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Quantitative evaluation of DNA methylation patterns for ALVE and TVB genes in a neoplastic disease susceptible and resistant chicken model.

Yu Y, Zhang H, Tian F, Bacon L, Zhang Y, Zhang W, Song J - PLoS ONE (2008)

Bottom Line: CpG methylation contents of the ALVE regions were significantly lower in line 6(3) than in line 7(2) in all tissues (P < 0.01) except the ALVE region 3/4 in liver.RNA expressions of ALVE regions 2 and 3 (PPT-U3) were significantly higher in line 6(3) than in line 7(2) (P < 0.01).The methylation level of the promoter region in the TVB was significantly different in blood (P < 0.05) and hypothalamus (P < 0.0001), respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal & Avian Sciences, University of Maryland, College Park, Maryland, USA.

ABSTRACT
Chicken endogenous viruses, ALVE (Avian Leukosis Virus subgroup E), are inherited as LTR (long terminal repeat) retrotransposons, which are negatively correlated with disease resistance, and any changes in DNA methylation may contribute to the susceptibility to neoplastic disease. The relationship between ALVE methylation status and neoplastic disease in the chicken is undefined. White Leghorn inbred lines 7(2) and 6(3) at the ADOL have been respectively selected for resistance and susceptibility to tumors that are induced by avian viruses. In this study, the DNA methylation patterns of 3 approximately 6 CpG sites of four conserved regions in ALVE, including one unique region in ALVE1, the promoter region in the TVB (tumor virus receptor of ALV subgroup B, D and E) locus, were analyzed in the two lines using pyrosequencing methods in four tissues, i.e., liver, spleen, blood and hypothalamus. A significant CpG hypermethylation level was seen in line 7(2) in all four tissues, e.g., 91.86 +/- 1.63% for ALVE region2 in blood, whereas the same region was hemimethylated (46.16 +/- 2.56%) in line 6(3). CpG methylation contents of the ALVE regions were significantly lower in line 6(3) than in line 7(2) in all tissues (P < 0.01) except the ALVE region 3/4 in liver. RNA expressions of ALVE regions 2 and 3 (PPT-U3) were significantly higher in line 6(3) than in line 7(2) (P < 0.01). The methylation levels of six recombinant congenic strains (RCSs) closely resembled to the background line 6(3) in ALVE-region 2, which imply the methylation pattern of ALVE-region 2 may be a biomarker in resistant disease breeding. The methylation level of the promoter region in the TVB was significantly different in blood (P < 0.05) and hypothalamus (P < 0.0001), respectively. Our data disclosed a hypermethylation pattern of ALVE that may be relevant for resistance against ALV induced tumors in chickens.

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Representative pyrograms for each gene region in line 72 and 63.The percentage on each CpG site is the methylation percentage of mC/(mC+C) on this site. mC: methylated cytosine, C: unmethylated cytosine. The sequence above the peak is the sequence to assay. The x axes are the dispensation nucleotides to the sequencing reaction based on the assayed sequences. The y axes show light emission obtained as relative light units. A: ALVE region1 in blood (the mean value of methylation percentage is 81.8±2.9 in line 72 and 13.2±1.2 in 63); B: ALVE region2 in blood (the mean value of methylation percentage is 85±1.6 in line 72 and 47.6±2.3 in 63); C: ALVE region3/4 in blood (the mean value of methylation percentage is 80.8±4.8 in line 72 and 68.8±6.7 in 63); D: Promoter region of TVB in Hypothalamus (the mean value of methylation percentage is 45.7±14.8 in line 72 and 37.0±14.1 in 63).
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pone-0001731-g002: Representative pyrograms for each gene region in line 72 and 63.The percentage on each CpG site is the methylation percentage of mC/(mC+C) on this site. mC: methylated cytosine, C: unmethylated cytosine. The sequence above the peak is the sequence to assay. The x axes are the dispensation nucleotides to the sequencing reaction based on the assayed sequences. The y axes show light emission obtained as relative light units. A: ALVE region1 in blood (the mean value of methylation percentage is 81.8±2.9 in line 72 and 13.2±1.2 in 63); B: ALVE region2 in blood (the mean value of methylation percentage is 85±1.6 in line 72 and 47.6±2.3 in 63); C: ALVE region3/4 in blood (the mean value of methylation percentage is 80.8±4.8 in line 72 and 68.8±6.7 in 63); D: Promoter region of TVB in Hypothalamus (the mean value of methylation percentage is 45.7±14.8 in line 72 and 37.0±14.1 in 63).

Mentions: The whole ALVE1 is a CpG island that including 813 CpG sites within 7.5 kp based on the BLAT with UCSC Genomic Browser (Figure 1). To determine the DNA methylation levels of ALVEs, we selected four conserved regions among ALVE1, 2 and 3 that cover the gag gene, PPT region and U3 regions of 5′ and 3′ LTR, then quantitative evaluated the methylation differences between inbred chicken line 63 and line 72 using pyrosequencing method in four different tissues (blood, liver, spleen and hypothalamus) (Figure 1). Figure 2A, 2B and 2C show the typical methylation pyrograms for the six CpG sites in ALVE region 1 (p19 matrix protein in gag gene), the five CpG sites in ALVE region 2 (Direction Repeat Sequence, DRS, adjacent to 3′ LTR of ALVE), and the four CpG sites in ALVE region 3/4 (U3 region in 5′ and 3′ LTR of ALVE) in blood of line 72 and line 63, respectively. The averages from five individuals showed that hypermethylated CpG sites were identified in blood of line 72 , 85.56±2.66% for ALVE region 1 and 91.86±1.63% for ALVE region 2, whereas the same regions are hypomethylated (10.05±2.56%) and hemimethylated (46.16±3.41%) in the line 63 (Figure 2). The measurements using pyrosequencing technique for each conserved region of ALVE were proved to be highly reproducible between biological replicates. Moreover, the methylation percentages of region 2 in ALVE from bisulfite cloning and sequencing methods were very comparable to that from pyrosequencing as shown in Figure S2, which validated the pyrosequencing data.


Quantitative evaluation of DNA methylation patterns for ALVE and TVB genes in a neoplastic disease susceptible and resistant chicken model.

Yu Y, Zhang H, Tian F, Bacon L, Zhang Y, Zhang W, Song J - PLoS ONE (2008)

Representative pyrograms for each gene region in line 72 and 63.The percentage on each CpG site is the methylation percentage of mC/(mC+C) on this site. mC: methylated cytosine, C: unmethylated cytosine. The sequence above the peak is the sequence to assay. The x axes are the dispensation nucleotides to the sequencing reaction based on the assayed sequences. The y axes show light emission obtained as relative light units. A: ALVE region1 in blood (the mean value of methylation percentage is 81.8±2.9 in line 72 and 13.2±1.2 in 63); B: ALVE region2 in blood (the mean value of methylation percentage is 85±1.6 in line 72 and 47.6±2.3 in 63); C: ALVE region3/4 in blood (the mean value of methylation percentage is 80.8±4.8 in line 72 and 68.8±6.7 in 63); D: Promoter region of TVB in Hypothalamus (the mean value of methylation percentage is 45.7±14.8 in line 72 and 37.0±14.1 in 63).
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Related In: Results  -  Collection

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pone-0001731-g002: Representative pyrograms for each gene region in line 72 and 63.The percentage on each CpG site is the methylation percentage of mC/(mC+C) on this site. mC: methylated cytosine, C: unmethylated cytosine. The sequence above the peak is the sequence to assay. The x axes are the dispensation nucleotides to the sequencing reaction based on the assayed sequences. The y axes show light emission obtained as relative light units. A: ALVE region1 in blood (the mean value of methylation percentage is 81.8±2.9 in line 72 and 13.2±1.2 in 63); B: ALVE region2 in blood (the mean value of methylation percentage is 85±1.6 in line 72 and 47.6±2.3 in 63); C: ALVE region3/4 in blood (the mean value of methylation percentage is 80.8±4.8 in line 72 and 68.8±6.7 in 63); D: Promoter region of TVB in Hypothalamus (the mean value of methylation percentage is 45.7±14.8 in line 72 and 37.0±14.1 in 63).
Mentions: The whole ALVE1 is a CpG island that including 813 CpG sites within 7.5 kp based on the BLAT with UCSC Genomic Browser (Figure 1). To determine the DNA methylation levels of ALVEs, we selected four conserved regions among ALVE1, 2 and 3 that cover the gag gene, PPT region and U3 regions of 5′ and 3′ LTR, then quantitative evaluated the methylation differences between inbred chicken line 63 and line 72 using pyrosequencing method in four different tissues (blood, liver, spleen and hypothalamus) (Figure 1). Figure 2A, 2B and 2C show the typical methylation pyrograms for the six CpG sites in ALVE region 1 (p19 matrix protein in gag gene), the five CpG sites in ALVE region 2 (Direction Repeat Sequence, DRS, adjacent to 3′ LTR of ALVE), and the four CpG sites in ALVE region 3/4 (U3 region in 5′ and 3′ LTR of ALVE) in blood of line 72 and line 63, respectively. The averages from five individuals showed that hypermethylated CpG sites were identified in blood of line 72 , 85.56±2.66% for ALVE region 1 and 91.86±1.63% for ALVE region 2, whereas the same regions are hypomethylated (10.05±2.56%) and hemimethylated (46.16±3.41%) in the line 63 (Figure 2). The measurements using pyrosequencing technique for each conserved region of ALVE were proved to be highly reproducible between biological replicates. Moreover, the methylation percentages of region 2 in ALVE from bisulfite cloning and sequencing methods were very comparable to that from pyrosequencing as shown in Figure S2, which validated the pyrosequencing data.

Bottom Line: CpG methylation contents of the ALVE regions were significantly lower in line 6(3) than in line 7(2) in all tissues (P < 0.01) except the ALVE region 3/4 in liver.RNA expressions of ALVE regions 2 and 3 (PPT-U3) were significantly higher in line 6(3) than in line 7(2) (P < 0.01).The methylation level of the promoter region in the TVB was significantly different in blood (P < 0.05) and hypothalamus (P < 0.0001), respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal & Avian Sciences, University of Maryland, College Park, Maryland, USA.

ABSTRACT
Chicken endogenous viruses, ALVE (Avian Leukosis Virus subgroup E), are inherited as LTR (long terminal repeat) retrotransposons, which are negatively correlated with disease resistance, and any changes in DNA methylation may contribute to the susceptibility to neoplastic disease. The relationship between ALVE methylation status and neoplastic disease in the chicken is undefined. White Leghorn inbred lines 7(2) and 6(3) at the ADOL have been respectively selected for resistance and susceptibility to tumors that are induced by avian viruses. In this study, the DNA methylation patterns of 3 approximately 6 CpG sites of four conserved regions in ALVE, including one unique region in ALVE1, the promoter region in the TVB (tumor virus receptor of ALV subgroup B, D and E) locus, were analyzed in the two lines using pyrosequencing methods in four tissues, i.e., liver, spleen, blood and hypothalamus. A significant CpG hypermethylation level was seen in line 7(2) in all four tissues, e.g., 91.86 +/- 1.63% for ALVE region2 in blood, whereas the same region was hemimethylated (46.16 +/- 2.56%) in line 6(3). CpG methylation contents of the ALVE regions were significantly lower in line 6(3) than in line 7(2) in all tissues (P < 0.01) except the ALVE region 3/4 in liver. RNA expressions of ALVE regions 2 and 3 (PPT-U3) were significantly higher in line 6(3) than in line 7(2) (P < 0.01). The methylation levels of six recombinant congenic strains (RCSs) closely resembled to the background line 6(3) in ALVE-region 2, which imply the methylation pattern of ALVE-region 2 may be a biomarker in resistant disease breeding. The methylation level of the promoter region in the TVB was significantly different in blood (P < 0.05) and hypothalamus (P < 0.0001), respectively. Our data disclosed a hypermethylation pattern of ALVE that may be relevant for resistance against ALV induced tumors in chickens.

Show MeSH
Related in: MedlinePlus