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

<div><p>The TBX18 transcription factor is a crucial developmental regulator of several organ systems in mice, and loss of its transcriptional repression activity causes dilative nephropathies in humans. The molecular complexes with which TBX18 regulates transcription are poorly understood prompting us to use an unbiased proteomic approach to search for protein interaction partners. Using overexpressed dual tagged TBX18 as bait, we identified by tandem purification and subsequent LC-MS analysis TBX18 binding proteins in 293 cells. Clustering of functional annotations of the identified proteins revealed a highly significant enrichment of transcriptional cofactors and homeobox transcription factors. Using nuclear recruitment assays as well as GST pull-downs, we validated CBFB, GAR1, IKZF2, NCOA5, SBNO2 and CHD7 binding to the T-box of TBX18 <i>in vitro</i>. From these transcriptional cofactors, CBFB, CHD7 and IKZF2 enhanced the transcriptional repression of TBX18, while NCOA5 and SBNO2 dose-dependently relieved it. All tested homeobox transcription factors interacted with the T-box of TBX18 in pull-down assays, with members of the <i>Pbx</i> and <i>Prrx</i> subfamilies showing coexpression with <i>Tbx18</i> in the developing ureter of the mouse. In summary, we identified and characterized new TBX18 binding partners that may influence the transcriptional activity of TBX18 <i>in vivo</i>.</p></div

TBX18 colocalizes with most of the candidate transcriptional cofactors in the nucleus of 293 cells.

<p>(A) 293 cells were transfected with expression constructs for MYC-tagged GAR1, IKZF2, NCOA5, SSXB2 or SUV39H2 (<i>green)</i> in the presence of HA-tagged TBX18 lacking the nuclear localization signal (TBX18ΔNLS, <i>red</i>), or with expression constructs for HA-tagged BASP1, CHD7 or SBNO2 (red) in the presence of MYC-tagged TBX18ΔNLS (green). Immunofluorescence analysis shows that NLS-deficient TBX18 protein is efficiently shuttled from the cytoplasm to the nucleus by all candidate proteins except SUV39H2. (B) 293 cells were transfected with expression constructs for MYC-tagged RCOR3 or CBFB (<i>green)</i> in the presence of HA-tagged full-length TBX18 protein (TBX18ΔNLS, <i>red</i>). Immunofluorescence analysis shows that TBX18 protein recruits RCOR3 and CBFB from the cytoplasm into the nucleus. DAPI, 4,6-diamidino-2-phenylindole nuclear counterstain. Scale bar length is 25 μm.</p

Homeobox transcription factors directly bind to the T-box of TBX18 and are coexpressed with <i>Tbx18</i> during ureter development.

<p>(A) Autoradiographic analysis of pull-down assays of GST and of fusion proteins of GST with N-, N+T (NT)-, T-, and C-domains of TBX18 from <i>E</i>. <i>coli</i> extracts, and reticulocyte lysates programmed for <i>in vitro</i> translation of ³⁵S-labelled full-length candidate transcriptional cofactors. DUXBL1, GSC, PAX1, PBX1, PBX4 and PRRX2 interact with the T-box of TBX18. (B) Comparative RNA <i>in situ</i> hybridization analysis on transverse sections of the proximal ureter of E12.5 embryos of <i>Tbx18</i> and of genes encoding homeobox transcription factor candidates and related subfamily members. ue, ureteric epithelium, um, ureteric mesenchyme. Scale bar length is 25 μm.</p

Candidate transcriptional cofactors modify the transcriptional repression activity of TBX18.

<p>(A) Luciferase assays from extracts of 293 cells cotransfected with 250 ng of the <i>pGL3</i>.<i>Prom</i>.<i>Tbx18BS2</i> reporter plasmid, 250 ng of the TBX18 expression plasmid <i>pcDNA3</i>.<i>Tbx18</i> and 25, 250 or 500 ng of plasmids for expression of the transcriptional cofactors. Luciferase activity is normalized to a cotransfection of the reporter plasmid with an empty <i>pcDNA3</i> expression vector. TBX18 represses luciferase activity to about 50% of the control value. CBFB, CHD7, IKZF2, RCOR3 further augment repression by TBX18; NCOA5, SBNO2 and SSXB2 relieve TBX18 repression activity. Values are displayed as mean ± sd. * P≤0.05 ** P≤0.01 ***P≤0.001; two-tailed Student's t-test. (For statistical values see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200964#pone.0200964.s010" target="_blank">S6 Table</a>). (B) Summary of TBX18 binding and activation assays for transcriptional cofactor candidates. Three cofactors (CBFB, CHD7, IKZF2) reliably interact with the T-box of TBX18 and repress transcription. Two cofactors interact with the TBX18 T-box but relieve repression.</p

TBX18 does not bind to candidate interaction partners at the centrosome.

<p>(A) Autoradiographic analysis of pull-down assays of GST and fusion proteins of GST with N-, N+T- (NT), T-, and C-domains of TBX18 from <i>E</i>. <i>coli</i> extracts, and reticulocyte lysates programmed for <i>in vitro</i> translation of ³⁵S-labelled full-length candidate centrosomal proteins. B9D2, HAUS8, MAP9, PARP3, SYBU and TPX2 interact with the T-box of TBX18; B9D2 and SYBU additionally bind to the C-, HAUS8 to the N-terminal domain. (B) Immunofluorescent staining against epitope tags of candidate centrosomal proteins expressed in 293 cells reveals nuclear localization of B9D2 and TPX2, cytoplasmic localization of HAUS8 and ASAP, and confinement of PARP3 to cellular protrusions. (C) 293 cells were transfected with expression constructs for MYC-tagged B9D2 and TPX2 (<i>green)</i> in the presence of HA-tagged TBX18 lacking the nuclear localization signal (TBX18ΔNLS, <i>red</i>), or with expression constructs for HA-tagged HAUS8, ASAP and PARP3 (<i>red</i>) in the presence of MYC-tagged full-length TBX18 (<i>green</i>). Immunofluorescence analysis shows that NLS-deficient TBX18 protein is efficiently shuttled from the cytoplasm to the nucleus by B9D2 and TPX2 while the extranuclear localization of HAUS8, ASAP and PARP3 is unaffected by coexpression of full-length TBX18. (D) 293 cells were transfected with an expression construct for MYC-tagged TBX18 (<i>green)</i>. Co-immunofluorescence analysis shows that TBX18 protein does not localize to the centrosome marked by TUBG expression (<i>red</i>) but to the nucleus. Scale bar length is 25 μm.</p

Mass spectrometry analysis identifies novel TBX18 interacting proteins in 293 cells.

<p>(A) Diagram of the identification strategy. A construct encoding mouse TBX18 protein fused to a N-terminal triple FLAG tag followed by a biotinylation signal peptide while also encoding the bacterial BirA enzyme required for biotinylation was transfected into 293 cells. Protein complexes containing TBX18 were purified from nuclear cell extracts by a two-step affinity purification strategy using Anti-FLAG (red) and anti-Biotin chromatography (green). The purified protein complexes were resolved by SDS-PAGE. Silver staining of a sample aliquot is displayed, arrowheads show IgG heavy and light chains. After extraction, they were subjected to LC-MS analysis. Proteins were functionally classified and clustered. (B) Gene annotation enrichment analysis uncovered clusters of transcriptional cofactors, transcription factors of the homeobox family, and centrosomal proteins. Gene ontology analysis is provided. UNIPROT: Uniprot accession number. Database annotations are UP: Uniprot, GO: Gene Ontology, IP: Interpro, SM: Smart.</p

TBX18 physically interacts with candidate transcriptional cofactors preferentially via its T-box.

<p>(A) Western blot analysis of pull-down assays performed with GST and fusion proteins of GST with N-, N+T (NT), T-, and C-domains of TBX18 obtained from <i>E</i>. <i>coli</i> extracts, and protein extracts from 293 cells transfected with MYC- or HA-tagged full-length expression constructs of candidate transcriptional cofactors. Detection was performed with anti-MYC immunohistochemistry for CBFB, GAR1, IKZF2, NCOA5, RCOR3, SSXB2 and SUV39H2, and anti-HA immunohistochemistry for NKX2.5, BASP1, SBNO2 and CHD7. (B) Autoradiographic analysis of pull-down assays performed with the same GST-TBX18 fusion proteins and reticulocyte lysates programmed for <i>in vitro</i> translation of ³⁵S-labelled full-length candidate transcriptional cofactors. CBFB, GAR1, IKZF2, SBNO2, NCOA5 and CHD7 interact with the T-box of TBX18 as does the control protein NKX2.5. Due to the large size of CHD7, subfragments were expressed by <i>in vitro</i> translation and used in the pull-down assay. CHD7-1, amino acid residues 1–799; CHD7-2, 732–1567; CHD7-3, 1533–2380; CHD7-4, 2325–2997.</p

Lack of Genetic Interaction between <i>Tbx18</i> and <i>Tbx2/Tbx20</i> in Mouse Epicardial Development

<div><p>The epicardium, the outermost layer of the heart, is an essential source of cells and signals for the formation of the cardiac fibrous skeleton and the coronary vasculature, and for the maturation of the myocardium during embryonic development. The molecular factors that control epicardial mobilization and differentiation, and direct the epicardial-myocardial cross-talk are, however, insufficiently understood. The T-box transcription factor gene <i>Tbx18</i> is specifically expressed in the epicardium of vertebrate embryos. Loss of <i>Tbx18</i> is dispensable for epicardial development, but may influence coronary vessel maturation. In contrast, over-expression of an activator version of TBX18 severely impairs epicardial development by premature differentiation of epicardial cells into SMCs indicating a potential redundancy of <i>Tbx18</i> with other repressors of the T-box gene family. Here, we show that <i>Tbx2</i> and <i>Tbx20</i> are co-expressed with <i>Tbx18</i> at different stages of epicardial development. Using a conditional gene targeting approach we find that neither the epicardial loss of <i>Tbx2</i> nor the combined loss of <i>Tbx2</i> and <i>Tbx18</i> affects epicardial development. Similarly, we observed that the heterozygous loss of <i>Tbx20</i> with and without additional loss of <i>Tbx18</i> does not impact on epicardial integrity and mobilization in mouse embryos. Thus, <i>Tbx18</i> does not function redundantly with <i>Tbx2</i> or <i>Tbx20</i> in epicardial development.</p></div

Phenotypic analysis of hearts with combined loss of T<i>bx18</i> and <i>Tbx2</i> in the epicardium at E18.5.

<p>(A) Histological analysis by hematoxylin and eosin staining of transverse sections of <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/fl</i> hearts reveals a dilatation of the atria in comparison to <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/+</i> or <i>Tbx18</i>^<i>cre/+</i><i>;Tbx2</i>^<i>fl/fl</i> or control (<i>Tbx18</i>^<i>cre/+</i>) hearts. Atrial dilatation is occasionally seen in <i>Tbx18</i>-deficient mice as well (not shown). The ventricular compartment of <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/fl</i> hearts appears indistinguishable from control hearts (n = 2). The scale bars are 500 μm. (B) Immunofluorescence analysis of ACTA2 and TAGLN expression shows normal differentiation of coronary SMCs and their localization to coronary arteries (CAr). Capillary density, although not quantified, appears unaffected by the loss of <i>Tbx18</i>, <i>Tbx2</i> or both genes in the epicardium as visualized by immunofluorescence against EMCN. The presence of POSTN in the myocardium confirms the formation of cardiac fibroblasts from epicardial cells in all mutants. Two specimens per genotype were analyzed. Scale bars are 100 μm. Epi, epicardium; LA, left atrium; LV, left ventricle; Peri, pericardium; RA, right atrium; RV, right ventricle.</p

Phenotypic analysis of hearts with combined loss of <i>Tbx18</i> and <i>Tbx2</i> in the epicardium at E14.5.

<p>(A) Histological analysis of one to two embryos per genotype by hematoxylin and eosin staining of transverse heart sections does not reveal any gross morphological defects in <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/fl</i> double mutant hearts compared to control (<i>Tbx18</i>^<i>cre/+</i>) or compound mutant (<i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/+</i> or <i>Tbx18</i>^<i>cre/+</i><i>;Tbx2</i>^<i>fl/fl</i>) embryos. The scale bars are 500 μm. The black arrows point to pericardial defects observed in <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/fl</i> and <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/+</i> hearts. (B) Higher magnification of the right ventricle shows a tightly attached epicardium on top of the heart in all genotypes. The scale bars are 20 μm (n = 1). (C) Immunofluorescence analysis of GFP and WT1 expression confirms epicardial integrity and subepicardial as well as myocardial localization of epicardium-derived cells in all genotypes. The scale bars represent 50 μm. The in-growing vasculature, visualized by EMCN immunofluorescence, has almost reached the apex of the right ventricle (white arrows). TAGLN is not expressed in the epicardium as emphasized by double immunofluorescence with COLIV (grey arrowheads). In contrast, NOTCH3 expression is found in the epicardium of <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/fl</i> and <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/+</i> mice and occasionally in <i>Tbx18</i>^<i>cre/+</i><i>;Tbx2</i>^<i>fl/fl</i> and control hearts (white arrowheads). Double staining with the <i>Tbx18</i>-lineage marker GFP indicates NOTCH3-positive cells in the epicardium of <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/fl</i> and <i>Tbx18</i>^{<i>cre/GFP</i>}<i>;Tbx2</i>^<i>fl/+</i> mice. Dashed lines mark the border between epicardium and myocardium. Scale bars in NOTCH3 and TAGLN single staining are 50 μm, and 20 μm in the double staining of these markers with GFP or COLIV. Two specimens per genotype and stage were analyzed by immunostaining. Epi, epicardium; LA, left atrium; LV, left ventricle; Peri, pericardium; RA, right atrium; RV, right ventricle.</p