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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/RAgAZGmKKw_vwJnyN4xbpVE2idhPjnZCtN_yA_5fQKots#_6 http://resource.belframework.org/belframework/20131211/knowledge/large_corpus.bel http://purl.org/pav/version 20131211 http://www.tkuhn.ch/bel2nanopub/RAgAZGmKKw_vwJnyN4xbpVE2idhPjnZCtN_yA_5fQKots#_5 http://www.w3.org/ns/prov#value Sequence analysis of the immunoprecipitated Tie2 and Foxf1 promoter regions confirmed the presence of several of the recently identified HOXA13 binding sites (Figure 10B) [71]. Next, using an electrophoretic mobility shift assay (EMSA), the HOXA13 DNA binding domain was confirmed to bind the promoter regions detected by the ChIP assay in a concentration-dependent manner (Figure 10C). Quantitation of HOXA13's affinity for the Tie2 and Foxf1 ChIP-positive regions using fluorescence polarization anisotropy revealed high affinity for the binding sites present in Tie2 (Kd = 27±1.4 nM and 22 nM±1.6 nM) and Foxf1 (Kd = 48±4 nM) compared to a control sequence lacking the HOXA13 binding site (Kd = 250±22 nM) (Figure 10D and 10E). Next, the capacity of HOXA13 to regulate gene expression through the 140 base-pair Tie2 and 121 base-pair Foxf1 ChIP-positive DNA fragments was examined (Figure 10F). The pGL3-Basic vector was selected for this analysis based on previous studies that confirm its capacity to assess promoter/enhancer activity in vitro, including previous characterizations of HOXA13's capacity to regulate transcription from minimal promoter elements [72], [74]–[78]. In the absence of the Tie2 or Foxf1 DNA elements, the empty pGL3-basic luciferase plasmid exhibited only a minor increase in luciferase expression when co-transfected with a Hoxa13 expression plasmid (Figure 10F). Similarly, the same luciferase vector containing either the Tie2 or the Foxf1 ChIP-positive regions also exhibited minimal luciferase expression in the absence of HOXA13 (Figure 10F). Co-transfection with a Hoxa13 expression vector stimulated luciferase expression from these minimal promoter elements resulting in low but significant increases in normalized luciferase expression: 3.7 fold for Tie2 and 3.2 fold for Foxf1 http://www.tkuhn.ch/bel2nanopub/RAgAZGmKKw_vwJnyN4xbpVE2idhPjnZCtN_yA_5fQKots#_5 http://www.w3.org/ns/prov#wasQuotedFrom http://www.ncbi.nlm.nih.gov/pubmed/18483557 http://www.tkuhn.ch/bel2nanopub/RAgAZGmKKw_vwJnyN4xbpVE2idhPjnZCtN_yA_5fQKots#_6 http://www.w3.org/2000/01/rdf-schema#label Selventa http://www.tkuhn.ch/bel2nanopub/RAgAZGmKKw_vwJnyN4xbpVE2idhPjnZCtN_yA_5fQKots#assertion http://www.w3.org/ns/prov#hadPrimarySource http://www.ncbi.nlm.nih.gov/pubmed/18483557 http://www.tkuhn.ch/bel2nanopub/RAgAZGmKKw_vwJnyN4xbpVE2idhPjnZCtN_yA_5fQKots#assertion http://www.w3.org/ns/prov#wasDerivedFrom http://resource.belframework.org/belframework/20131211/knowledge/large_corpus.bel http://www.tkuhn.ch/bel2nanopub/RAgAZGmKKw_vwJnyN4xbpVE2idhPjnZCtN_yA_5fQKots#assertion http://www.w3.org/ns/prov#wasDerivedFrom http://www.tkuhn.ch/bel2nanopub/RAgAZGmKKw_vwJnyN4xbpVE2idhPjnZCtN_yA_5fQKots#_5 http://www.tkuhn.ch/bel2nanopub/RAgAZGmKKw_vwJnyN4xbpVE2idhPjnZCtN_yA_5fQKots#pubinfo http://www.tkuhn.ch/bel2nanopub/RAgAZGmKKw_vwJnyN4xbpVE2idhPjnZCtN_yA_5fQKots http://purl.org/dc/terms/created 2014-07-03T14:33:21.351+02:00 http://www.tkuhn.ch/bel2nanopub/RAgAZGmKKw_vwJnyN4xbpVE2idhPjnZCtN_yA_5fQKots http://purl.org/pav/createdBy http://orcid.org/0000-0001-6818-334X http://www.tkuhn.ch/bel2nanopub/RAgAZGmKKw_vwJnyN4xbpVE2idhPjnZCtN_yA_5fQKots http://purl.org/pav/createdBy http://orcid.org/0000-0002-1267-0234