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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 3’ core hexad sequence for RXRα binding.(A) Tandem AGGTCA sequences are required for detecting RXRα DNA binding. The indicated biotin-labeled DNA was incubated with recombinant RXRα. (B) Determination of the optimal DNA sequences for RXRα binding. Biotin-labeled double-stranded oligonucleotides comprising DR1 shown in Fig 6A were incubated with recombinant RXRα and unlabeled competitors (10- and 30-fold excess of biotin-labeled DNA). The competitors had all 4 possible nucleotides at the indicated positions (+1 to +6). All signals without competitors are identical among the four panels at each position. A dotted line indicates a discontinued signal but they are derived from identical blots/membranes. (C-D) The effect of non-preferred nucleotides in the 3’ core hexad sequence for RXRα binding on PPARα-induced transcriptional activation. Reporter gene assays were performed with the indicated sequences of PPRE (n = 6–9).
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pone.0134996.g006: The 3’ core hexad sequence for RXRα binding.(A) Tandem AGGTCA sequences are required for detecting RXRα DNA binding. The indicated biotin-labeled DNA was incubated with recombinant RXRα. (B) Determination of the optimal DNA sequences for RXRα binding. Biotin-labeled double-stranded oligonucleotides comprising DR1 shown in Fig 6A were incubated with recombinant RXRα and unlabeled competitors (10- and 30-fold excess of biotin-labeled DNA). The competitors had all 4 possible nucleotides at the indicated positions (+1 to +6). All signals without competitors are identical among the four panels at each position. A dotted line indicates a discontinued signal but they are derived from identical blots/membranes. (C-D) The effect of non-preferred nucleotides in the 3’ core hexad sequence for RXRα binding on PPARα-induced transcriptional activation. Reporter gene assays were performed with the indicated sequences of PPRE (n = 6–9).

Mentions: RXR binds to the 3’ core hexad sequence of the PPRE. To identify the optimal 3’ core hexad sequence, we performed DNA pull-down assays using recombinant RXRα and biotin-labeled DNA. As shown in Fig 6A, biotin-labeled DNA that included the sequence identified as the putative PPRE in the heart (Fig 1H) but that did not contain the PPARα binding half-site (PPRE(A)3’) was used first. RXRα did not significantly bind to the PPRE(A)3’ containing only the single AGGTCA sequence, although RXRα did significantly bind to PPRE(A), which is composed of two AGGTCA sequences. The proximal sequence of the RXR binding site of the core hexad element of the putative PPRE (Fig 1H) may coincidently comprise a certain sequence that reduces RXRα DNA binding. However, the same result was observed when the perfect DR1 and a single AGGTCA sequence were introduced into a sequential A sequence. These results suggest that binding of RXRα to DNA is highly dependent on dimer formation, despite the fact that monomeric PPARα strongly binds to DNA under the same experimental conditions.


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 3’ core hexad sequence for RXRα binding.(A) Tandem AGGTCA sequences are required for detecting RXRα DNA binding. The indicated biotin-labeled DNA was incubated with recombinant RXRα. (B) Determination of the optimal DNA sequences for RXRα binding. Biotin-labeled double-stranded oligonucleotides comprising DR1 shown in Fig 6A were incubated with recombinant RXRα and unlabeled competitors (10- and 30-fold excess of biotin-labeled DNA). The competitors had all 4 possible nucleotides at the indicated positions (+1 to +6). All signals without competitors are identical among the four panels at each position. A dotted line indicates a discontinued signal but they are derived from identical blots/membranes. (C-D) The effect of non-preferred nucleotides in the 3’ core hexad sequence for RXRα binding on PPARα-induced transcriptional activation. Reporter gene assays were performed with the indicated sequences of PPRE (n = 6–9).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134996.g006: The 3’ core hexad sequence for RXRα binding.(A) Tandem AGGTCA sequences are required for detecting RXRα DNA binding. The indicated biotin-labeled DNA was incubated with recombinant RXRα. (B) Determination of the optimal DNA sequences for RXRα binding. Biotin-labeled double-stranded oligonucleotides comprising DR1 shown in Fig 6A were incubated with recombinant RXRα and unlabeled competitors (10- and 30-fold excess of biotin-labeled DNA). The competitors had all 4 possible nucleotides at the indicated positions (+1 to +6). All signals without competitors are identical among the four panels at each position. A dotted line indicates a discontinued signal but they are derived from identical blots/membranes. (C-D) The effect of non-preferred nucleotides in the 3’ core hexad sequence for RXRα binding on PPARα-induced transcriptional activation. Reporter gene assays were performed with the indicated sequences of PPRE (n = 6–9).
Mentions: RXR binds to the 3’ core hexad sequence of the PPRE. To identify the optimal 3’ core hexad sequence, we performed DNA pull-down assays using recombinant RXRα and biotin-labeled DNA. As shown in Fig 6A, biotin-labeled DNA that included the sequence identified as the putative PPRE in the heart (Fig 1H) but that did not contain the PPARα binding half-site (PPRE(A)3’) was used first. RXRα did not significantly bind to the PPRE(A)3’ containing only the single AGGTCA sequence, although RXRα did significantly bind to PPRE(A), which is composed of two AGGTCA sequences. The proximal sequence of the RXR binding site of the core hexad element of the putative PPRE (Fig 1H) may coincidently comprise a certain sequence that reduces RXRα DNA binding. However, the same result was observed when the perfect DR1 and a single AGGTCA sequence were introduced into a sequential A sequence. These results suggest that binding of RXRα to DNA is highly dependent on dimer formation, despite the fact that monomeric PPARα strongly binds to DNA under the same experimental conditions.

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.