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5-Lipoxygenase DNA methylation and mRNA content in the brain and heart of young and old mice.

Dzitoyeva S, Imbesi M, Ng LW, Manev H - Neural Plast. (2009)

Bottom Line: Aging also increased 5-LOX DNA methylation and this effect was site- (i.e., enzyme) and tissue-specific.Generally, DNMT1 and DNMT3a mRNA content was lower in the brain regions compared to the heart; the only effect of aging was observed in the mRNA content of DNMT3a, which was decreased in the heart of old mice.These results indicate a complex tissue-specific and aging-dependent interplay between the DNA methylation system and 5-LOX mRNA content.

View Article: PubMed Central - PubMed

Affiliation: The Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, 1601 West Taylor Street, MC912, Chicago, IL 60612, USA.

ABSTRACT
The expression of 5-lipoxygenase (5-LOX) is affected by aging and regulated by epigenetic mechanisms including DNA methylation. We used methylation-sensitive restriction endonucleases (AciI, BstUI, HpaII, and HinP1I) to assess 5-LOX DNA methylation in brain and heart tissue samples from young (2 months) and old (22 months) mice. We also measured mRNA content for 5-LOX and the DNA methyltransferases DNMT1 and DNMT3a. In young mice, the 5-LOX mRNA content was significantly greater in the heart compared to the brain; 5-LOX DNA methylation was lower, except in the AciI assay in which it was higher in the heart. Aging decreased 5-LOX mRNA content in the heart and increased it in the brain. Aging also increased 5-LOX DNA methylation and this effect was site- (i.e., enzyme) and tissue-specific. Generally, DNMT1 and DNMT3a mRNA content was lower in the brain regions compared to the heart; the only effect of aging was observed in the mRNA content of DNMT3a, which was decreased in the heart of old mice. These results indicate a complex tissue-specific and aging-dependent interplay between the DNA methylation system and 5-LOX mRNA content. Interpretation of this data must take into account that the tissue samples contained a mixture of various cell types.

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Related in: MedlinePlus

An example of the SYBR Green RD-qPCR assay of 5-LOX DNA methylation (shown are graphs obtained from 10 cerebellar samples). The samples were digested with the methylation-sensitive endonucleases (AciI, BstUI, HinP1I, and HpaII) as described in Material and Methods. The PCR reaction for the promoter-5′UTR (10 samples each: AciI = blue, BstUI = red, HinP1I = green, and HpaII = gray) and corresponding input control regions (shown in yellow) were carried out in separate tubes. Panel A shows the dissociation curve data, which indicate the presence of only one PCR product (peak) for each specific set of primers (fluorescence (first derivative of the raw  fluorescence reading multiplied by  −1) on the Y-axis versus the PCR product melting temperature (°C) on the X-axis). Panel B shows examples of the amplification plots used for calculating the quantitative data (the amplification plots fluorescence (baseline-corrected raw fluorescence) on the Y-axis versus cycle number on the X-axis). In this assay, the threshold cycle is inversely proportional to the log of the initial copy number. In other words, the more template that is present initially, the fewer the number of cycles required for the fluorescence signal to be detectable above background.
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fig2: An example of the SYBR Green RD-qPCR assay of 5-LOX DNA methylation (shown are graphs obtained from 10 cerebellar samples). The samples were digested with the methylation-sensitive endonucleases (AciI, BstUI, HinP1I, and HpaII) as described in Material and Methods. The PCR reaction for the promoter-5′UTR (10 samples each: AciI = blue, BstUI = red, HinP1I = green, and HpaII = gray) and corresponding input control regions (shown in yellow) were carried out in separate tubes. Panel A shows the dissociation curve data, which indicate the presence of only one PCR product (peak) for each specific set of primers (fluorescence (first derivative of the raw fluorescence reading multiplied by −1) on the Y-axis versus the PCR product melting temperature (°C) on the X-axis). Panel B shows examples of the amplification plots used for calculating the quantitative data (the amplification plots fluorescence (baseline-corrected raw fluorescence) on the Y-axis versus cycle number on the X-axis). In this assay, the threshold cycle is inversely proportional to the log of the initial copy number. In other words, the more template that is present initially, the fewer the number of cycles required for the fluorescence signal to be detectable above background.

Mentions: Genomic DNA was extracted from the above-noted tissues. They were homogenized in homogenizing buffer (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 25 mM EDTA, 0.5% SDS, 0.1 mg/ml proteinase K), 100 μl per 10 mg of tissue, and incubated 4-5 hours at 37°C. The DNA was purified with phenol, phenol-chloroform (pH 8.0), precipitated with isopropanol, and dissolved in H2O. The DNA methylation assay was designed as follows: The promoter-exon-intron structure of the Mus musculus 5-LOX gene was reconstructed by aligning the 5-LOX (ALOX5) mRNA (NM_009662), the promoter, the 5′ untranslated region (UTR), and partial coding region sequences (AF393814) with the mouse genome sequence. This procedure identified a sequence of about 53 kb. Although the mouse 5-LOX gene promoter does not contain typical CpG islands, the mouse 5-LOX promoter-5′UTR-first exon, the adjacent intron portion, and the 3′ end of the gene contain the highest C and G densities. The 5′UTR-first exon and the adjacent intron region (comprising 519 nucleotides) contain 66% of all C and G nucleotides. Furthermore, this region contains 41 CpG dinucleotides; 14 are located upstream from the ATG translation start codon and 27 are downstream (Figure 1). Multiple methylation-sensitive endonuclease recognition sites were located in that portion. Thus, we investigated their methylation rate (i.e., upstream and downstream from the ATG translation start codon) with the aid of four methylation-sensitive endonucleases—AciI (C ↓ CGC), BstUI (CG ↓ CG), HinP1I (G ↓ CGC), and HpaII (C ↓ CGG) (their respective recognition sequences cutting sites are shown in parentheses). To measure the 5-LOX DNA methylation levels, we used the restriction digest-quantitative PCR (SYBR Green RD-qPCR). The selected approach utilizes the ability of methylation-sensitive endonucleases to digest only unmethylated recognition sites, and their inability to act on sites with methylated cytosine. Thus, if the targeted *CpG is methylated, the site is blocked for the enzyme's endonuclease activity and as a result, greater amounts of templates are available for the action of the Taq DNA polymerase. The assay was performed as follows: 1 μg of genomic DNA was used in a restriction digest reaction for each of the four endonucleases. Digested DNA samples were diluted with water and an aliquot (100 ng DNA) was used for qPCR (Stratagene) with the Maxima SYBR Green qPCR Master Mix (Fermentas) according to the manufacturer's protocol. The following primers were used: forward 5′-agagaaggatgcgttggaaggt-3′ and reverse 5′-gactccgggcaagtgagtgct -3′. These primers amplify the 238 nt region upstream of the first ATG translation start codon. This region contains two recognition sites for each of 4 endonucleases selected for the assay. Primers for the exon-intron part were as follows: forward 5′-agtcatgccctcctacacggtca-3′, reverse 5′-agtcatgccctcctacacggtca-3′. They amplify a 344 bp fragment. For the input control, we used the 394 nt region in the first intron because this region does not contain recognition sites for the selected methylation-sensitive endonucleases. This region was amplified with the following primers: forward 5′-tgatgtggctggcctcttatgtga-3′, reverse 5′-actgggactgagtgcaggaaatgt-3′. The qPCR reactions were run with 2 different primer sets (target and input) in separate tubes and the coefficient of variation for the relative amount of a target sequence was calculated [36]. An example of the qPCR assay performance is shown in Figure 2.


5-Lipoxygenase DNA methylation and mRNA content in the brain and heart of young and old mice.

Dzitoyeva S, Imbesi M, Ng LW, Manev H - Neural Plast. (2009)

An example of the SYBR Green RD-qPCR assay of 5-LOX DNA methylation (shown are graphs obtained from 10 cerebellar samples). The samples were digested with the methylation-sensitive endonucleases (AciI, BstUI, HinP1I, and HpaII) as described in Material and Methods. The PCR reaction for the promoter-5′UTR (10 samples each: AciI = blue, BstUI = red, HinP1I = green, and HpaII = gray) and corresponding input control regions (shown in yellow) were carried out in separate tubes. Panel A shows the dissociation curve data, which indicate the presence of only one PCR product (peak) for each specific set of primers (fluorescence (first derivative of the raw  fluorescence reading multiplied by  −1) on the Y-axis versus the PCR product melting temperature (°C) on the X-axis). Panel B shows examples of the amplification plots used for calculating the quantitative data (the amplification plots fluorescence (baseline-corrected raw fluorescence) on the Y-axis versus cycle number on the X-axis). In this assay, the threshold cycle is inversely proportional to the log of the initial copy number. In other words, the more template that is present initially, the fewer the number of cycles required for the fluorescence signal to be detectable above background.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2801004&req=5

fig2: An example of the SYBR Green RD-qPCR assay of 5-LOX DNA methylation (shown are graphs obtained from 10 cerebellar samples). The samples were digested with the methylation-sensitive endonucleases (AciI, BstUI, HinP1I, and HpaII) as described in Material and Methods. The PCR reaction for the promoter-5′UTR (10 samples each: AciI = blue, BstUI = red, HinP1I = green, and HpaII = gray) and corresponding input control regions (shown in yellow) were carried out in separate tubes. Panel A shows the dissociation curve data, which indicate the presence of only one PCR product (peak) for each specific set of primers (fluorescence (first derivative of the raw fluorescence reading multiplied by −1) on the Y-axis versus the PCR product melting temperature (°C) on the X-axis). Panel B shows examples of the amplification plots used for calculating the quantitative data (the amplification plots fluorescence (baseline-corrected raw fluorescence) on the Y-axis versus cycle number on the X-axis). In this assay, the threshold cycle is inversely proportional to the log of the initial copy number. In other words, the more template that is present initially, the fewer the number of cycles required for the fluorescence signal to be detectable above background.
Mentions: Genomic DNA was extracted from the above-noted tissues. They were homogenized in homogenizing buffer (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 25 mM EDTA, 0.5% SDS, 0.1 mg/ml proteinase K), 100 μl per 10 mg of tissue, and incubated 4-5 hours at 37°C. The DNA was purified with phenol, phenol-chloroform (pH 8.0), precipitated with isopropanol, and dissolved in H2O. The DNA methylation assay was designed as follows: The promoter-exon-intron structure of the Mus musculus 5-LOX gene was reconstructed by aligning the 5-LOX (ALOX5) mRNA (NM_009662), the promoter, the 5′ untranslated region (UTR), and partial coding region sequences (AF393814) with the mouse genome sequence. This procedure identified a sequence of about 53 kb. Although the mouse 5-LOX gene promoter does not contain typical CpG islands, the mouse 5-LOX promoter-5′UTR-first exon, the adjacent intron portion, and the 3′ end of the gene contain the highest C and G densities. The 5′UTR-first exon and the adjacent intron region (comprising 519 nucleotides) contain 66% of all C and G nucleotides. Furthermore, this region contains 41 CpG dinucleotides; 14 are located upstream from the ATG translation start codon and 27 are downstream (Figure 1). Multiple methylation-sensitive endonuclease recognition sites were located in that portion. Thus, we investigated their methylation rate (i.e., upstream and downstream from the ATG translation start codon) with the aid of four methylation-sensitive endonucleases—AciI (C ↓ CGC), BstUI (CG ↓ CG), HinP1I (G ↓ CGC), and HpaII (C ↓ CGG) (their respective recognition sequences cutting sites are shown in parentheses). To measure the 5-LOX DNA methylation levels, we used the restriction digest-quantitative PCR (SYBR Green RD-qPCR). The selected approach utilizes the ability of methylation-sensitive endonucleases to digest only unmethylated recognition sites, and their inability to act on sites with methylated cytosine. Thus, if the targeted *CpG is methylated, the site is blocked for the enzyme's endonuclease activity and as a result, greater amounts of templates are available for the action of the Taq DNA polymerase. The assay was performed as follows: 1 μg of genomic DNA was used in a restriction digest reaction for each of the four endonucleases. Digested DNA samples were diluted with water and an aliquot (100 ng DNA) was used for qPCR (Stratagene) with the Maxima SYBR Green qPCR Master Mix (Fermentas) according to the manufacturer's protocol. The following primers were used: forward 5′-agagaaggatgcgttggaaggt-3′ and reverse 5′-gactccgggcaagtgagtgct -3′. These primers amplify the 238 nt region upstream of the first ATG translation start codon. This region contains two recognition sites for each of 4 endonucleases selected for the assay. Primers for the exon-intron part were as follows: forward 5′-agtcatgccctcctacacggtca-3′, reverse 5′-agtcatgccctcctacacggtca-3′. They amplify a 344 bp fragment. For the input control, we used the 394 nt region in the first intron because this region does not contain recognition sites for the selected methylation-sensitive endonucleases. This region was amplified with the following primers: forward 5′-tgatgtggctggcctcttatgtga-3′, reverse 5′-actgggactgagtgcaggaaatgt-3′. The qPCR reactions were run with 2 different primer sets (target and input) in separate tubes and the coefficient of variation for the relative amount of a target sequence was calculated [36]. An example of the qPCR assay performance is shown in Figure 2.

Bottom Line: Aging also increased 5-LOX DNA methylation and this effect was site- (i.e., enzyme) and tissue-specific.Generally, DNMT1 and DNMT3a mRNA content was lower in the brain regions compared to the heart; the only effect of aging was observed in the mRNA content of DNMT3a, which was decreased in the heart of old mice.These results indicate a complex tissue-specific and aging-dependent interplay between the DNA methylation system and 5-LOX mRNA content.

View Article: PubMed Central - PubMed

Affiliation: The Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, 1601 West Taylor Street, MC912, Chicago, IL 60612, USA.

ABSTRACT
The expression of 5-lipoxygenase (5-LOX) is affected by aging and regulated by epigenetic mechanisms including DNA methylation. We used methylation-sensitive restriction endonucleases (AciI, BstUI, HpaII, and HinP1I) to assess 5-LOX DNA methylation in brain and heart tissue samples from young (2 months) and old (22 months) mice. We also measured mRNA content for 5-LOX and the DNA methyltransferases DNMT1 and DNMT3a. In young mice, the 5-LOX mRNA content was significantly greater in the heart compared to the brain; 5-LOX DNA methylation was lower, except in the AciI assay in which it was higher in the heart. Aging decreased 5-LOX mRNA content in the heart and increased it in the brain. Aging also increased 5-LOX DNA methylation and this effect was site- (i.e., enzyme) and tissue-specific. Generally, DNMT1 and DNMT3a mRNA content was lower in the brain regions compared to the heart; the only effect of aging was observed in the mRNA content of DNMT3a, which was decreased in the heart of old mice. These results indicate a complex tissue-specific and aging-dependent interplay between the DNA methylation system and 5-LOX mRNA content. Interpretation of this data must take into account that the tissue samples contained a mixture of various cell types.

Show MeSH
Related in: MedlinePlus