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LINE-1 methylation patterns of different loci in normal and cancerous cells.

Phokaew C, Kowudtitham S, Subbalekha K, Shuangshoti S, Mutirangura A - Nucleic Acids Res. (2008)

Bottom Line: Therefore, the loss of genome-wide methylation in cancerous cells occurs as a generalized process.However, different LINE-1 loci showed different incidences of HNSCC hypomethylation, which is a lower methylation level than NOE.In conclusion, even though the global hypomethylation process that occurs in cancerous cells can generally deplete LINE-1 methylation levels, LINE-1 methylation can be influenced differentially depending on where the particular sequences are located in the genome.

View Article: PubMed Central - PubMed

Affiliation: Inter-Department Program of BioMedical Sciences, Faculty of Graduate School, Chulalongkorn University, Bangkok 10330, Thailand.

ABSTRACT
This study evaluated methylation patterns of long interspersed nuclear element-1 (LINE-1) sequences from 17 loci in several cell types, including squamous cell cancer cell lines, normal oral epithelium (NOE), white blood cells and head and neck squamous cell cancers (HNSCC). Although sequences of each LINE-1 are homologous, LINE-1 methylation levels at each locus are different. Moreover, some loci demonstrate the different methylation levels between normal tissue types. Interestingly, in some chromosomal regions, wider ranges of LINE-1 methylation levels were observed. In cancerous cells, the methylation levels of most LINE-1 loci demonstrated a positive correlation with each other and with the genome-wide levels. Therefore, the loss of genome-wide methylation in cancerous cells occurs as a generalized process. However, different LINE-1 loci showed different incidences of HNSCC hypomethylation, which is a lower methylation level than NOE. Additionally, we report a closer direct association between two LINE-1s in different EPHA3 introns. Finally, hypermethylation of some LINE-1s can be found sporadically in cancer. In conclusion, even though the global hypomethylation process that occurs in cancerous cells can generally deplete LINE-1 methylation levels, LINE-1 methylation can be influenced differentially depending on where the particular sequences are located in the genome.

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COBRA for unique L1 sequence (CU-L1) and COBRA for genome-wide LINE-1s (COBRALINE1). COBRA was performed as previously described (30), with some modifications (4). (A) Schematic representation of CU-L1 and COBRALINE-1, showing LINE-1 sequence in relation to the 5′ unique sequence. AACCG and CCGA are LINE-1 sequences, following bisulfite treatment and PCR, unmethylated AACCG sequences are converted to AATTG (TasI site) and methylated sequences are converted from CCGA to TCGA (TaqI site). The amplicon sizes of CU-L1 are between 300 and 500 bp and for COBRALINE-1 are 160 bp. After digestion, COBRALINE-1 yielded two TasI-digested unmethylated L1 sequence fragments of 62 and 98 bp and one 80 bp TaqI-digested methylated L1 sequence. The LINE-1 methylation level was calculated as a percentage (the intensity of methylated LINE-1 divided by the sum of the unmethylated LINE-1 and the TaqI-positive amplicons). CU-L1 usually has additional TaqI site(s) and AATT sequences; therefore, there are additional methylated, unmethylated and control bands. Met and unmet are methylated and unmethylated sequences, respectively. Control bands are digested DNA fragments without candidate CpG restriction sequences. (B) A typical example of results from COBRALINE-1 and CU-L1. The ranges of intensity between methylated and unmethylated bands of CU-L1 were wider than COBRALINE-1. The unmet, met and control arrows indicate unmethylated, methylated and control LINE1 sequences, respectively. M is a standard size marker. The –ve is dH2O. Several samples from WSU-HN cells are shown.
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Figure 1: COBRA for unique L1 sequence (CU-L1) and COBRA for genome-wide LINE-1s (COBRALINE1). COBRA was performed as previously described (30), with some modifications (4). (A) Schematic representation of CU-L1 and COBRALINE-1, showing LINE-1 sequence in relation to the 5′ unique sequence. AACCG and CCGA are LINE-1 sequences, following bisulfite treatment and PCR, unmethylated AACCG sequences are converted to AATTG (TasI site) and methylated sequences are converted from CCGA to TCGA (TaqI site). The amplicon sizes of CU-L1 are between 300 and 500 bp and for COBRALINE-1 are 160 bp. After digestion, COBRALINE-1 yielded two TasI-digested unmethylated L1 sequence fragments of 62 and 98 bp and one 80 bp TaqI-digested methylated L1 sequence. The LINE-1 methylation level was calculated as a percentage (the intensity of methylated LINE-1 divided by the sum of the unmethylated LINE-1 and the TaqI-positive amplicons). CU-L1 usually has additional TaqI site(s) and AATT sequences; therefore, there are additional methylated, unmethylated and control bands. Met and unmet are methylated and unmethylated sequences, respectively. Control bands are digested DNA fragments without candidate CpG restriction sequences. (B) A typical example of results from COBRALINE-1 and CU-L1. The ranges of intensity between methylated and unmethylated bands of CU-L1 were wider than COBRALINE-1. The unmet, met and control arrows indicate unmethylated, methylated and control LINE1 sequences, respectively. M is a standard size marker. The –ve is dH2O. Several samples from WSU-HN cells are shown.

Mentions: Previously, we investigated the methylation of genome-wide LINE-1s by investigating the methylation status of two CpG dinucleotides at the 5′-end of LINE-1s (4). This study compares the methylation status of LINE-1s across different loci. Because there are variations in both lengths and sequences of LINE-1s, we compared methylation levels of LINE-1s between locations by comparing methylation levels of the same representative CpG dinucleotides: TaqI positive 80-bp methylated DNA fragments and TasI positive 98-bp unmethylated DNA fragments (Figure 1A). We selected a set of 17 full-length LINE-1s located within introns. All LINE-1s possessed both representative CpG dinucleotides. Selected LINE-1s (Supplementary Table S1), a schematic representation of the assay (Figure 1A) and typical examples of PCR results (Figure 1B) are shown. We modified the COBRA protocol (30) to devise a PCR protocol to evaluate LINE-1 methylation status in the entire genome (COBRALINE-1) and at a specific locus (CU-L1) by replacing the LINE-1 forward primer with an oligonucleotide locating on unique sequence 5′ to LINE-1. Briefly, DNA is treated with bisulfite to distinguish between methylated and unmethylated sequences before PCR (32). Treatment with bisulfite converts unmethylated cytosines, but not methylated cytosines, to uracils and then thymines after PCR. This generates detectable methylation-dependent changes in the restriction pattern of PCR-amplified LINE-1 sequences (Figure 1A). The modified DNA was amplified by 5′-UTR LINE-1 bisulfited sequence primers and then digested with TaqI and TasI restriction enzymes, which recognize methylated and unmethylated sequences, respectively. The level of LINE-1 methylation in each sample was calculated by dividing the measured intensity of TaqI digestible amplicons by the sum of the TasI and TaqI products (4) (Figure 1A).Figure 1.


LINE-1 methylation patterns of different loci in normal and cancerous cells.

Phokaew C, Kowudtitham S, Subbalekha K, Shuangshoti S, Mutirangura A - Nucleic Acids Res. (2008)

COBRA for unique L1 sequence (CU-L1) and COBRA for genome-wide LINE-1s (COBRALINE1). COBRA was performed as previously described (30), with some modifications (4). (A) Schematic representation of CU-L1 and COBRALINE-1, showing LINE-1 sequence in relation to the 5′ unique sequence. AACCG and CCGA are LINE-1 sequences, following bisulfite treatment and PCR, unmethylated AACCG sequences are converted to AATTG (TasI site) and methylated sequences are converted from CCGA to TCGA (TaqI site). The amplicon sizes of CU-L1 are between 300 and 500 bp and for COBRALINE-1 are 160 bp. After digestion, COBRALINE-1 yielded two TasI-digested unmethylated L1 sequence fragments of 62 and 98 bp and one 80 bp TaqI-digested methylated L1 sequence. The LINE-1 methylation level was calculated as a percentage (the intensity of methylated LINE-1 divided by the sum of the unmethylated LINE-1 and the TaqI-positive amplicons). CU-L1 usually has additional TaqI site(s) and AATT sequences; therefore, there are additional methylated, unmethylated and control bands. Met and unmet are methylated and unmethylated sequences, respectively. Control bands are digested DNA fragments without candidate CpG restriction sequences. (B) A typical example of results from COBRALINE-1 and CU-L1. The ranges of intensity between methylated and unmethylated bands of CU-L1 were wider than COBRALINE-1. The unmet, met and control arrows indicate unmethylated, methylated and control LINE1 sequences, respectively. M is a standard size marker. The –ve is dH2O. Several samples from WSU-HN cells are shown.
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Figure 1: COBRA for unique L1 sequence (CU-L1) and COBRA for genome-wide LINE-1s (COBRALINE1). COBRA was performed as previously described (30), with some modifications (4). (A) Schematic representation of CU-L1 and COBRALINE-1, showing LINE-1 sequence in relation to the 5′ unique sequence. AACCG and CCGA are LINE-1 sequences, following bisulfite treatment and PCR, unmethylated AACCG sequences are converted to AATTG (TasI site) and methylated sequences are converted from CCGA to TCGA (TaqI site). The amplicon sizes of CU-L1 are between 300 and 500 bp and for COBRALINE-1 are 160 bp. After digestion, COBRALINE-1 yielded two TasI-digested unmethylated L1 sequence fragments of 62 and 98 bp and one 80 bp TaqI-digested methylated L1 sequence. The LINE-1 methylation level was calculated as a percentage (the intensity of methylated LINE-1 divided by the sum of the unmethylated LINE-1 and the TaqI-positive amplicons). CU-L1 usually has additional TaqI site(s) and AATT sequences; therefore, there are additional methylated, unmethylated and control bands. Met and unmet are methylated and unmethylated sequences, respectively. Control bands are digested DNA fragments without candidate CpG restriction sequences. (B) A typical example of results from COBRALINE-1 and CU-L1. The ranges of intensity between methylated and unmethylated bands of CU-L1 were wider than COBRALINE-1. The unmet, met and control arrows indicate unmethylated, methylated and control LINE1 sequences, respectively. M is a standard size marker. The –ve is dH2O. Several samples from WSU-HN cells are shown.
Mentions: Previously, we investigated the methylation of genome-wide LINE-1s by investigating the methylation status of two CpG dinucleotides at the 5′-end of LINE-1s (4). This study compares the methylation status of LINE-1s across different loci. Because there are variations in both lengths and sequences of LINE-1s, we compared methylation levels of LINE-1s between locations by comparing methylation levels of the same representative CpG dinucleotides: TaqI positive 80-bp methylated DNA fragments and TasI positive 98-bp unmethylated DNA fragments (Figure 1A). We selected a set of 17 full-length LINE-1s located within introns. All LINE-1s possessed both representative CpG dinucleotides. Selected LINE-1s (Supplementary Table S1), a schematic representation of the assay (Figure 1A) and typical examples of PCR results (Figure 1B) are shown. We modified the COBRA protocol (30) to devise a PCR protocol to evaluate LINE-1 methylation status in the entire genome (COBRALINE-1) and at a specific locus (CU-L1) by replacing the LINE-1 forward primer with an oligonucleotide locating on unique sequence 5′ to LINE-1. Briefly, DNA is treated with bisulfite to distinguish between methylated and unmethylated sequences before PCR (32). Treatment with bisulfite converts unmethylated cytosines, but not methylated cytosines, to uracils and then thymines after PCR. This generates detectable methylation-dependent changes in the restriction pattern of PCR-amplified LINE-1 sequences (Figure 1A). The modified DNA was amplified by 5′-UTR LINE-1 bisulfited sequence primers and then digested with TaqI and TasI restriction enzymes, which recognize methylated and unmethylated sequences, respectively. The level of LINE-1 methylation in each sample was calculated by dividing the measured intensity of TaqI digestible amplicons by the sum of the TasI and TaqI products (4) (Figure 1A).Figure 1.

Bottom Line: Therefore, the loss of genome-wide methylation in cancerous cells occurs as a generalized process.However, different LINE-1 loci showed different incidences of HNSCC hypomethylation, which is a lower methylation level than NOE.In conclusion, even though the global hypomethylation process that occurs in cancerous cells can generally deplete LINE-1 methylation levels, LINE-1 methylation can be influenced differentially depending on where the particular sequences are located in the genome.

View Article: PubMed Central - PubMed

Affiliation: Inter-Department Program of BioMedical Sciences, Faculty of Graduate School, Chulalongkorn University, Bangkok 10330, Thailand.

ABSTRACT
This study evaluated methylation patterns of long interspersed nuclear element-1 (LINE-1) sequences from 17 loci in several cell types, including squamous cell cancer cell lines, normal oral epithelium (NOE), white blood cells and head and neck squamous cell cancers (HNSCC). Although sequences of each LINE-1 are homologous, LINE-1 methylation levels at each locus are different. Moreover, some loci demonstrate the different methylation levels between normal tissue types. Interestingly, in some chromosomal regions, wider ranges of LINE-1 methylation levels were observed. In cancerous cells, the methylation levels of most LINE-1 loci demonstrated a positive correlation with each other and with the genome-wide levels. Therefore, the loss of genome-wide methylation in cancerous cells occurs as a generalized process. However, different LINE-1 loci showed different incidences of HNSCC hypomethylation, which is a lower methylation level than NOE. Additionally, we report a closer direct association between two LINE-1s in different EPHA3 introns. Finally, hypermethylation of some LINE-1s can be found sporadically in cancer. In conclusion, even though the global hypomethylation process that occurs in cancerous cells can generally deplete LINE-1 methylation levels, LINE-1 methylation can be influenced differentially depending on where the particular sequences are located in the genome.

Show MeSH
Related in: MedlinePlus