Journal: Respiratory Research

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

doi: 10.1186/s12931-021-01679-y

Figure Lengend Snippet: ChIP-Seq analysis identifies genomic binding sites of TBX2 in E14.5 lungs. a , b Analysis of TBX2 ChIP-sequencing peaks with Genomic Regions Enrichment of Annotations Tool (GREAT, version 4.0.4). a Bar diagram showing the orientation and distance of TBX2 ChIP peaks to a transcription start site (TSS). b Functional annotation shows enrichment of genes associated with TBX2 ChIP peaks in clusters with annotated mouse phenotypes and biological processes sorted by − log10 binomial p-value. c De-novo motif analysis was performed in Galaxy using FIMO—Scan a set of sequences for motifs (Galaxy v4.11.1.0) Novel consensus sequences are highlighted in colored boxes and compared to known motifs with TomTom Motif Comparison Tool v5.1.1. One palindromic and one non-palindromic motif with similarities to a known TBX2 binding element in the Jaspar database were discovered with E-values of 5.9e−198 and 4.6e−152 (grey boxes). Additional novel palindromic sequences show similarities to a TBX21 binding site in the footprint database, E = 1.7e−252 (green box), an ETS (Jaspar database) and homeobox (footprint database) binding motif, E = 6.8e−497 (red box), and an HMG-Box binding site (footprintDB), E = 4.4e−300 (blue box). e Analysis of motif localization by GREAT discovered conjunct motifs for TBX2 (green), ETS/homeobox (red) and HMG-box proteins (blue) in TBX2 ChIP-Seq peaks. Motifs are colored as in c and colored boxes in d reflect spatial arrangement and interconnection of motifs on both DNA strands (+ and −)

Article Snippet: The DNA-containing supernatants were incubated with a ChIP grade anti-TBX2 antibody (1:50; sc-514291 X, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-Histone H3 (1:50; #9005, Cell Signaling Technology) or an IgG control (1:50; #9005, Cell Signaling Technology) for 1 h at room temperature, and together with ChIP-Grade ProteinG Magnetic Beads (#9006S, Cell Signaling Technology) overnight at 4 °C.

Techniques: ChIP-sequencing, Binding Assay, Functional Assay, Comparison

Journal: Respiratory Research

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

doi: 10.1186/s12931-021-01679-y

Figure Lengend Snippet: Microarray analysis identifies functional targets of TBX2 activity in E14.5 lungs. a Pie-chart summarizing the results of 4 individual transcriptional profiling experiments by microarrays of E14.5 control and Tbx2 -deficient lungs. b Intersection of the list of genes upregulated in the microarrays of E14.5 Tbx2- deficient lungs and the list of genes associated with TBX2 ChIP peaks in the E14.5 lung. c List of genes upregulated in the microarrays of E14.5 Tbx2 -deficient lungs and having a TBX2 ChIP-peak. Shown are the average fold changes (avgFC) of the 4 individual microarray data sets. d Functional annotation analysis by MouseMine websoftware identifies functional enrichment of terms related to stress response and growth control in the set of 39 genes upregulated in the microarrays of E14.5 Tbx2 -deficient lungs and having a TBX2 ChIP-peak. e RNA in situ hybridization analysis of Ccn4 and Il33 expression on sections of E14.5 control and Tbx2 -deficient lungs. f Scheme depicting the genomic loci of Ccn4 and Il33 . Binding peaks identified by ChIP-Seq analysis are indicated above. Black boxes indicate peaks further validated by ChIP-PCR. g ChIP-PCR-validation of peaks in Il33 and Ccn4 as indicated in ( f ). Lanes were loaded as indicated

Article Snippet: The DNA-containing supernatants were incubated with a ChIP grade anti-TBX2 antibody (1:50; sc-514291 X, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-Histone H3 (1:50; #9005, Cell Signaling Technology) or an IgG control (1:50; #9005, Cell Signaling Technology) for 1 h at room temperature, and together with ChIP-Grade ProteinG Magnetic Beads (#9006S, Cell Signaling Technology) overnight at 4 °C.

Techniques: Microarray, Functional Assay, Activity Assay, Control, RNA In Situ Hybridization, Expressing, Binding Assay, ChIP-sequencing, Biomarker Discovery

Journal: Respiratory Research

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

doi: 10.1186/s12931-021-01679-y

Figure Lengend Snippet: LC–MS/MS identifies TBX2 interaction partner in E14.5 lungs. a Diagram depicting the strategy to identify TBX2 interacting proteins in embryonic lungs. Tissue of E14.5 wildtype formaldehyde fixed lungs was homogenized, cells were lysed, and nuclei extracted. Protein complexes containing TBX2 were purified with an α-TBX2 antibody. Subsequent LC–MS/MS analysis and statistical filtering (Student's t -test difference of ≥ 2) revealed an enrichment of 219 proteins within the α-TBX2 fraction compared to the control lacking the α-TBX2 antibody. Manual exclusion of mitochondrial, proteasomal, and ribosomal proteins as well as hemoglobins, immunoglobins and non-nuclear proteins lead to a list of 119 candidate proteins. Of these, 22 were associated with the GO term “transcriptional regulation”, 7 with the terms “histone/histone modification”. 7 proteins were in the intersection of both GO term lists. b , c List of enriched proteins associated with the GO term “transcription regulation” ( b ) and “histones” or “histone modification” ( c ) according to DAVID functional analysis. d STRING analysis of interactions of the candidate proteins shown in ( b ) and ( c ). Three clusters were identified using MCL clustering with an inflation parameter of 2, an interaction score of high confidence (0.700) and deactivating the interaction source "textmining"

Article Snippet: The DNA-containing supernatants were incubated with a ChIP grade anti-TBX2 antibody (1:50; sc-514291 X, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-Histone H3 (1:50; #9005, Cell Signaling Technology) or an IgG control (1:50; #9005, Cell Signaling Technology) for 1 h at room temperature, and together with ChIP-Grade ProteinG Magnetic Beads (#9006S, Cell Signaling Technology) overnight at 4 °C.

Techniques: Liquid Chromatography with Mass Spectroscopy, Purification, Control, Modification, Functional Assay

Journal: Respiratory Research

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

doi: 10.1186/s12931-021-01679-y

Figure Lengend Snippet: Interaction candidates are coexpressed with TBX2 in the pulmonary mesenchyme and interact in HEK293 cells. a Co-immunofluorescence analysis of candidate interaction partners (red) and TBX2 (green) on frontal sections of the right lung of E14.5 Tbx2 cre/ + embryos. Antigens are color-coded and nuclei were counterstained with DAPI (blue). Insets or selected regions in overview images are magnified in rows 2,4 and 6. b In situ proximity ligation assay of TBX2 and candidate interaction partners on 10 µm frontal sections of E14.5 wildtype and Tbx2 cre/fl mutant lungs. Direct interaction is visualized by small red fluorescent dots. Larger more diffuse orange stains are due to auto-fluorescence of blood cells. Nuclei are counterstained with DAPI (blue). c Western blot analysis of co-immunoprecipitation experiments for verification of TBX2 interaction with candidate proteins on 10% SDS polyacrylamide gels. Detection was performed with an anti-TBX2 primary antibody and developed with chemoluminescence-IHC. Arrows indicate TBX2 bands. Lanes were loaded as follows: No antibody: IP without specific antibody resembling negative IP-control; 5% input: 5% of crude cell extract before precipitation; empty: no protein loaded; IP: co-immunoprecipitate with antibody for specific candidate. Expected molecular weight for TBX2.HA approx. 76.2 kDa

Article Snippet: The DNA-containing supernatants were incubated with a ChIP grade anti-TBX2 antibody (1:50; sc-514291 X, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-Histone H3 (1:50; #9005, Cell Signaling Technology) or an IgG control (1:50; #9005, Cell Signaling Technology) for 1 h at room temperature, and together with ChIP-Grade ProteinG Magnetic Beads (#9006S, Cell Signaling Technology) overnight at 4 °C.

Techniques: Immunofluorescence, In Situ, Proximity Ligation Assay, Mutagenesis, Fluorescence, Western Blot, Immunoprecipitation, Control, Molecular Weight

Journal: Respiratory Research

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

doi: 10.1186/s12931-021-01679-y

Figure Lengend Snippet: Scheme of the molecular interactions of TBX2 in repression of target genes in the pulmonary mesenchyme. Grey discs represent nucleosomes. DNA is displayed as black line. Dotted line indicates unknown length of DNA fragment and possible long distance. Circles for proteins do not represent the real size. TBX2 binds in concert with HMGB2 and PBX1 to specific DNA sites in enhancer regions. Binding to PBX1 is probably indirect via unknown proteins, binding to HMGB2 is direct and may aid in chromatin bending and bringing enhancer regions to the proximity of transcription start sites. TBX2/DNMT1 binding may induce DNA methylation to repress gene expression. TBX2 and CBX3 directly bind and both associate with the NuRD complex for chromatin remodeling. TBX2/MYBBP1 interaction may strengthen the association with the NuRD complex to repress target genes. TBX2 directly interacts with components of the NuRD complex, namely HDACs for chromatin deacetylation and the chromatin remodeling complex CHD4. TBX2 interaction with CBX3, HDACs and CHD4 collectively induces chromatin compaction. RBBP4/7 binds to chromatin to consolidate binding of the NuRD-complex to target gene DNA

Article Snippet: The DNA-containing supernatants were incubated with a ChIP grade anti-TBX2 antibody (1:50; sc-514291 X, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-Histone H3 (1:50; #9005, Cell Signaling Technology) or an IgG control (1:50; #9005, Cell Signaling Technology) for 1 h at room temperature, and together with ChIP-Grade ProteinG Magnetic Beads (#9006S, Cell Signaling Technology) overnight at 4 °C.

Techniques: Binding Assay, DNA Methylation Assay, Gene Expression