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Figure 5


Fig. 5. Binding of TR to TREKCNQ4 analyzed by EMSA. (A) Sequence and localization of TRE-1KCNQ4, TRE-2KCNQ4, and TRE-3KCNQ4 relative to the transcriptional start site. Hexamers are underlined and nucleotides matching consensus sequence are in bold. (B) EMSA of the three TREKCNQ4. Recombinant chicken TR{alpha} (cTR{alpha}) shifts [32P]TRE-3 (lane 4), but not [32P]TRE-1 (lane 2) or [32P]TRE-2 (lane 3) to positions similar to [32P]DR4 (lane 1), which correspond to a higher cTR{alpha} homodimer and a lower cTR{alpha} monomer complex. In competitor experiments, the interaction between cTR{alpha} and [32P]TRE-3 (lane 4) is significantly reduced in the presence of an excess of unlabeled competitor oligomer TRE3KCNQ4 (lane 5) and DR4 (lane 6) but not in the presence of unlabeled TFIID-specific oligomer (lane 7). Mutations in the 5' half of unlabeled TRE-3KCNQ4 (TRE-3mut1) have no influence on the capability of cTR{alpha} to shift the [32P]TRE-3 (lane 8). Mutations in the 3' half of TRE-3KCNQ4 (TRE-3mut2) still abolish the interaction (lane 9) to a similar degree as unlabeled TRE-3KCNQ4 or DR4 competitor oligomers (lanes 5, 6). (C) Sequence annotation of the mutated TRE-3KCNQ4 oligomers used as competitors. Mutated residues are highlighted. (D) Functional analysis of the putative human KCNQ4 promoter and the TRE-3KCNQ4 in reporter gene assays. Introduction of the human KCNQ4 promoter into a promoterless vector leads to 4.3-fold induction of reporter gene expression compared with the promoterless vector (4.3-fold ±0.3 s.d.; n=4; white column). Insertion of the TRE-3KCNQ4 upstream of the human KCNQ4 promoter leads to a decreased 2.1-fold induction of reporter gene expression (2.1±0.5 s.d., ***P<0.05; n=4) in the absence of ligands (black column) which could be overcome upon addition of 150 nM T3 and 1.5 µM ATRA restoring promoter activity to 4.1-fold induction of reporter gene expression (4.1±1.1 s.d., ***P<0.05; n=3; gray column). (E) Compared with control conditions (lane 1) addition of 600 nM T3 (lane 2) impairs the binding of cTR{alpha} homodimers (higher shift band) to [32P]TRE3KCNQ4 whereas monomer binding is unaffected.





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