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SMAD3 prevents binding of NKX2.1 and FOXA1 to the SpB promoter through its MH1 and MH2 domains.

Minoo P, Hu L, Zhu N, Borok Z, Bellusci S, Groffen J, Kardassis D, Li C - Nucleic Acids Res. (2007)

Bottom Line: In this study, we found that SMAD3 interacts through its MAD domains, MH1 and MH2 with NKX2.1 and FOXA1 proteins.Both in vitro studies and in vivo ChIP assays show that interaction of SMAD3 MH1 and MH2 domains with NKX2.1 and FOXA1 results in reduced binding of NKX2.1 and FOXA1 to their cognate DNA-binding sites, and diminished promoter occupancy within the SpB promoter.Thus, these studies reveal for the first time a mechanism of TGF-beta-induced SpB gene repression that involves interactions between specific SMAD3 domains and the corresponding functional sites on NKX2.1 and FOXA1 transcription factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Division of Neonatology, Will Rogers Institute Pulmonary Research Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA. minoo@usc.edu

ABSTRACT
Mechanisms of gene repression by transforming growth factor-beta (TGF-beta) are not well understood. TGF-beta represses transcription of pulmonary surfactant protein-B gene in lung epithelial cells. Repression is mediated by SMAD3 through interactions with NKX2.1 and FOXA1, two key transcription factors that are positive regulators of SpB transcription. In this study, we found that SMAD3 interacts through its MAD domains, MH1 and MH2 with NKX2.1 and FOXA1 proteins. The sites of interaction on NKX2.1 are located within the NH2 and COOH domains, known to be involved in transactivation function. In comparison, weaker interaction of FOXA1 winged helix, and the NH(2)-terminal domains was documented with SMAD3. Both in vitro studies and in vivo ChIP assays show that interaction of SMAD3 MH1 and MH2 domains with NKX2.1 and FOXA1 results in reduced binding of NKX2.1 and FOXA1 to their cognate DNA-binding sites, and diminished promoter occupancy within the SpB promoter. Thus, these studies reveal for the first time a mechanism of TGF-beta-induced SpB gene repression that involves interactions between specific SMAD3 domains and the corresponding functional sites on NKX2.1 and FOXA1 transcription factors.

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Related in: MedlinePlus

Physical interactions between SMAD3 and functional domains of NKX2.1. GAL4 or VP16 fused NKX2.1 and its various functional domains were used in mammalian two-hybrid assays to determine the site of interactions with SMAD3. Vp16 or Gal4 plasmids were used either alone or in combination with other expression vectors as control. Luciferase reading from combination of empty Vp16 and Gal4 plasmids were adjusted to unity and used for normalization of all experimental values. Representative results from three independent experiments are shown. Asterisks denote P < 0.05.
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Figure 3: Physical interactions between SMAD3 and functional domains of NKX2.1. GAL4 or VP16 fused NKX2.1 and its various functional domains were used in mammalian two-hybrid assays to determine the site of interactions with SMAD3. Vp16 or Gal4 plasmids were used either alone or in combination with other expression vectors as control. Luciferase reading from combination of empty Vp16 and Gal4 plasmids were adjusted to unity and used for normalization of all experimental values. Representative results from three independent experiments are shown. Asterisks denote P < 0.05.

Mentions: To identify the specific domains on the NKX2.1 molecule that mediate the NKX2.1–SMAD3 interactions, we constructed Gal4-fusion plasmids containing one of the three functional domains on the NKX2.1 protein; the NH2-terminus, the homeodomain and the COOH-terminus (Figure 3, Panel A). Each of the latter domains is thought to render distinct roles in transcriptional activation function of NKX2.1. The use of the mammalian two-hybrid assay in A549 cells showed little, if any physical interactions between SMAD3 and the DNA binding, homeodomain region of NKX2.1 (Figure 3, Panel B). In contrast, as evidenced by the level of luciferase production, both the NH2- and the COOH-terminal ends of NKX2.1 interacted strongly with SMAD3 indicating the two regions flanking the NKX2.1 homeodomain to be the sites of interaction involved in inhibition of DNA-binding function.Figure 3.


SMAD3 prevents binding of NKX2.1 and FOXA1 to the SpB promoter through its MH1 and MH2 domains.

Minoo P, Hu L, Zhu N, Borok Z, Bellusci S, Groffen J, Kardassis D, Li C - Nucleic Acids Res. (2007)

Physical interactions between SMAD3 and functional domains of NKX2.1. GAL4 or VP16 fused NKX2.1 and its various functional domains were used in mammalian two-hybrid assays to determine the site of interactions with SMAD3. Vp16 or Gal4 plasmids were used either alone or in combination with other expression vectors as control. Luciferase reading from combination of empty Vp16 and Gal4 plasmids were adjusted to unity and used for normalization of all experimental values. Representative results from three independent experiments are shown. Asterisks denote P < 0.05.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2248754&req=5

Figure 3: Physical interactions between SMAD3 and functional domains of NKX2.1. GAL4 or VP16 fused NKX2.1 and its various functional domains were used in mammalian two-hybrid assays to determine the site of interactions with SMAD3. Vp16 or Gal4 plasmids were used either alone or in combination with other expression vectors as control. Luciferase reading from combination of empty Vp16 and Gal4 plasmids were adjusted to unity and used for normalization of all experimental values. Representative results from three independent experiments are shown. Asterisks denote P < 0.05.
Mentions: To identify the specific domains on the NKX2.1 molecule that mediate the NKX2.1–SMAD3 interactions, we constructed Gal4-fusion plasmids containing one of the three functional domains on the NKX2.1 protein; the NH2-terminus, the homeodomain and the COOH-terminus (Figure 3, Panel A). Each of the latter domains is thought to render distinct roles in transcriptional activation function of NKX2.1. The use of the mammalian two-hybrid assay in A549 cells showed little, if any physical interactions between SMAD3 and the DNA binding, homeodomain region of NKX2.1 (Figure 3, Panel B). In contrast, as evidenced by the level of luciferase production, both the NH2- and the COOH-terminal ends of NKX2.1 interacted strongly with SMAD3 indicating the two regions flanking the NKX2.1 homeodomain to be the sites of interaction involved in inhibition of DNA-binding function.Figure 3.

Bottom Line: In this study, we found that SMAD3 interacts through its MAD domains, MH1 and MH2 with NKX2.1 and FOXA1 proteins.Both in vitro studies and in vivo ChIP assays show that interaction of SMAD3 MH1 and MH2 domains with NKX2.1 and FOXA1 results in reduced binding of NKX2.1 and FOXA1 to their cognate DNA-binding sites, and diminished promoter occupancy within the SpB promoter.Thus, these studies reveal for the first time a mechanism of TGF-beta-induced SpB gene repression that involves interactions between specific SMAD3 domains and the corresponding functional sites on NKX2.1 and FOXA1 transcription factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Division of Neonatology, Will Rogers Institute Pulmonary Research Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA. minoo@usc.edu

ABSTRACT
Mechanisms of gene repression by transforming growth factor-beta (TGF-beta) are not well understood. TGF-beta represses transcription of pulmonary surfactant protein-B gene in lung epithelial cells. Repression is mediated by SMAD3 through interactions with NKX2.1 and FOXA1, two key transcription factors that are positive regulators of SpB transcription. In this study, we found that SMAD3 interacts through its MAD domains, MH1 and MH2 with NKX2.1 and FOXA1 proteins. The sites of interaction on NKX2.1 are located within the NH2 and COOH domains, known to be involved in transactivation function. In comparison, weaker interaction of FOXA1 winged helix, and the NH(2)-terminal domains was documented with SMAD3. Both in vitro studies and in vivo ChIP assays show that interaction of SMAD3 MH1 and MH2 domains with NKX2.1 and FOXA1 results in reduced binding of NKX2.1 and FOXA1 to their cognate DNA-binding sites, and diminished promoter occupancy within the SpB promoter. Thus, these studies reveal for the first time a mechanism of TGF-beta-induced SpB gene repression that involves interactions between specific SMAD3 domains and the corresponding functional sites on NKX2.1 and FOXA1 transcription factors.

Show MeSH
Related in: MedlinePlus