Charakterisierung des Gens Fbxl22 und seiner Funktion während der Muskelentwicklung der Maus

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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

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

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

Hypoxia and the anticoagulants dalteparin and acetylsalicylic acid affect human placental amino acid transport.

BACKGROUND: Anticoagulants, e.g. low-molecular weight heparins (LMWHs) and acetylsalicylic acid (ASA) are prescribed to women at risk for pregnancy complications that are associated with impaired placentation and placental hypoxia. Beyond their role as anticoagulants these compounds exhibit direct effects on trophoblast but their impact on placental function is unknown. The amino acid transport systems A and L, which preferably transfer essential amino acids, are well-described models to study placental nutrient transport. We aimed to examine the effect of hypoxia, LMWHs and ASA on the activity of the placental amino acid transport systems A and L and associated signalling mechanisms. METHODS: The uptake of C14-MeAIB (system A) or H3-leucin (system L) was investigated after incubation of primary villous fragments isolated from term placentas. Villous tissue was incubated at 2% O2 (hypoxia), 8% O2 and standard culture conditions (21% O2) or at 2% O2 and 21% O2 with dalteparin or ASA. Activation of the JAK/STAT or mTOR signalling pathways was determined by Western analysis of total and phosphorylated STAT3 or Raptor. RESULTS: Hypoxia decreased system A mediated MeAIB uptake and increased system L mediated leucine uptake compared to standard culture conditions (21% O2). This was accompanied by an impairment of STAT3 and a stimulation of Raptor signalling. System L activity increased at 8% O2. Dalteparin treatment reduced system A and system L activity under normoxic conditions and ASA (1 mM) decreased system A and L transporter activity under normoxic and hypoxic conditions. CONCLUSIONS: Our data underline the dependency of placental function on oxygen supply. LMWHs and ASA are not able to reverse the effects of hypoxia on placental amino acid transport. These findings and the uncovering of the signalling mechanisms in more detail will help to understand the impact of LMWHs and ASA on placental function and fetal growth

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

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