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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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Effects of in vivo siRNA knockdown of transcriptional factors on LDL receptor gene expression. Studies were performed in NR rats. Six sites, two in each liver lobe, were injected with the WT LDLR promoter construct and the indicated siRNAs or saline (negative control), followed by electroporation of the injection site. A. Liver punches were excised 24 hours later for lysate preparation followed by luciferase assays. B. Liver samples adjacent to the electroporation sites were used in the preparation of RNA samples that were analyzed using real-time PCR. In both panels, the data are reported as mean ± SEM for at least eight animals per treatment condition. p Values were obtained by comparing to the activity of the WT construct in the presence of saline.
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f0025: Effects of in vivo siRNA knockdown of transcriptional factors on LDL receptor gene expression. Studies were performed in NR rats. Six sites, two in each liver lobe, were injected with the WT LDLR promoter construct and the indicated siRNAs or saline (negative control), followed by electroporation of the injection site. A. Liver punches were excised 24 hours later for lysate preparation followed by luciferase assays. B. Liver samples adjacent to the electroporation sites were used in the preparation of RNA samples that were analyzed using real-time PCR. In both panels, the data are reported as mean ± SEM for at least eight animals per treatment condition. p Values were obtained by comparing to the activity of the WT construct in the presence of saline.

Mentions: To evaluate the relative involvement of endogenous TRβ1, PPARα, and USF-2 in mediating transcriptional activation of the hepatic LDL receptor gene by T3, siRNAs to knockdown these transcriptional factors were utilized. The siRNAs were introduced by in vivo electroporation together with the WT LDL receptor promoter construct into euthyroid rats. The LDL receptor promoter activity obtained in the presence of the siRNAs was directly compared with the promoter activity seen in the presence of saline. As shown in Fig. 5A, the receptor promoter activity was reduced to about 25% of control in the presence of siRNAs against TRβ1. LDL receptor mRNA levels were reduced to about 35% of control by the same siRNAs (Fig. 5B). Knockdown of PPARα and USF-2 using siRNAs significantly reduced LDL receptor promoter activity to about 35% of control (Fig. 5A), while LDL receptor mRNA levels were reduced to about 50% of control (Fig. 5B).


Using in vivo electroporation to identify hepatic LDL receptor promoter elements and transcription factors mediating activation of transcription by T 3
Effects of in vivo siRNA knockdown of transcriptional factors on LDL receptor gene expression. Studies were performed in NR rats. Six sites, two in each liver lobe, were injected with the WT LDLR promoter construct and the indicated siRNAs or saline (negative control), followed by electroporation of the injection site. A. Liver punches were excised 24 hours later for lysate preparation followed by luciferase assays. B. Liver samples adjacent to the electroporation sites were used in the preparation of RNA samples that were analyzed using real-time PCR. In both panels, the data are reported as mean ± SEM for at least eight animals per treatment condition. p Values were obtained by comparing to the activity of the WT construct in the presence of saline.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121208&req=5

f0025: Effects of in vivo siRNA knockdown of transcriptional factors on LDL receptor gene expression. Studies were performed in NR rats. Six sites, two in each liver lobe, were injected with the WT LDLR promoter construct and the indicated siRNAs or saline (negative control), followed by electroporation of the injection site. A. Liver punches were excised 24 hours later for lysate preparation followed by luciferase assays. B. Liver samples adjacent to the electroporation sites were used in the preparation of RNA samples that were analyzed using real-time PCR. In both panels, the data are reported as mean ± SEM for at least eight animals per treatment condition. p Values were obtained by comparing to the activity of the WT construct in the presence of saline.
Mentions: To evaluate the relative involvement of endogenous TRβ1, PPARα, and USF-2 in mediating transcriptional activation of the hepatic LDL receptor gene by T3, siRNAs to knockdown these transcriptional factors were utilized. The siRNAs were introduced by in vivo electroporation together with the WT LDL receptor promoter construct into euthyroid rats. The LDL receptor promoter activity obtained in the presence of the siRNAs was directly compared with the promoter activity seen in the presence of saline. As shown in Fig. 5A, the receptor promoter activity was reduced to about 25% of control in the presence of siRNAs against TRβ1. LDL receptor mRNA levels were reduced to about 35% of control by the same siRNAs (Fig. 5B). Knockdown of PPARα and USF-2 using siRNAs significantly reduced LDL receptor promoter activity to about 35% of control (Fig. 5A), while LDL receptor mRNA levels were reduced to about 50% of control (Fig. 5B).

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 − 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.

No MeSH data available.


Related in: MedlinePlus