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An Ideal PPAR Response Element Bound to and Activated by PPARα.

Tzeng J, Byun J, Park JY, Yamamoto T, Schesing K, Tian B, Sadoshima J, Oka S - PLoS ONE (2015)

Bottom Line: The single nucleotide substitution, which reduces binding of RXRα to DNA, attenuated PPARα-induced transcriptional activation, but this is not always true for PPARα.Using the definition of the PPRE sequence, novel PPREs were successfully identified.Taken altogether, the provided PPRE sequence definition contributes to the understanding of PPARα signaling by identifying PPARα direct target genes with functional PPARα response elements.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Molecular Medicine, Rutgers Biomedical Health Sciences, Newark, NJ 07103, United States of America.

ABSTRACT
Peroxisome proliferator-activated receptor-α (PPARα), a nuclear receptor, plays an important role in the transcription of genes involved in fatty acid metabolism through heterodimerization with the retinoid x receptor (RXR). The consensus sequence of the PPAR response element (PPRE) is composed of two AGGTCA-like sequences directionally aligned with a single nucleotide spacer. PPARα and RXR bind to the 5' and 3' hexad sequences, respectively. However, the precise sequence definition of the PPRE remains obscure, and thus, the consensus sequence currently available remains AGGTCANAGGTCA with unknown redundancy. The vague PPRE sequence definition poses an obstacle to understanding how PPARα regulates fatty acid metabolism. Here we show that, rather than the generally accepted 6-bp sequence, PPARα actually recognized a 12-bp DNA sequence, of which the preferred binding sequence was WAWVTRGGBBAH. Additionally, the optimized RXRα hexad binding sequence was RGKTYA. Thus, the optimal PPARα/RXRα heterodimer binding sequence was WAWVTRGGBBAHRGKTYA. The single nucleotide substitution, which reduces binding of RXRα to DNA, attenuated PPARα-induced transcriptional activation, but this is not always true for PPARα. Using the definition of the PPRE sequence, novel PPREs were successfully identified. Taken altogether, the provided PPRE sequence definition contributes to the understanding of PPARα signaling by identifying PPARα direct target genes with functional PPARα response elements.

No MeSH data available.


The effect of PPRE orientation.(A) Schematic representation of the reporter gene constructs. Both orientations of the PPRE consensus sequences indicated were connected to the luciferase gene with minimal promoter. PPARα and RXR binding hexad sequences are indicated. (B) Both orientations of PPRE relative to the luciferase gene were equally activated in primary cultured myocytes stimulated with WY14643 (n = 5–6). (C) Forced expression of PPARα preferentially activates the major direction of PPRE in H9c2 cells (n = 7–9). A solid triangle indicates statistical significance compared with PPRE(←) transfected with the same dose of PPARα expression vector.
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pone.0134996.g002: The effect of PPRE orientation.(A) Schematic representation of the reporter gene constructs. Both orientations of the PPRE consensus sequences indicated were connected to the luciferase gene with minimal promoter. PPARα and RXR binding hexad sequences are indicated. (B) Both orientations of PPRE relative to the luciferase gene were equally activated in primary cultured myocytes stimulated with WY14643 (n = 5–6). (C) Forced expression of PPARα preferentially activates the major direction of PPRE in H9c2 cells (n = 7–9). A solid triangle indicates statistical significance compared with PPRE(←) transfected with the same dose of PPARα expression vector.

Mentions: To investigate the effect of the orientation of PPRE, a reporter assay was performed with a luciferase reporter construct driven by a 32-bp minimum promoter including the PPRE sequence with 5’ and 3’ 7-bp extended sequences, as shown in Fig 2A. Since PPARα is not significantly expressed in an immortalized cell line, we used primary cultured rat neonatal cardiac myocytes to examine the effect of the PPRE sequence on transcriptional activation induced by ligand-stimulated endogenous PPARα. At the same time, we used the H9c2 rat cardiac myocyte cell line to examine the effect of the PPRE sequence on transcriptional activation induced by exogenous PPARα overexpression to reduce animal usage. As shown in Fig 2B, both orientations of the PPRE were equally activated by WY14,647, an artificial ligand for PPARα, in primary culture cardiac myocytes. In contrast, overexpression of PPARα more strongly activated transcription when the orientation of the sequential PPARα and RXR binding site were opposite that of the luciferase gene body (Fig 2C). Thus, ligand-stimulated PPARα equally activates transcription in both directions, whereas PPARα-overexpression-induced transcriptional activation is sensitive to the orientation of the PPRE.


An Ideal PPAR Response Element Bound to and Activated by PPARα.

Tzeng J, Byun J, Park JY, Yamamoto T, Schesing K, Tian B, Sadoshima J, Oka S - PLoS ONE (2015)

The effect of PPRE orientation.(A) Schematic representation of the reporter gene constructs. Both orientations of the PPRE consensus sequences indicated were connected to the luciferase gene with minimal promoter. PPARα and RXR binding hexad sequences are indicated. (B) Both orientations of PPRE relative to the luciferase gene were equally activated in primary cultured myocytes stimulated with WY14643 (n = 5–6). (C) Forced expression of PPARα preferentially activates the major direction of PPRE in H9c2 cells (n = 7–9). A solid triangle indicates statistical significance compared with PPRE(←) transfected with the same dose of PPARα expression vector.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4524655&req=5

pone.0134996.g002: The effect of PPRE orientation.(A) Schematic representation of the reporter gene constructs. Both orientations of the PPRE consensus sequences indicated were connected to the luciferase gene with minimal promoter. PPARα and RXR binding hexad sequences are indicated. (B) Both orientations of PPRE relative to the luciferase gene were equally activated in primary cultured myocytes stimulated with WY14643 (n = 5–6). (C) Forced expression of PPARα preferentially activates the major direction of PPRE in H9c2 cells (n = 7–9). A solid triangle indicates statistical significance compared with PPRE(←) transfected with the same dose of PPARα expression vector.
Mentions: To investigate the effect of the orientation of PPRE, a reporter assay was performed with a luciferase reporter construct driven by a 32-bp minimum promoter including the PPRE sequence with 5’ and 3’ 7-bp extended sequences, as shown in Fig 2A. Since PPARα is not significantly expressed in an immortalized cell line, we used primary cultured rat neonatal cardiac myocytes to examine the effect of the PPRE sequence on transcriptional activation induced by ligand-stimulated endogenous PPARα. At the same time, we used the H9c2 rat cardiac myocyte cell line to examine the effect of the PPRE sequence on transcriptional activation induced by exogenous PPARα overexpression to reduce animal usage. As shown in Fig 2B, both orientations of the PPRE were equally activated by WY14,647, an artificial ligand for PPARα, in primary culture cardiac myocytes. In contrast, overexpression of PPARα more strongly activated transcription when the orientation of the sequential PPARα and RXR binding site were opposite that of the luciferase gene body (Fig 2C). Thus, ligand-stimulated PPARα equally activates transcription in both directions, whereas PPARα-overexpression-induced transcriptional activation is sensitive to the orientation of the PPRE.

Bottom Line: The single nucleotide substitution, which reduces binding of RXRα to DNA, attenuated PPARα-induced transcriptional activation, but this is not always true for PPARα.Using the definition of the PPRE sequence, novel PPREs were successfully identified.Taken altogether, the provided PPRE sequence definition contributes to the understanding of PPARα signaling by identifying PPARα direct target genes with functional PPARα response elements.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Molecular Medicine, Rutgers Biomedical Health Sciences, Newark, NJ 07103, United States of America.

ABSTRACT
Peroxisome proliferator-activated receptor-α (PPARα), a nuclear receptor, plays an important role in the transcription of genes involved in fatty acid metabolism through heterodimerization with the retinoid x receptor (RXR). The consensus sequence of the PPAR response element (PPRE) is composed of two AGGTCA-like sequences directionally aligned with a single nucleotide spacer. PPARα and RXR bind to the 5' and 3' hexad sequences, respectively. However, the precise sequence definition of the PPRE remains obscure, and thus, the consensus sequence currently available remains AGGTCANAGGTCA with unknown redundancy. The vague PPRE sequence definition poses an obstacle to understanding how PPARα regulates fatty acid metabolism. Here we show that, rather than the generally accepted 6-bp sequence, PPARα actually recognized a 12-bp DNA sequence, of which the preferred binding sequence was WAWVTRGGBBAH. Additionally, the optimized RXRα hexad binding sequence was RGKTYA. Thus, the optimal PPARα/RXRα heterodimer binding sequence was WAWVTRGGBBAHRGKTYA. The single nucleotide substitution, which reduces binding of RXRα to DNA, attenuated PPARα-induced transcriptional activation, but this is not always true for PPARα. Using the definition of the PPRE sequence, novel PPREs were successfully identified. Taken altogether, the provided PPRE sequence definition contributes to the understanding of PPARα signaling by identifying PPARα direct target genes with functional PPARα response elements.

No MeSH data available.