tinman and Pannier activate the enhancer in vitro and Tinman is capable of binding to its consensus sequence within the enhancer.(A)Activation of the -2775/-2432 Mef2-lacZ in S2 cells by Tin, Pnr or Tin combined with Pnr. Tin activated the reporter moderately, while activation by Pnr was not significantly above negative controls. When combined, activation of the reporter was increased significantly above that achieved by Tin alone. (B)Electrophoretic mobility shift assay to determine if Tin and Pnr could bind to consensus sites. Free probe had a high mobility when combined with unprogrammed lysate (Un). A complex of probe plus protein was formed in the presence of Tin lysate, which was competed by 300X excess of nonradioactive wild-type sequence (wt comp) but not by 300X excess of nonradioactive mutant sequence (mut comp).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520567&req=5

pone.0132965.g002: Tinman and Pannier activate the enhancer in vitro and Tinman is capable of binding to its consensus sequence within the enhancer.(A)Activation of the -2775/-2432 Mef2-lacZ in S2 cells by Tin, Pnr or Tin combined with Pnr. Tin activated the reporter moderately, while activation by Pnr was not significantly above negative controls. When combined, activation of the reporter was increased significantly above that achieved by Tin alone. (B)Electrophoretic mobility shift assay to determine if Tin and Pnr could bind to consensus sites. Free probe had a high mobility when combined with unprogrammed lysate (Un). A complex of probe plus protein was formed in the presence of Tin lysate, which was competed by 300X excess of nonradioactive wild-type sequence (wt comp) but not by 300X excess of nonradioactive mutant sequence (mut comp).
Mentions: To test the ability of candidate factors to regulate the Mef2 enhancer, we transfected Drosophila S2 cells with the enhancer fused to a lacZ reporter gene, along with plasmids containing the cDNAs of either tin, or pnr, or both factors. After incubation for 48hr, cell lysates were prepared and reporter activity was determined using a quantitative ßGal assay. There was moderate but significant activation of the Mef2-lacZ construct by Tin, while Pnr on its own was unable to significantly activate the enhancer. When Tin and Pnr were combined, activation was more than additive, suggesting that the two factors might work synergistically to activate Mef2 in the heart (Fig 2A).

Bottom Line: We found that mesodermal over-expression of Tinman and Pannier resulted in approximately 20% of embryos with ectopic Hand and Sulphonylurea receptor (Sur) expression.By adding MEF2 alongside Tinman and Pannier, a dramatic expansion in the expression of Hand and Sur was observed in almost all embryos analyzed.Two additional cardiac markers were also expanded in their expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of New Mexico, Albuquerque, NM 87131-1091, United States of America.

ABSTRACT
Expression of the MADS domain transcription factor Myocyte Enhancer Factor 2 (MEF2) is regulated by numerous and overlapping enhancers which tightly control its transcription in the mesoderm. To understand how Mef2 expression is controlled in the heart, we identified a late stage Mef2 cardiac enhancer that is active in all heart cells beginning at stage 14 of embryonic development. This enhancer is regulated by the NK-homeodomain transcription factor Tinman, and the GATA transcription factor Pannier through both direct and indirect interactions with the enhancer. Since Tinman, Pannier and MEF2 are evolutionarily conserved from Drosophila to vertebrates, and since their vertebrate homologs can convert mouse fibroblast cells to cardiomyocytes in different activator cocktails, we tested whether over-expression of these three factors in vivo could ectopically activate known cardiac marker genes. We found that mesodermal over-expression of Tinman and Pannier resulted in approximately 20% of embryos with ectopic Hand and Sulphonylurea receptor (Sur) expression. By adding MEF2 alongside Tinman and Pannier, a dramatic expansion in the expression of Hand and Sur was observed in almost all embryos analyzed. Two additional cardiac markers were also expanded in their expression. Our results demonstrate the ability to initiate ectopic cardiac fate in vivo by the combination of only three members of the conserved Drosophila cardiac transcription network, and provide an opportunity for this genetic model system to be used to dissect the mechanisms of cardiac specification.

No MeSH data available.


Related in: MedlinePlus