Abstract
Thyroid transcription factor-1 (TTF-1, also known as NKX2-1) is a tissue-specific transcription factor in lung epithelial cells. Although TTF-1 inhibits the epithelial-to-mesenchymal transition induced by transforming growth factor-β (TGF-β) in lung adenocarcinoma cells, the mechanism through which TTF-1 inhibits the functions of TGF-β is unknown. Here we show that TTF-1 disrupts the nuclear Smad3-Smad4 complex without affecting the nuclear localization of phospho-Smad3. Genome-wide analysis by chromatin immunoprecipitation followed by sequencing revealed that TTF-1 colocalizes with Smad3 on chromatin and alters Smad3-binding patterns throughout the genome, while TTF-1 generally inhibits Smad4 binding to chromatin. Moreover, Smad3 binds to chromatin together with TTF-1, but not with Smad4, at some Smad3-binding regions when TGF-β signaling is absent, and knockdown of Smad4 expression does not attenuate Smad3 binding in these regions. Thus, TTF-1 may compete with Smad4 for interaction with Smad3, and in the presence of TTF-1, Smad3 regulates the transcription of certain genes independently of Smad4. These findings provide a new model of regulation of TGF-β-Smad signaling by TTF-1.
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Acknowledgements
We are grateful to Kaori Shiina, Hiroko Meguro and Keiko Yuki for technical assistance, as well as to members of the Miyazono laboratory for discussion and advice. This research was supported by KAKENHI (grants-in-aid for scientific research) on Innovative Area (Integrative Research on Cancer Microenvironment Network, 22112002 to KM) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and Scientific Research (S), 20221009 to HA and Research Activity Start-up, 24890039 to KI from MEXT. This research was also supported by the Ministry of Health, Labor, and Welfare of Japan (a grant to DK), and the Genome Network Project from MEXT (a grant to HA). DK is supported by a grant from Mochida Memorial Foundation for Medical and Pharmaceutical Research and Project for Development of Innovative Research on Cancer Therapeutics from MEXT. The authors declare no competing interests.
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Supplementary information
Supplementary information, Figure S1
Co-localization of TTF-1 and Smad3 determined by in situ PLA assay. (PDF 598 kb)
Supplementary information, Figure S2
Validation of the change of Smad3 binding to the PAI-l promoter region in H441 cells transfected with siTTF-1. (PDF 350 kb)
Supplementary information, Figure S3
Validation of the anti-TTF-1 antibody and ChIP-seq analyses of TTF-1 binding to chromatin. (PDF 451 kb)
Supplementary information, Figure S4
Effect of TTF-1 siRNA on TGF-β-induced transcriptional regulation of SNAI1 and SNAI2 in H441 cells. (PDF 612 kb)
Supplementary information, Figure S5
Co-occupation of TTF-1 with Smad2 and Smad3 at LM03 locus. (PDF 302 kb)
Supplementary information, Figure S6
Effect of TGF-β on TTF-1 binding. (PDF 808 kb)
Supplementary information, Figure S7
Effect of exogenously expressed TTF-1 on cellular response of A549 cells. (PDF 817 kb)
Supplementary information, Table S1
Binding peaks of Smad3 in TGF-β-stimulated H441 cells treated with Control siRNA. (PDF 2715 kb)
Supplementary information, Table S2
Binding peaks of Smad3 in TGF-β-stimulated H441 cells treated with TTF-1 siRNA. (PDF 4217 kb)
Supplementary information, Table S3
Binding peaks of Smad4 in TGF-β-stimulated H441 cells treated with Control siRNA. (PDF 95 kb)
Supplementary information, Table S4
Binding peaks of Smad4 in TGF-β-stimulated H441 cells treated with TTF-1 siRNA. (PDF 564 kb)
Supplementary information, Table S5
Binding peaks of TTF-1 in TGF-β-unstimulated H441 cells. (PDF 9152 kb)
Supplementary information, Table S6
Binding peaks of TTF-1 in TGF-β-stimulated H441 cells. (PDF 6295 kb)
Supplementary information, Table S7
Genes that are expressed in H441 cells and up-regulated 1.5-fold by TGF-β stimulation, and whose expression is increased two-fold by TTF-1 knockdown. (PDF 744 kb)
Supplementary information, Table S8
Genes that have STRL near their regions, and whose expression is reduced by TGF-β stimulation. (PDF 369 kb)
Supplementary information, Table S9
Binding peaks of Smad3 in TGF-β-stimulated A549 cells. (XLS 171 kb)
Supplementary information, Data S1
Materials and Methods (PDF 53 kb)
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Isogaya, K., Koinuma, D., Tsutsumi, S. et al. A Smad3 and TTF-1/NKX2-1 complex regulates Smad4-independent gene expression. Cell Res 24, 994–1008 (2014). https://doi.org/10.1038/cr.2014.97
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DOI: https://doi.org/10.1038/cr.2014.97
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