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All rights reserved. http://resource.belframework.org/belframework/20131211/knowledge/large_corpus.bel http://purl.org/dc/elements/1.1/title BEL Framework Large Corpus Document http://resource.belframework.org/belframework/20131211/knowledge/large_corpus.bel http://purl.org/pav/authoredBy http://www.tkuhn.ch/bel2nanopub/RAlw9fjznLnqsVBkmr1LDZhkYSTSUDA_cHexsYpNQEWjg#_6 http://resource.belframework.org/belframework/20131211/knowledge/large_corpus.bel http://purl.org/pav/version 20131211 http://www.tkuhn.ch/bel2nanopub/RAlw9fjznLnqsVBkmr1LDZhkYSTSUDA_cHexsYpNQEWjg#_5 http://www.w3.org/ns/prov#value This activity was totally lost when the experiment was carried out in NIH 3T3 cells stably expressing p160 (Fig. 4B). We also tested whether p160 inhibited the DNA-binding activity of Prep1-Pbx1, using EMSA with in vitro-translated proteins and the b2-PP2 oligonucleotide from the HoxB2 enhancer (see Materials and Methods). In vitro-translated Prep1 and Pbx1a formed a retarded complex that was not observed with Prep1 or Pbx1 alone, as expected (18). No binding was observed with p160 alone. Preincubation with in vitro-translated p160 specifically decreased the binding of Prep1-Pbx1a complexes (see Fig. S5 in the supplemental material). Finally, transient-transfection experiments showed that expression of either p160 or p67 also inhibited the transcription-activating effect of Prep1, Pbx1, and HoxB1 on the luciferase activity of the HoxB1 and HoxB2 enhancer constructs (see Fig. S4 in the supplemental material). These data are consistent with a model in which p160 and/or p67 interacts with Prep1, preventing the binding of Prep1 to Pbx1 and to DNA and thus the Prep1-Pbx1 transcriptional activity. To explore whether p160 overexpression inhibited endogenous Prep1 activity, we turned to NT2-D1 teratocarcinoma cells, which can be induced by RA to differentiate and to transcribe the HoxB genes (45). We first used EMSA and inhibition by specific antibodies to determine the nature of the proteins binding the enhancer of the HoxB2 gene (oligonucleotide b2-PP2; see Materials and Methods). As shown in Fig. 4C, control cell extracts (lane 1) produced a single band. In cells treated with 10 µM RA, two major bands appeared (lane 2), one comigrating with the single band in control cells and an upper band. The lower band was inhibited by Prep1 and Pbx1 antibodies (lanes 3 and 4) and hence corresponds mostly to a Prep1-Pbx1 dimer. The upper band was inhibited by Prep1, Pbx1 (not Pbx2), and HoxB1 antibodies and hence was mostly represented by the Prep1-Pbx1-HoxB1 trimer, as expected (18). We then tested pools of cells stably expressing p160 or empty vector to compare their DNA-binding activities (with or without treatment with 10 µM RA) and their RA-induced expression of HoxB2. As shown in the EMSA in Fig. 4D, overexpression of p160 decreased the intensity of the shifted band in uninduced cells and even more in RA-treated cells (i.e., the binding due to Prep1-Pbx1 dimers and Prep1-Pbx1-HoxB1 trimers). Expression of p160 had no effect on the level of endogenous Prep1 (Fig. 4D) or Pbx1b (not shown) and did not change the DNA-binding activity of the unrelated SP1 transcription factor. The functional significance of the effect observed by EMSA was seen when we determined by real-time PCR the level of HoxB2 mRNA induced by RA. As shown in Fig. 4E, HoxB2 mRNA induction was decreased by about 45% in the cells expressing p160. Overall, these experiments allowed us to conclude that the ectopic expression of p160 inhibits Prep1 DNA binding and transcriptional activities both in vitro and in vivo, in agreement with the hypothesis that p160 competes with Pbx1 for Prep1 binding. http://www.tkuhn.ch/bel2nanopub/RAlw9fjznLnqsVBkmr1LDZhkYSTSUDA_cHexsYpNQEWjg#_5 http://www.w3.org/ns/prov#wasQuotedFrom http://www.ncbi.nlm.nih.gov/pubmed/17875935 http://www.tkuhn.ch/bel2nanopub/RAlw9fjznLnqsVBkmr1LDZhkYSTSUDA_cHexsYpNQEWjg#_6 http://www.w3.org/2000/01/rdf-schema#label Selventa http://www.tkuhn.ch/bel2nanopub/RAlw9fjznLnqsVBkmr1LDZhkYSTSUDA_cHexsYpNQEWjg#assertion http://www.w3.org/ns/prov#hadPrimarySource http://www.ncbi.nlm.nih.gov/pubmed/17875935 http://www.tkuhn.ch/bel2nanopub/RAlw9fjznLnqsVBkmr1LDZhkYSTSUDA_cHexsYpNQEWjg#assertion http://www.w3.org/ns/prov#wasDerivedFrom http://resource.belframework.org/belframework/20131211/knowledge/large_corpus.bel http://www.tkuhn.ch/bel2nanopub/RAlw9fjznLnqsVBkmr1LDZhkYSTSUDA_cHexsYpNQEWjg#assertion http://www.w3.org/ns/prov#wasDerivedFrom http://www.tkuhn.ch/bel2nanopub/RAlw9fjznLnqsVBkmr1LDZhkYSTSUDA_cHexsYpNQEWjg#_5 http://www.tkuhn.ch/bel2nanopub/RAlw9fjznLnqsVBkmr1LDZhkYSTSUDA_cHexsYpNQEWjg#pubinfo http://www.tkuhn.ch/bel2nanopub/RAlw9fjznLnqsVBkmr1LDZhkYSTSUDA_cHexsYpNQEWjg http://purl.org/dc/terms/created 2014-07-03T14:33:12.471+02:00 http://www.tkuhn.ch/bel2nanopub/RAlw9fjznLnqsVBkmr1LDZhkYSTSUDA_cHexsYpNQEWjg http://purl.org/pav/createdBy http://orcid.org/0000-0001-6818-334X http://www.tkuhn.ch/bel2nanopub/RAlw9fjznLnqsVBkmr1LDZhkYSTSUDA_cHexsYpNQEWjg http://purl.org/pav/createdBy http://orcid.org/0000-0002-1267-0234