Identification Of Target Genes Mediated By Thyroid Hormone Receptor TR?A Regulated By Thyroid Hormone Of Paralichthys Olivaceus

Thyroid hormone(TH)plays an extremely significant role in the regulation of metamorphosis of Paralichthys olivaceus,which directly controls whether the larvae could finish metamorphosis and its process through thyroid hormone receptors(TRs).Studies have demonstrated that TR combines with retinoid X receptor or other nuclear receptors to form heterodimers which bind to thyroid hormone response elements(TREs)in the promoter region of target genes,thereby regulating their transcription.To identify the target genes directly regulated by the TH-TRs signaling pathway,in this report,thyroid hormone receptor TR?A was taken as the research object,and p3×Flag-TR?A was co-transfected with the recombinant reporter gene expression vector(containing 5'-flanking sequence deleted fragments of the candidate targets)in HEK293 T cells.Genes of atoh8 and Syt-1-like were identified as target genes regulated by thyroid hormone mediated by TRs by the double luciferase reporter assay.The main results are as follows:1.Screening of candidate targets regulated by thyroid hormone mediated by thyroid hormone receptor TRsBased on screening of transcriptome and TREs database and combined with TREs identification types,atoh8(Gene ID: 109627718)and Syt-1-like(Gene ID: 109624206)were initially determined as candidate targets regulated by thyroid hormone mediated by TRs of Paralichthys olivaceus.Analysis of promoter transcriptional activity and transcription factor binding sites prediction indicated that the 5'-flanking sequence of atoh8 gene was predicted to have three TREs,also including other transcription factor binding sites,such as GATA-1,E2 F,GR,GAGA,MyoD,Cart-1,E2F4,RAR?,RAR?,and Pax-4a;three upstream primers Pro-atoh8-F1/F2/F3 and one common downstream primer Pro-atoh8-R were designed according to the location of TREs,and deleted fragments containing 3,2,and 1 TREs sites can be obtained by PCR amplification.The 5'-flanking sequence of Syt-1-like gene was predicted to have two TREs,including other transcription factor binding sites,such as ZEB,HIP-1;three upstream primers Pro-Syt-1-like-F1/F2/F3 and a common downstream primer Pro-Syt-1-like-R were designed according to the location of TREs,and deleted fragments containing 2,1 and 0 TREs can be obtain by PCR amplification.In this section,by PCR amplification,regarding the 5'-flanking sequences of the atoh8 gene,the deleted fragments of 1517 bp,1333bp,and 708 bp were actually obtained;with respect to the 5'-flanking sequence of the Syt-1-like gene,the deleted fragments of 1692 bp,1079bp and 791 bp were actually obtained;the recombinant plasmids pGL3-Pro-atoh8-1517/1333/708 and pGL3-Pro-Syt1-like-1692/1079/791 were successfully constructed and used for cell transfection.2.Construction of eukaryotic expression vector of Paralichthys olivaceus TR?A and expression and purification of fusion proteinIn this section,the CDS region of TR?A gene was cloned by RT-PCR,and the recombinant eukaryotic expression vector p3×Flag-TR?A was constructed.This recombinant plasmid was transiently transfected into HEK293 T cells,and then results detected by RT-PCR,real-time quantitative PCR and Western Blot indicated that the TR?A of Paralichthys olivaceus was successfully transcribed and translated in the mammalian protein expression system;and the cell lysate transfected with the recombinant plasmid was purified by G1 affinity chromatography and filtered to obtain the pure fusion protein 3×Flag-TR?A.The Flag purification tag is fused at the N-terminus of the target protein;because the molecular weight of the 3×Flag tag is relatively small,the protein activity is hardly affected;the purified product can be directly used for the study of protein function.In this section,the 3×Flag-TR?A fusion protein was successfully expressed and purified by mammalian cell protein expression system for the first time,which laid a material foundation for structural analysis and functional research.3.Interaction analysis between TR?A and 5'-flanking sequences of candidate targetsTo detect whether TR?A interacts with the 5'-flanking sequences of candidate targets(atoh8 and Syt-1-like),that is,to verify whether the candidate targets were directly regulated by thyroid hormone mediated by TR?A,dual luciferase reporter assay was performed in HEK293 T cells.p3×Flag-TR?A was co-transfected with recombinant reporter gene expression vector to explore the effect of different lengths of 5?-flanking sequence and different treatments to the same 5?-flanking sequence on the value in the ratio of firefly luciferase to Renilla luciferase,namely,to check the effect on the transcriptional activity of the promoter.With regard to atoh8 gene,three sets of experiments were designed:(1)the effect of the 5'-flanking sequence of different lengths(1517bp,1333 bp,708bp)on the promoter activity of the atoh8 gene;(2)the effect of the different treatments(TR?A,TR?A+T3)to Pro-atoh8-1517 on the promoter activity;(3)the effect of different treatments(TR?A,TR?A+T3)to Pro-atoh8-1333 on the promoter activity;(4)the effect of different treatments(TR?A,TR?A+T3)to Pro-atoh8-708 on the promoter activity.The results manifested that pGL3-Pro-atoh8-1517 contains two TRE sites,and the TR?A receptor via the DNA binding domain(DBD)binds to the TRE sequence specific to the-1497~-688 5' regulatory region of the atoh8 gene to regulate transcription level.Thus,it is demonstrated that atoh8 is a direct target gene for TR?A-mediated TH regulation.In terms of Syt-1-like gene,four sets of experiments were designed:(1)the effect of the 5'-flanking sequence of different lengths(1692bp,1079 bp,791bp)on the promoter activity of the Syt-1-like gene;(2)the effect of the different treatments(TR?A,TR?A+T3)to Pro-Syt-1-like-1692 on the promoter activity;(3)the effect of different treatments(TR?A,TR?A+T3)to Pro-Syt-1-like-1079 on the promoter activity,(4)the effect of different treatments(TR?A,TR?A+T3)to Pro-Syt-1-like-791 on the promoter activity.The results elucidates that pGL3-Pro-Syt1-like-1692 contains two TRE sites at least,and the TR?A receptor via the DNA binding domain(DBD)binds to the TRE sequence specific to the-1520~-1 5' regulatory region of the Syt-1-like gene to regulate transcription level.Hence,it is demonstrated that Syt-1-like is a direct target gene for TR?A-mediated TH regulation.4.Candidate target genes expression during the metamorphosis of larvaeTo verify the response to TH of candidate target genes(atoh8,Syt-1-like),real-time quantitative PCR was used to detect the expression changes of atoh8 and Syt-1-like in NC,TH and TU groups during the metamorphosis of larvae.It was shown that in the early stage of metamorphosis(21dph),the expression level of atoh8 gene in TH group was higher than that in the corresponding NC group,yet it was not significant;in the peak of metamorphosis(28dph),the group of TU treatment was significantly lower than the corresponding NC group;it was suggested that atoh8 has a weak response effect on TH.The expression level of Syt-1-like gene in the TH group in the early metamorphosis(21dph)stage is significantly higher than that in the corresponding NC group,and the expression level of corresponding TU group Syt-1-like gene decreased but not significantly,which fully indicated that the Syt-1-like gene was significantly regulated by TH in the early metamorphosis of larvae,suggesting that Syt-1-like is a target gene mediated by thyroid hormone receptor regulated by TH.Based on the above results,it's reasonable to conclude that atoh8 and Syt-1-like are target genes mediated by thyroid hormone receptor regulated by thyroid hormone.At a specific developmental stage(early metamorphosis),the former is a low level of weak regulation;the latter is a high level of strong regulation,and the difference in thyroid hormone response of both genes may be related to gene function.Conclusion: The receptor of TR?A binds to the TRE sequence specific to the-1497~-688 promoter region of the atoh8 gene,and-1520~-1 promoter region of the Syt-1-like gene to regulate the transcription of these genes,respectively.Therefore,atoh8 and Syt-1-like are target genes for TR?A-mediated direct regulation of the thyroid hormone.This study provides a basis for further exploration of the signaling pathway mediated by thyroid hormone receptor TR?A regulated by thyroid hormone.