| The transcription factor FOXO1 has many biological functions such as energy metabolism.FOXO1 interacts with promoters through its DNA-binding domain and regulates the activity of target genes,thus exerting its biological functions.The laboratory previously found that the presence of FOXO1 lacking the DNA-binding domain(FOXO1ΔDBD)inhibited the function of wild-type FOXO1 and affected the metabolic level of animals,but the molecular mechanism of its effect was unknown.In this dissertation,a series of researches have been carried out from the aspects of molecular biology,cell biology and animal physiology.1.Phenotypic analysis of FoxO1ΔDBD transgenic mice was first performed.The results showed that the plasma levels of leptin and norepinephrine in transgenic mice were significantly lower than those in wild-type mice(P<0.05),but there was no significant difference in epinephrine.In brown adipose tissue(BAT),the type 2 iodothyronine deiodinase(DIO2)gene,the uncoupling protein 1(UCP1)gene,and the lipocatabolism-related genes long-chain acyl-Co A dehydrogenase(LCAD)and The transcription level of the fatty acid β-oxidation rate-limiting enzyme acetyl-Co A carboxylase β(ACC2)gene was significantly higher than that in wild-type mice(P<0.05),while the lipid synthesis-related genes fatty acid synthase(FASN),diacylglycerol acyltransferase(DGAT1)and thyroid hormone response protein(Thrsp)expression had no significant difference.In order to detect the thermogenic function of BAT,wild-type and transgenic animals were subjected to cold-stimulation experiments.The results showed that the triglyceride(TG)content and lipid droplet size in BAT were significantly larger than those in wild-type mice when coldstimulated at 4 °C for 8 h.mice(P<0.05);the expression level of dio2 was still significantly higher than that in wild-type mice(P<0.05),while ucp1 had no significant difference compared with wild-type mice,and the lipid anabolism-related genes FASN,DGAT1 and Thrsp were significantly lower than those in wild-type mice(P<0.05).In wild-type mice,the transcription level of the lipocatabolism-related gene ACC2 was significantly lower than that in wild-type mice(P<0.05);The content of free triiodothyronine(T3)and tetraiodine thyroxine(T4)in the plasma of transgenic mice was significantly higher than that of wild-type mice(P<0.05).The results showed that the biological effects of thyroid hormones and fat catabolism in BAT were enhanced in transgenic mice,consistent with previous findings of increased energy expenditure in transgenic mice.The energy expenditure of mice is closely related to the oxidative phosphorylation on the mitochondrial respiratory chain in BAT,and the increased expression of ucp1 in the BAT of FoxO1ΔDBD transgenic mice leads to the enhanced uncoupling of oxidative phosphorylation and promotes thermogenesis,which is one of the important reasons for increasing energy consumption.Thyroid hormone T3 can promote the expression of ucp1,while the function of DIO2 is to catalyze the deiodination of T4 into biologically active T3,indicating that DIO2 is an important factor indirectly regulating the effect of UCP1.To this end,this paper focuses on the regulation of the dio2 gene promoter by transgenic FoxO1ΔDBD.The results are as follows:2.FOXO1ΔDBD interferes with the binding of FOXO1 to the dio2 promoter.The potential binding sites of FOXO1 and dio2 promoter were predicted by bioinformatics analysis,which were divided into 6 regions and corresponding 6 pairs of PCR primers were synthesized;Four 16-week-old wild-type and transgenic mice of F14 generation were selected,and the chromatin in BAT after cross-linking and ultrasonic fragmentation was used for chromatin immunoprecipitation(Ch IP).The results showed that the binding strength of FOXO1 in transgenic mice was significantly lower than that in wild-type mice in two regions of dio2 transcription start site-635 ~-444 bp(region 2)and-1251 ~-1051 bp(region 5)(P<0.05).3.The effect of deletion of region 2 and region 5 on the activity of dio2 promoter.Genomic DNA was extracted from mouse liver,the DNA sequence of dio2 transcription initiation site-1628 ~ +100bp was cloned by gene recombination technology,and assembled in p GL3-basic to form 12 luciferase reporter gene detection vectors containing different promoter truncations,DNA sequencing results showed that various vectors were successfully constructed;after transfection of Hek293 cells:-479 ~-30 bp deletion reduced the promoter activity by 90%(P<0.05).In order to verify the effect of region 2 and region 5 on the activity of the dio2 promoter,the activity detection of the promoters with simultaneous deletion or respective deletion of the two regions was carried out(P < 0.05).Overexpression of FOXO1 had no significant effect on the promoter activity,indicating that the regulation of FOXO1 on the dio2 promoter depends on these two regions;while overexpression of FOXO1ΔDBD significantly increased its activity by 30%(P < 0.05),indicating that FOXO1ΔDBD can be activated by other in a way that affects promoter activity.4.FOXO1ΔDBD affects the nuclear localization of FOXO1.FOXO1 and FOXO1ΔDBD were overexpressed in Hek293 cells.The relative contents were detected by Western Blot,and immunofluorescence was observed by confocal microscopy.The nuclear localization of Fox O1 and FOXO1ΔDBD was analyzed.The results of both experiments showed that FOXO1 was nuclear-exported under the stimulation of serum,but the nuclear-exporting effect of FOXO1 was weakened after the addition of FOXO1ΔDBD,indicating that FOXO1ΔDBD affected the nuclear localization of FOXO1.5.The effect of FOXO1ΔDBD on dio2 promoter activity in the presence or absence of drugs.Different traditional Chinese medicine extracts obtained by grinding powder dissolution or high temperature leaching were added to the cell culture medium,and the full-length dio2 promoter(-1628 ~ +100)was transfected into Hek293 cells.When dio2 promoter activity was detected in the presence or absence of overexpressed FOXO1 and FOXO1ΔDBD.The results showed that berberine hydrochloride,Coptis chinensis and Scutellaria baicalensis could promote the activity of dio2 promoter;even in the presence of the above drugs,overexpression of FOXO1 inhibited the activity of dio2 promoter(P<0.05),while FOXO1ΔDBD had a significant promoting effect.(P<0.05).Conclusion: FOXO1ΔDBD can significantly up-regulate the expression of ucp1 and dio2 in the BAT of mice,increase the blood level of T3,and inhibit fat synthesis and increase lipolysis,indicating that the transgene promotes the energy consumption of mice through the thyroid hormone pathway and lipid metabolism pathway.;The presence of FOXO1ΔDBD significantly reduced the binding effect of FOXO1 and two regions on the dio2 promoter in BAT,thereby releasing the inhibitory effect of FOXO1 on dio2,which was verified at the cellular level;truncation experiments proved that-479 The ~-30 bp region is required for the activity of the dio2 promoter.Meanwhile,region 2 and region 5 are the key regions for FOXO1 to regulate the activity of the dio2 promoter;FOXO1ΔDBD affects the nuclear localization of FOXO1,which may affect the function of FOXO1;Berberine hydrochloride,Coptis chinensis and Scutellaria baicalensis can significantly increase the activity of dio2 promoter.In the presence of these drugs,the inhibitory effect of FOXO1 on the dio2 promoter and the promotion effect of FOXO1ΔDBD still exist.In conclusion,this paper studies the regulation of FOXO1ΔDBD on dio2 promoter activity and the mechanism of action of animal energy metabolism at the cellular level,which will provide a reference for the field of energy metabolism research and metabolic disease research. |
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