The Transactive Activity Of The Novel Thyroid Hormone Receptor β△ And The Relationship Between Its P-box Amino Acid Sequence And Transaction

Objective:To study the transactivation of a novel thyroid hormone receptor isoform, TRβ△, on target genes through thyroid hormone response element(TRE),and whether TRβ△had the characteristics of transcription factor. Used the two way of mutation to change the P-box amino acid sequence in the DBD (DNA-binding domain, DBD) of TRβ△, and then research whether TRβ△still had the function of binding DNA and transactivation after mutation.At last it clarified that it was important for the binding DNA and transactivation of TRβ△receptor to maintain the conservation of its P-box amino acid sequence in DBD.Methods:The thyroid hormone receptorβ1 andβ△gene were amplified respectively from pETDuet-TRβ1 and pETDuet-TRβ△by PCR and sub-cloned into pcDNA3.1. The multi-copy recombinant eukaryotic expression plasmid pcDNA3.1-TRβ1 and pcDNA3.1-TRβ△were constructed. A short of sequence of the pGL3-Promoter would mutate to a kind of TRE-PAL (palindromic) with designing a pair of special primers. The reporter gene vector plasmid pGL3-Promoter-PAL TRE was constructed. The recombinant reporter gene vector pGL3-promoter-PAL TRE was transiently co-transfected into COS-7 cells with pcDNA3.1-TRβ1 and pcDNA3.1-TRβ△respectively. Cells were treated with DMEM with or without T3, then the activity of luciferase was detected.The control group is that:the reporter gene vector pGL3-promoter was transiently co-transfected into COS-7 cells with pcDNA3.1-TRβ1 and pcDNA3.1-TRβ△respectively, pGL3-promoter-PAL TRE was transfected into COS-7 cells lonely.The DBD P-box amino acid sequence in TRβ△was mutated respectively to different sequence used the way of site-directed mutation. EMSA was performed after the labeled DR4 respectively combined with two kinds of mutation proteins and the dimerization complex of mutation proteins with RXR protein.The recombinant reporter gene vector pGL3-promoter-PAL TRE was transiently co-transfected into COS-7 cells with two kinds of mutant plasmids. Cells were treated with DMEM with or without T3, then the activity of luciferase was detected. Results:The new multi-copy recombinant eukaryotic expression plasmid pcDNA3.1-TRβ1 and pcDNA3.1-TRβ△were successfully constructed, which was confirmed by PCR. The result of sequencing and double digesting of recombined plasmid were completely correct. The reporter gene vector plasmid pGL3-Promoter-PAL was successfully constructed, which was confirmed by the result of sequencing and double digesting of recombined plasmid. The recombinant reporter gene vector pGL3-promoter-PAL TRE was transiently co-transfected into COS-7 cells with pcDNA3.1-TRβ1 and pcDNA3.1-TRβ△respectively. It can be detected very high activity of luciferase after transfection for 48h.The expression of reporter gene could be respectively increased up to 16 and 14 times by T3 after pGL3-promoter-PAL TRE with pcDNA3.1-TRβ1 and pcDNA3.1-TRβ△co-transfected into cells.The mutant plasmids pcDNA3.1-TRβ△mutl and pcDNA3.1-TRβ△mut2 were successfully constructed, which were confirmed by the result of sequencing. The DBD P-box amino acid sequence in pcDNA3.1-TRβ△mutl was mutated to CEGCKG,the same as TRβ1. The P-box amino acid sequence in pcDNA3.1-TRβ△mut2 was mutated to CGACKP. TRPAmutl protein and the dimerization complex of TRβ△mutl protein with RXR protein can all combine with labeled DR4. The TRβ△mut2 protein itself can not combine with labeled DR4, but RXR protein can combine with DR4, only the dimerization complex of TRβ△mut2 protein with RXR protein can combine with DR4.pGL3-promoter-PAL TRE was transiently co-transfected into COS-7 cells with pcDNAS.l-TRβ△mutl and pcDNA3.1-TRβ△mut2 respectively. The activity of luciferase of former was 7 times higher than the latter after transfection for 48h. The expression of reporter gene could be respectively increased up to 7 and 2.5 times by T3 after pGL3-promoter-PAL TRE with pcDNA3.1-TRβ△mut1 and pcDNA3.1-TRβ△mut2 co-transfected into cells.Conclusion:A novel thyroid hormone receptor isoforms TRβ△the same as TRβ1 had the transactivation effect on target genes through the TRE.The expression of reporter gene could be increased by T3. TRβ△was a functional transcription factor induced by T3. The DBD P-box amino acid sequence in TRβ△and the original P-box in TRβ1 had the same transactivation effect. The DBD P-box amino acid sequence in TRβ△was mutated to the sequence in TRβ1 with more than 30aa behind the P-box in TRβ1,it still had the transactivation effect on target genes through the TRE. The expression of reporter gene could be increased by T3. TRβ△Amutl protein and the dimerization complex of TRβ△mutl protein with RXR protein can all combine with labeled DR4.But two the most important amino acid in DBD P-box were changed, transactivation effect was lose. TRβ△mut2 protein itself can not combine with labeled DR4.It was important for the binding DNA and transactivation of TRβ△receptor to maintain the conservation of its P-box amino acid sequence in DBD.