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Aberrant methylation and associated transcriptional mobilization of Alu elements contributes to genomic instability in hypoxia.

Pal A, Srivastava T, Sharma MK, Mehndiratta M, Das P, Sinha S, Chattopadhyay P - J. Cell. Mol. Med. (2010)

Bottom Line: Because a majority of the cytosine-phosphate-guanine (CpG) islands are found within the repeat elements of DNA, and are usually methylated under normoxic conditions, we suggested that retrotransposable Alu or short interspersed nuclear elements (SINEs) which show altered methylation and associated changes of gene expression during hypoxia, could be associated with genomic instability.U87MG glioblastoma cells were cultured in 0.1% O₂ for 6 weeks and compared with cells cultured in 21% O₂ for the same duration.Our results show that aberrant methylation leading to increased transcription of SINE and reverse transcriptase associated LINE elements could lead to increased genomic instability in hypoxia.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.

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(A) Change in percentage (%) of site-specific CpG methylation at SINE locus (i) at chromosome 16 in U87MG, (ii) at chromosome 16 in SaOS2, (iii) at chromosome 7 in U87MG, (iv) at chromosome 7 in SaOS2. Change is calculated for test group as follows: N(-)H: percentage methylation in normal minus percentage methylation in hypoxia; N(-)R: percentage methylation in normal minus percentage methylation in hypoxia; H(-)HR: percentage methylation in hypoxia minus percentage methylation in revert. x-axis: CpG sites; y-axis: percentage change in methylation. (B) Global methylation assay: LINE-COBRA was done in two biological replicates of normoxia (N), hypoxia (H) and reverted samples (HR) of U87MG and SaOS2 cells and the average value is depicted. In the representative picture shown, the band of 160 bp shows the unmethylated fraction and 80 bp is of methylated fraction. Densitometry shows global hypomethylation in hypoxia samples as compared to normoxia controls in both U87MG (P= 0.033) and SaOS2 (P= 0.032) cells. (C) DNMT1 expression: Western blot of U87MG cells exposed to various durations of 0.1% hypoxia showed a marked decrease in DNMT1 levels in 6-week hypoxia (H) as compared to N. The sudden increase in DNMT1 expression in 1 hr hypoxia was followed by reduced expression in 24 hrs, 1 week and 6 weeks. The IDV values are normalized to b-actin levels.
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fig03: (A) Change in percentage (%) of site-specific CpG methylation at SINE locus (i) at chromosome 16 in U87MG, (ii) at chromosome 16 in SaOS2, (iii) at chromosome 7 in U87MG, (iv) at chromosome 7 in SaOS2. Change is calculated for test group as follows: N(-)H: percentage methylation in normal minus percentage methylation in hypoxia; N(-)R: percentage methylation in normal minus percentage methylation in hypoxia; H(-)HR: percentage methylation in hypoxia minus percentage methylation in revert. x-axis: CpG sites; y-axis: percentage change in methylation. (B) Global methylation assay: LINE-COBRA was done in two biological replicates of normoxia (N), hypoxia (H) and reverted samples (HR) of U87MG and SaOS2 cells and the average value is depicted. In the representative picture shown, the band of 160 bp shows the unmethylated fraction and 80 bp is of methylated fraction. Densitometry shows global hypomethylation in hypoxia samples as compared to normoxia controls in both U87MG (P= 0.033) and SaOS2 (P= 0.032) cells. (C) DNMT1 expression: Western blot of U87MG cells exposed to various durations of 0.1% hypoxia showed a marked decrease in DNMT1 levels in 6-week hypoxia (H) as compared to N. The sudden increase in DNMT1 expression in 1 hr hypoxia was followed by reduced expression in 24 hrs, 1 week and 6 weeks. The IDV values are normalized to b-actin levels.

Mentions: From the consensus SINE locus of chromosome 16, 23 CpG sites were chosen for bisulphite PCR sequencing. In U87MG, there was statistically significant decrease in methylation status after exposure to long-term hypoxic conditions which did not change even after reverting back to normoxic conditions. Seventeen sites showed average decrease in methylation by 9.76% (from 91.37% to 81.63%) (P= 0.01) in hypoxia with respect to normoxia (Fig. 3A-i) (Fig. S2). Major hypomethylation was seen at two specific CpG sites (site14: from 91.6% to 47.8% and site17: from 77.1% to 34.2%). Of these two sites, site 17 showed no change in methylation on reversion to normoxia, while at site 14 there was some increase in methylation in revertent (62.8%). The average percentage of methylation of these 17 sites in revertents was 81.5%, i.e. there was almost no change (0.05%) in methylation in comparison to hypoxia (P= 0.97) (Fig. 3A-i).


Aberrant methylation and associated transcriptional mobilization of Alu elements contributes to genomic instability in hypoxia.

Pal A, Srivastava T, Sharma MK, Mehndiratta M, Das P, Sinha S, Chattopadhyay P - J. Cell. Mol. Med. (2010)

(A) Change in percentage (%) of site-specific CpG methylation at SINE locus (i) at chromosome 16 in U87MG, (ii) at chromosome 16 in SaOS2, (iii) at chromosome 7 in U87MG, (iv) at chromosome 7 in SaOS2. Change is calculated for test group as follows: N(-)H: percentage methylation in normal minus percentage methylation in hypoxia; N(-)R: percentage methylation in normal minus percentage methylation in hypoxia; H(-)HR: percentage methylation in hypoxia minus percentage methylation in revert. x-axis: CpG sites; y-axis: percentage change in methylation. (B) Global methylation assay: LINE-COBRA was done in two biological replicates of normoxia (N), hypoxia (H) and reverted samples (HR) of U87MG and SaOS2 cells and the average value is depicted. In the representative picture shown, the band of 160 bp shows the unmethylated fraction and 80 bp is of methylated fraction. Densitometry shows global hypomethylation in hypoxia samples as compared to normoxia controls in both U87MG (P= 0.033) and SaOS2 (P= 0.032) cells. (C) DNMT1 expression: Western blot of U87MG cells exposed to various durations of 0.1% hypoxia showed a marked decrease in DNMT1 levels in 6-week hypoxia (H) as compared to N. The sudden increase in DNMT1 expression in 1 hr hypoxia was followed by reduced expression in 24 hrs, 1 week and 6 weeks. The IDV values are normalized to b-actin levels.
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Related In: Results  -  Collection

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fig03: (A) Change in percentage (%) of site-specific CpG methylation at SINE locus (i) at chromosome 16 in U87MG, (ii) at chromosome 16 in SaOS2, (iii) at chromosome 7 in U87MG, (iv) at chromosome 7 in SaOS2. Change is calculated for test group as follows: N(-)H: percentage methylation in normal minus percentage methylation in hypoxia; N(-)R: percentage methylation in normal minus percentage methylation in hypoxia; H(-)HR: percentage methylation in hypoxia minus percentage methylation in revert. x-axis: CpG sites; y-axis: percentage change in methylation. (B) Global methylation assay: LINE-COBRA was done in two biological replicates of normoxia (N), hypoxia (H) and reverted samples (HR) of U87MG and SaOS2 cells and the average value is depicted. In the representative picture shown, the band of 160 bp shows the unmethylated fraction and 80 bp is of methylated fraction. Densitometry shows global hypomethylation in hypoxia samples as compared to normoxia controls in both U87MG (P= 0.033) and SaOS2 (P= 0.032) cells. (C) DNMT1 expression: Western blot of U87MG cells exposed to various durations of 0.1% hypoxia showed a marked decrease in DNMT1 levels in 6-week hypoxia (H) as compared to N. The sudden increase in DNMT1 expression in 1 hr hypoxia was followed by reduced expression in 24 hrs, 1 week and 6 weeks. The IDV values are normalized to b-actin levels.
Mentions: From the consensus SINE locus of chromosome 16, 23 CpG sites were chosen for bisulphite PCR sequencing. In U87MG, there was statistically significant decrease in methylation status after exposure to long-term hypoxic conditions which did not change even after reverting back to normoxic conditions. Seventeen sites showed average decrease in methylation by 9.76% (from 91.37% to 81.63%) (P= 0.01) in hypoxia with respect to normoxia (Fig. 3A-i) (Fig. S2). Major hypomethylation was seen at two specific CpG sites (site14: from 91.6% to 47.8% and site17: from 77.1% to 34.2%). Of these two sites, site 17 showed no change in methylation on reversion to normoxia, while at site 14 there was some increase in methylation in revertent (62.8%). The average percentage of methylation of these 17 sites in revertents was 81.5%, i.e. there was almost no change (0.05%) in methylation in comparison to hypoxia (P= 0.97) (Fig. 3A-i).

Bottom Line: Because a majority of the cytosine-phosphate-guanine (CpG) islands are found within the repeat elements of DNA, and are usually methylated under normoxic conditions, we suggested that retrotransposable Alu or short interspersed nuclear elements (SINEs) which show altered methylation and associated changes of gene expression during hypoxia, could be associated with genomic instability.U87MG glioblastoma cells were cultured in 0.1% O₂ for 6 weeks and compared with cells cultured in 21% O₂ for the same duration.Our results show that aberrant methylation leading to increased transcription of SINE and reverse transcriptase associated LINE elements could lead to increased genomic instability in hypoxia.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.

Show MeSH
Related in: MedlinePlus