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Using in vivo electroporation to identify hepatic LDL receptor promoter elements and transcription factors mediating activation of transcription by T 3

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ABSTRACT

The technique of in vivo electroporation was adapted to investigate the promoter elements and transcription factors mediating the rapid induction of hepatic LDL receptor expression in response to thyroid hormone. Direct comparisons between wild type and mutant promoter constructs were made within the same animal. It was demonstrated that both TREs at bp − 612 and − 156 were required for the l-triiodothyronine (T3) response. ChIP analysis showed that binding of TRβ1 to the − 612 and − 156 TREs was markedly stimulated by T3in vivo. Introduction of siRNAs against TRβ1/RXRα with LDL receptor promoter-luciferase construct by in vivo electroporation demonstrated that these transcription factors play the major physiological role in the activation of hepatic LDL receptor transcription. The findings agree with those made by transfecting H4IIE cells in vitro thus validating this technique for in vivo studies of mechanisms of transcriptional regulation. The findings reported herein also indicated, for the first time, that PPARα and USF-2 were required for maximum transcriptional activation of the LDL receptor in response to T3 treatment.

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In vivo contributions of the − 612 and − 156 TREs to the T3-dependent activation of the LDLR promoter. For this experiment, 10 μg of the WT, − 612Mt, and DbMt LDLR promoter constructs were electroporated into the livers of Hx and Hx + T3 rats. Two doses of T3 were given 72 and 24 hours before electroporation as described under Materials and methods. Lysate preparation and luciferase assays were carried out. The data are presented as mean LDLR promoter activity ± SEM for at least four electroporation sites per each treatment condition. p Values were obtained by comparing to the WT construct.
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f0015: In vivo contributions of the − 612 and − 156 TREs to the T3-dependent activation of the LDLR promoter. For this experiment, 10 μg of the WT, − 612Mt, and DbMt LDLR promoter constructs were electroporated into the livers of Hx and Hx + T3 rats. Two doses of T3 were given 72 and 24 hours before electroporation as described under Materials and methods. Lysate preparation and luciferase assays were carried out. The data are presented as mean LDLR promoter activity ± SEM for at least four electroporation sites per each treatment condition. p Values were obtained by comparing to the WT construct.

Mentions: The in vivo contributions of the TREs located at bp − 612 and − 156, relative to the transcription start site, to T3 stimulation of LDL receptor transcription was evaluated by introducing receptor promoter constructs into rat livers by in vivo electroporation. The rat wild-type (WT), − 156 mutant (− 156Mt), − 612 mutant (− 612Mt), and double mutant (DbMt) LDL receptor promoter constructs were introduced into separate liver lobes of normal (NR), hypophysectomized (Hx), and T3-treated hypophysectomized (Hx + T3) rats, for direct comparisons within the same animal. As shown in Fig. 2A, the promoter activity of the WT construct was 39% lower in the Hx than in the NR rats. Treating with a single dose of T3 (after electroporation) 16 hours before euthanization caused a significant (p < 0.01) induction (2.4-fold) in LDL receptor promoter activity as compared to the activity observed in the Hx animals (Fig. 2A). These changes in LDL receptor promoter activity directly correlated with changes in free T3 (fT3) (Fig. 2B) and hepatic LDL receptor mRNA levels (Fig. 2C). Fig. 3A illustrates the results for the mutation analysis experiment. For this specific experiment, the Hx rats were treated with two injections of T3, 72 and 24 hours prior to electroporation, as indicated under Materials and methods. As shown, the activity of the WT construct was increased 8.42-fold in response to two doses of T3, when compared to the LDL receptor promoter activity seen in the Hx animals (Fig. 3). Mutating the − 612 TRE reduced the T3-dependent stimulation of the LDL receptor promoter about 49% (Fig. 3). Similarly to the results obtained in H4IIE cells (Lopez et al., 2007), mutating the − 156 TRE had no significant effect on the T3 dependent activation of the LDL receptor promoter (data not shown). When both TREs were mutated, the stimulation by T3 was reduced by 76% (Fig. 3).


Using in vivo electroporation to identify hepatic LDL receptor promoter elements and transcription factors mediating activation of transcription by T 3
In vivo contributions of the − 612 and − 156 TREs to the T3-dependent activation of the LDLR promoter. For this experiment, 10 μg of the WT, − 612Mt, and DbMt LDLR promoter constructs were electroporated into the livers of Hx and Hx + T3 rats. Two doses of T3 were given 72 and 24 hours before electroporation as described under Materials and methods. Lysate preparation and luciferase assays were carried out. The data are presented as mean LDLR promoter activity ± SEM for at least four electroporation sites per each treatment condition. p Values were obtained by comparing to the WT construct.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
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f0015: In vivo contributions of the − 612 and − 156 TREs to the T3-dependent activation of the LDLR promoter. For this experiment, 10 μg of the WT, − 612Mt, and DbMt LDLR promoter constructs were electroporated into the livers of Hx and Hx + T3 rats. Two doses of T3 were given 72 and 24 hours before electroporation as described under Materials and methods. Lysate preparation and luciferase assays were carried out. The data are presented as mean LDLR promoter activity ± SEM for at least four electroporation sites per each treatment condition. p Values were obtained by comparing to the WT construct.
Mentions: The in vivo contributions of the TREs located at bp − 612 and − 156, relative to the transcription start site, to T3 stimulation of LDL receptor transcription was evaluated by introducing receptor promoter constructs into rat livers by in vivo electroporation. The rat wild-type (WT), − 156 mutant (− 156Mt), − 612 mutant (− 612Mt), and double mutant (DbMt) LDL receptor promoter constructs were introduced into separate liver lobes of normal (NR), hypophysectomized (Hx), and T3-treated hypophysectomized (Hx + T3) rats, for direct comparisons within the same animal. As shown in Fig. 2A, the promoter activity of the WT construct was 39% lower in the Hx than in the NR rats. Treating with a single dose of T3 (after electroporation) 16 hours before euthanization caused a significant (p < 0.01) induction (2.4-fold) in LDL receptor promoter activity as compared to the activity observed in the Hx animals (Fig. 2A). These changes in LDL receptor promoter activity directly correlated with changes in free T3 (fT3) (Fig. 2B) and hepatic LDL receptor mRNA levels (Fig. 2C). Fig. 3A illustrates the results for the mutation analysis experiment. For this specific experiment, the Hx rats were treated with two injections of T3, 72 and 24 hours prior to electroporation, as indicated under Materials and methods. As shown, the activity of the WT construct was increased 8.42-fold in response to two doses of T3, when compared to the LDL receptor promoter activity seen in the Hx animals (Fig. 3). Mutating the − 612 TRE reduced the T3-dependent stimulation of the LDL receptor promoter about 49% (Fig. 3). Similarly to the results obtained in H4IIE cells (Lopez et al., 2007), mutating the − 156 TRE had no significant effect on the T3 dependent activation of the LDL receptor promoter (data not shown). When both TREs were mutated, the stimulation by T3 was reduced by 76% (Fig. 3).

View Article: PubMed Central - PubMed

ABSTRACT

The technique of in vivo electroporation was adapted to investigate the promoter elements and transcription factors mediating the rapid induction of hepatic LDL receptor expression in response to thyroid hormone. Direct comparisons between wild type and mutant promoter constructs were made within the same animal. It was demonstrated that both TREs at bp &minus;&nbsp;612 and &minus;&nbsp;156 were required for the l-triiodothyronine (T3) response. ChIP analysis showed that binding of TR&beta;1 to the &minus;&nbsp;612 and &minus;&nbsp;156 TREs was markedly stimulated by T3in vivo. Introduction of siRNAs against TR&beta;1/RXR&alpha; with LDL receptor promoter-luciferase construct by in vivo electroporation demonstrated that these transcription factors play the major physiological role in the activation of hepatic LDL receptor transcription. The findings agree with those made by transfecting H4IIE cells in vitro thus validating this technique for in vivo studies of mechanisms of transcriptional regulation. The findings reported herein also indicated, for the first time, that PPAR&alpha; and USF-2 were required for maximum transcriptional activation of the LDL receptor in response to T3 treatment.

No MeSH data available.


Related in: MedlinePlus