The DNA-binding Of The Novel Thyroid Hormone Receptor Isoform-TRβ△

Thyroid hormone receptors (TRs) belong to the steroid/thyroid hormone receptor superfamily of ligand-dependent transcription factors and are distributed in almost all organs of the body. TRs often modulate transcriptional activity of target genes via heterodimerized with the 9-cis retinoic acid receptor (RXR). On positive thyroid response elements (TREs), RXR favors binding of the TR-RXR complex to DNA and stimulates transcription. TRs are coded by two genes, TRa and TRβ. Because of different transcript start position and alternative splicing, each subtype has several isoforms, including TRal, TRa2, TRa3, TR△α1, TRAa2, TRβ1, TRβ2, TRβ3, TRAβ3. TRPA is the unique extended-pattern TR isoforms which was found by our laboratory as a novel TRβisoform. It's mRNA/cDNA is only 108bps longer than that of TRβ1. The only difference between TRβ△and TRβ1 is an additional 36 amino-acid residues of TRβ△.Objective:In order to detect the novel thyroid hormone receptor isoform-TRPA could bind with RXRa as forming a heterodimer, and TRβ△/RXRa heterodimer has biological activity of binding target DNA, some experiments are disigned as following.Methods:1. Construction of recombinant expression vector pETDuet-1/rRXRa. RXRa target gene was obtained from recombinant plasmid pETDuet-1/rTRβ△/rRXRa,and then inserted into prokaryotic expression vector pETDuet-1, and tested by DNA sequencing.2. The recombinant plasmids were transformed into E.coliBL21(DE3) and expressed.The recombinant plasmid pETDuet-1/rRXRαwas transformed into E.coli BL21(DE3) and induced with IPTG(1mmol/L) at 37℃for 6h. It's amount and molecular weight of the recombinant protein were tested by SDS-PAGE and Western blot. At the same time we also completed induced expression, SDS-PAGE and Western blot of pETDuet-1/rTRβ1 and pETDuet-1/rTRβ△.3. Heterodimers of TRβ1/RXRa and TRβ△/RXRa were detected by Co-immunoprecipitation. Complexes of TRβ1-RXRa and TRβ△-RXRa were co-immunoprecipitated with Rabbit polyclonal to Retinoid X Receptor alpha or anti-His antibody.The immunocomplexes were detected by Western blot with anti-His or Retinoid X Receptor alpha antibody to identify the formation of heterodimers.4. Biological activity assay of TRβ△. First TRβ1 and TRβ△were incubated separately with RXRa at room temperature,then their biological activity of binding target DNA was confirmed by electrophoretic mobility shift assay (EMSA).Results:1. We constructed recombinant expression vector pETDuet-1/rRXRa.2. We expressed recombinant RXRa protein. RXRa,TRβ1 and TRβ△fusion proteins were expressed in Ecoli. Fusion proteins were analyzed by SDS-PAGE and Western blot.The result showed that fusion proteins were stained near 55kD (RXRa 50kD,TR(3156kD and TRβ△5kKD) and fusion proteins were mainly in inclusion bodies. The yield of RXRa,TRβ1 and TRβ△reached 213.8mg/L,251.0mg/L.161.9mg/L medium and accounted for 32.2%,30.5%,28.8% of total protein in E.coli BL21(DE3).3. Inclusion body of RXRa, TRβ1 and TRβ△dissolved in a high concentration of urea solution and accounted for the major part of the supernatant.4. Co-immunoprecipitation confirmed that recombinant protein TRβ△, like TRβ1, could bind with RXRa as forming a heterodimer.5. Complexes of RXRa with TRβ1 or TRβ△resulted in binding of TRβ1/RXRa and TRβ△/RXRa heterodimers, could bind DNA when incubated with the element. RXRa, TRβ1 and TRβ△could also bind DNA when incubated alone with the element.Conclusion:In this study we constructed recombinant expression vector pETDuet-1/rRXRαand expressed fusion protein RXRa. Our result shows that TRβ△, like TRβ1, could bind with RXRa as forming a heterodimer and complexes of TRβ△/RXRa and TRβ1/RXRa could bind with DNA. Interestingly, TRβ△,TRβ1 and RXRa also have the biological activity of binding target DNA when incubated alone with the element. Our results are different from Williams's report. Williams reported that RXRa and TRβ1 failed to bind DNA when incubated alone with the element, and coincubation of RXR with TRβ1 resulted in binding of TRβ1-RXR heterodimers. Therefore, it is need to study the DNA-binding of TRβ△,TRβ1,and even RXRa alone furthermore.