Proteomic analysis identifies transcriptional cofactors and homeobox transcription factors as TBX18 binding proteins

Rivera-Reyes, Reginaldo; Kleppa, Marc-Jens; Kispert, Andreas

doi:10.1371/journal.pone.0200964

Cited by 12 publications

(17 citation statements)

References 63 publications

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“…We used the following expression vectors for transfections in HEK293 cells: pcDNA3.huTBX2.HA encoding N-terminally HA-tagged full-length human TBX2; pCS2.Pbx1b encoding full-length mouse PBX1B , (gift from Heike Pöpperl, Institute for Biophysical Chemistry, Hannover Medical School, Germany); pd2EGFP-N1 (EGFP-expression vector) [ 35 ]; pCMV6-Entry.Cbx3-Myc-DDK encoding full-length mouse CBX3 (#MR224357, OriGene, Rockville, Maryland, USA); pCMV-SPORT6.Hdac1 encoding full-length mouse HDAC1 (#4217199, Sourcebioscience, Nottingham, UK); pCMV6-Entry.Hdac2-Myc-DDK encoding full-length mouse HDAC2 (#MR226709, OriGene); pCMV-SPORT6.Chd4 encoding full-length mouse CHD4 (#6489649, Sourcebioscience); pCMV-Entry.Hmgb2-GFP encoding full-length mouse HMGB2 (#MR202276, OriGene).…”

Section: Methodsmentioning

confidence: 99%

Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung

et al. 2021

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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.

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Section: Methodsmentioning

confidence: 99%

Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung

et al. 2021

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“…Approximately 1x10 8 Flp-In™ T-REx™ 293 cells stably expressing human TFs were induced with 2 μg/ml of tetracycline (AP-MS and BioID) and 50 μM biotin (BioID) for 24 hours. The cells were pelleted using centrifugation, snap frozen in liquid nitrogen and stored at -80°C.…”

Section: Affinity Purification and Mass Spectrometry Analysismentioning

confidence: 99%

“…Complex and multilayer regulation of transcription involves not only direct binding of TFs to a target gene's regulatory element(s) but there exists a complicated interplay between TFs and TF binding proteins. These include several cofactors, the Mediator complex, basal transcription machinery, TFactivity modulating enzymes (such as phosphatases and kinases), dimerization partners, subunits and inhibitory proteins [5][6][7][8] . Moreover, as the chromosomal DNA is packed into chromatin to prevent uncontrolled transcription, TFs also interact with several chromatin remodeling proteins.…”

Section: Introductionmentioning

confidence: 99%

Human transcription factor protein interaction networks

Göös

Kinnunen

Yadav

et al. 2021

Preprint

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In participation of transcriptional regulation, transcription factors (TFs) interact with several other proteins. Here, we identified 7233 and 2176 protein-protein interactions for 110 different human TFs through proximity-dependent biotinylation (BioID) and affinity purification mass spectrometry (AP-MS), respectively. The BioID analysis resulted more high-confident interactions, highlighting the transient and dynamic nature of many of the TF interactions. Using clustering and correlation analyses, we identified subgroups of TFs associated with specific biological functions, such as RNA-splicing, actin signalling or chromatin remodeling. We also observed 203 TF-TF interactions, of which 175 were interactions with Nuclear Factor 1 (NFI) -family members, indicating uncharacterized cross-talk between NFI signalling and numerous other TF signalling. Moreover, TF interactions with basal transcription machinery were mainly observed through TFIID and SAGA complexes. This study, not only, provides a rich resource of human TF interactions, but also act as starting point directing future studies aimed at understanding TF mediated transcription.

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“…Using overexpressed dual tagged TBX18 as bait, we identified by tandem purification and subsequent liquid chromatographymass spectrometry (LC-MS) analysis several transcriptional cofactors and homeobox transcription factors as preferred binding partners in 293 cells. The transcriptional cofactors CBFB, GAR1, IKZF2, NCOA5, SBNO2 and CHD7 and all identified homeobox proteins (members of the DUX, GSC, PAX, PBX1 and PRRX families) bound to the T-box of TBX18 in vitro and interacted with TBX18 upon coexpression in 293 cell nuclei [27].…”

Section: Introductionmentioning

confidence: 99%

“…The pd2EGFP-N1 plasmid (Clontech, Mountain View, CA, USA) was independently transfected to check for efficiency of transfection, which was verified by epifluorescence microscopy. Analysis of TBX18 mRNA expression in human cell lines was done by semiquantitative RT-PCR with normalization against GAPDH exactly as described before [27].…”

Section: Introductionmentioning

confidence: 99%

Proteomic analysis identifies ZMYM2 as endogenous binding partner of TBX18 protein in 293 and A549 cells

Lüdtke

Kleppa

Rivera-Reyes

et al. 2022

Biochemical Journal

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The TBX18 transcription factor regulates patterning and differentiation programs in the primordia of many organs yet the molecular complexes in which TBX18 resides to exert its crucial transcriptional function in these embryonic contexts have remained elusive. Here, we used 293 and A549 cells as an accessible cell source to search for endogenous protein interaction partners of TBX18 by an unbiased proteomic approach. We tagged endogenous TBX18 by CRISPR/Cas9 targeted genome editing with a triple FLAG peptide, and identified by anti-FLAG affinity purification and subsequent LC-MS analysis the ZMYM2 protein to be statistically enriched together with TBX18 in both 293 and A549 nuclear extracts. Using a variety of assays, we confirmed binding of TBX18 to ZMYM2, a component of the CoREST transcriptional corepressor complex. Tbx18 is coexpressed with Zmym2 in the mesenchymal compartment of the developing ureter of the mouse, and mutations in TBX18and in ZMYM2 were recently linked to congenital anomalies in the kidney and urinary tract (CAKUT) in line with a possible in vivo relevance of TBX18-ZMYM2 protein interaction in ureter development.

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Proteomic analysis identifies transcriptional cofactors and homeobox transcription factors as TBX18 binding proteins

Cited by 12 publications

References 63 publications

Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung

Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung

Human transcription factor protein interaction networks

Proteomic analysis identifies ZMYM2 as endogenous binding partner of TBX18 protein in 293 and A549 cells

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