Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung
Abstract Background Tbx2 encodes a transcriptional repressor implicated in the development of numerous organs in mouse. During lung development TBX2 maintains the proliferation of mesenchymal progenitors, and hence, epithelial proliferation and branching morphogenesis. The pro-proliferative function...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2021-03-01
|
| Series: | Respiratory Research |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12931-021-01679-y |
| _version_ | 1818862610008768512 |
|---|---|
| author | Timo H. Lüdtke Irina Wojahn Marc-Jens Kleppa Jasper Schierstaedt Vincent M. Christoffels Patrick Künzler Andreas Kispert |
| author_facet | Timo H. Lüdtke Irina Wojahn Marc-Jens Kleppa Jasper Schierstaedt Vincent M. Christoffels Patrick Künzler Andreas Kispert |
| author_sort | Timo H. Lüdtke |
| collection | DOAJ |
| description | Abstract Background Tbx2 encodes a transcriptional repressor implicated in the development of numerous organs in mouse. During lung development TBX2 maintains the proliferation of mesenchymal progenitors, and hence, epithelial proliferation and branching morphogenesis. The pro-proliferative function was traced to direct repression of the cell-cycle inhibitor genes Cdkn1a and Cdkn1b, as well as of genes encoding WNT antagonists, Frzb and Shisa3, to increase pro-proliferative WNT signaling. Despite these important molecular insights, we still lack knowledge of the DNA occupancy of TBX2 in the genome, and of the protein interaction partners involved in transcriptional repression of target genes. Methods We used chromatin immunoprecipitation (ChIP)-sequencing and expression analyses to identify genomic DNA-binding sites and transcription units directly regulated by TBX2 in the developing lung. Moreover, we purified TBX2 containing protein complexes from embryonic lung tissue and identified potential interaction partners by subsequent liquid chromatography/mass spectrometry. The interaction with candidate proteins was validated by immunofluorescence, proximity ligation and individual co-immunoprecipitation analyses. Results We identified Il33 and Ccn4 as additional direct target genes of TBX2 in the pulmonary mesenchyme. Analyzing TBX2 occupancy data unveiled the enrichment of five consensus sequences, three of which match T-box binding elements. The remaining two correspond to a high mobility group (HMG)-box and a homeobox consensus sequence motif. We found and validated binding of TBX2 to the HMG-box transcription factor HMGB2 and the homeobox transcription factor PBX1, to the heterochromatin protein CBX3, and to various members of the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex including HDAC1, HDAC2 and CHD4. Conclusion Our data suggest that TBX2 interacts with homeobox and HMG-box transcription factors as well as with the NuRD chromatin remodeling complex to repress transcription of anti-proliferative genes in the pulmonary mesenchyme. |
| first_indexed | 2024-12-19T10:02:36Z |
| format | Article |
| id | doaj.art-da418b249c7b4155b293e1e7ce76a75f |
| institution | Directory Open Access Journal |
| issn | 1465-993X |
| language | English |
| last_indexed | 2024-12-19T10:02:36Z |
| publishDate | 2021-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Respiratory Research |
| spelling | doaj.art-da418b249c7b4155b293e1e7ce76a75f2022-12-21T20:26:36ZengBMCRespiratory Research1465-993X2021-03-0122111710.1186/s12931-021-01679-yCombined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lungTimo H. Lüdtke0Irina Wojahn1Marc-Jens Kleppa2Jasper Schierstaedt3Vincent M. Christoffels4Patrick Künzler5Andreas Kispert6Institut Für Molekularbiologie, Medizinische Hochschule HannoverInstitut Für Molekularbiologie, Medizinische Hochschule HannoverInstitut Für Molekularbiologie, Medizinische Hochschule HannoverInstitut Für Molekularbiologie, Medizinische Hochschule HannoverDepartment of Anatomy, Embryology and Physiology, Academic Medical Center, University of AmsterdamInstitut Für Pflanzengenetik, Leibniz Universität HannoverInstitut Für Molekularbiologie, Medizinische Hochschule HannoverAbstract Background Tbx2 encodes a transcriptional repressor implicated in the development of numerous organs in mouse. During lung development TBX2 maintains the proliferation of mesenchymal progenitors, and hence, epithelial proliferation and branching morphogenesis. The pro-proliferative function was traced to direct repression of the cell-cycle inhibitor genes Cdkn1a and Cdkn1b, as well as of genes encoding WNT antagonists, Frzb and Shisa3, to increase pro-proliferative WNT signaling. Despite these important molecular insights, we still lack knowledge of the DNA occupancy of TBX2 in the genome, and of the protein interaction partners involved in transcriptional repression of target genes. Methods We used chromatin immunoprecipitation (ChIP)-sequencing and expression analyses to identify genomic DNA-binding sites and transcription units directly regulated by TBX2 in the developing lung. Moreover, we purified TBX2 containing protein complexes from embryonic lung tissue and identified potential interaction partners by subsequent liquid chromatography/mass spectrometry. The interaction with candidate proteins was validated by immunofluorescence, proximity ligation and individual co-immunoprecipitation analyses. Results We identified Il33 and Ccn4 as additional direct target genes of TBX2 in the pulmonary mesenchyme. Analyzing TBX2 occupancy data unveiled the enrichment of five consensus sequences, three of which match T-box binding elements. The remaining two correspond to a high mobility group (HMG)-box and a homeobox consensus sequence motif. We found and validated binding of TBX2 to the HMG-box transcription factor HMGB2 and the homeobox transcription factor PBX1, to the heterochromatin protein CBX3, and to various members of the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex including HDAC1, HDAC2 and CHD4. Conclusion Our data suggest that TBX2 interacts with homeobox and HMG-box transcription factors as well as with the NuRD chromatin remodeling complex to repress transcription of anti-proliferative genes in the pulmonary mesenchyme.https://doi.org/10.1186/s12931-021-01679-yTbx2Pulmonary mesenchymeLung developmentNuRDHDACCBX3 |
| spellingShingle | Timo H. Lüdtke Irina Wojahn Marc-Jens Kleppa Jasper Schierstaedt Vincent M. Christoffels Patrick Künzler Andreas Kispert Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung Respiratory Research Tbx2 Pulmonary mesenchyme Lung development NuRD HDAC CBX3 |
| title | Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung |
| title_full | Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung |
| title_fullStr | Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung |
| title_full_unstemmed | Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung |
| title_short | Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung |
| title_sort | combined genomic and proteomic approaches reveal dna binding sites and interaction partners of tbx2 in the developing lung |
| topic | Tbx2 Pulmonary mesenchyme Lung development NuRD HDAC CBX3 |
| url | https://doi.org/10.1186/s12931-021-01679-y |
| work_keys_str_mv | AT timohludtke combinedgenomicandproteomicapproachesrevealdnabindingsitesandinteractionpartnersoftbx2inthedevelopinglung AT irinawojahn combinedgenomicandproteomicapproachesrevealdnabindingsitesandinteractionpartnersoftbx2inthedevelopinglung AT marcjenskleppa combinedgenomicandproteomicapproachesrevealdnabindingsitesandinteractionpartnersoftbx2inthedevelopinglung AT jasperschierstaedt combinedgenomicandproteomicapproachesrevealdnabindingsitesandinteractionpartnersoftbx2inthedevelopinglung AT vincentmchristoffels combinedgenomicandproteomicapproachesrevealdnabindingsitesandinteractionpartnersoftbx2inthedevelopinglung AT patrickkunzler combinedgenomicandproteomicapproachesrevealdnabindingsitesandinteractionpartnersoftbx2inthedevelopinglung AT andreaskispert combinedgenomicandproteomicapproachesrevealdnabindingsitesandinteractionpartnersoftbx2inthedevelopinglung |