PwRn1, a novel Ty3/gypsy-like retrotransposon of Paragonimus westermani: molecular characters and its differentially preserved mobile potential according to host chromosomal polyploidy

<p>Abstract</p> <p>Background</p> <p>Retrotransposons have been known to involve in the remodeling and evolution of host genome. These reverse transcribing elements, which show a complex evolutionary pathway with diverse intermediate forms, have been comprehensively analyzed from a wide range of host genomes, while the information remains limited to only a few species in the phylum Platyhelminthes.</p> <p>Results</p> <p>A LTR retrotransposon and its homologs with a strong phylogenetic affinity toward <it>CsRn1 </it>of <it>Clonorchis sinensis </it>were isolated from a trematode parasite <it>Paragonimus westermani </it>via a degenerate PCR method and from an insect species <it>Anopheles gambiae </it>by <it>in silico </it>analysis of the whole mosquito genome, respectively. These elements, designated <it>PwRn1 </it>and <it>AgCR-1 </it>– <it>AgCR-14 </it>conserved unique features including a t-RNA^Trpprimer binding site and the unusual CHCC signature of Gag proteins. Their flanking LTRs displayed >97% nucleotide identities and thus, these elements were likely to have expanded recently in the trematode and insect genomes. They evolved heterogeneous expression strategies: a single fused ORF, two separate ORFs with an identical reading frame and two ORFs overlapped by -1 frameshifting. Phylogenetic analyses suggested that the elements with the separate ORFs had evolved from an ancestral form(s) with the overlapped ORFs. The mobile potential of <it>PwRn1 </it>was likely to be maintained differentially in association with the karyotype of host genomes, as was examined by the presence/absence of intergenomic polymorphism and mRNA transcripts.</p> <p>Conclusion</p> <p>Our results on the structural diversity of <it>CsRn1</it>-like elements can provide a molecular tool to dissect a more detailed evolutionary episode of LTR retrotransposons. The <it>PwRn1</it>-associated genomic polymorphism, which is substantial in diploids, will also be informative in addressing genomic diversification following inter-/intra-specific hybridization in <it>P. westermani </it>populations.</p

The 5'-end transitional CpGs between the CpG islands and retroelements are hypomethylated in association with loss of heterozygosity in gastric cancers

BACKGROUND: A loss of heterozygosity (LOH) represents a unilateral chromosomal loss that reduces the dose of highly repetitive Alu, L1, and LTR retroelements. The aim of this study was to determine if the LOH events can affect the spread of retroelement methylation in the 5'-end transitional area between the CpG islands and their nearest retroelements. METHODS: The 5'-transitional area of all human genes (22,297) was measured according to the nearest retroelements to the transcription start sites. For 50 gastric cancer specimens, the level of LOH events on eight cancer-associated chromosomes was estimated using the microsatellite markers, and the 5'-transitional CpGs of 20 selected genes were examined by methylation analysis using the bisulfite-modified DNA. RESULTS: The extent of the transitional area was significantly shorter with the nearest Alu elements than with the nearest L1 and LTR elements, as well as in the extragenic regions containing a higher density of retroelements than in the intragenic regions. The CpG islands neighbouring a high density of Alu elements were consistently hypomethylated in both normal and tumor tissues. The 5'-transitional methylated CpG sites bordered by a low density of Alu elements or the L1 and LTR elements were hypomethylated more frequently in the high-level LOH cases than in the low-level LOH cases. CONCLUSION: The 5'-transitional methylated CpG sites not completely protected by the Alu elements were hypomethylated in association with LOH events in gastric cancers. This suggests that an irreversible unbalanced decrease in the genomic dose reduces the spread of L1 methylation in the 5'-end regions of genes

Chromosomal Losses are Associated with Hypomethylation of the Gene-Control Regions in the Stomach with a Low Number of Active Genes

Transitional-CpG methylation between unmethylated promoters and nearby methylated retroelements plays a role in the establishment of tissue-specific transcription. This study examined whether chromosomal losses reducing the active genes in cancers can change transitional-CpG methylation and the transcription activity in a cancer-type-dependent manner. The transitional-CpG sites at the CpG-island margins of nine genes and the non-island-CpG sites round the transcription start sites of six genes lacking CpG islands were examined by methylation-specific polymerase chain reaction (PCR) analysis. The number of active genes in normal and cancerous tissues of the stomach, colon, breast, and nasopharynx were analyzed using the public data in silico. The CpG-island margins and non-island CpG sites tended to be hypermethylated and hypomethylated in all cancer types, respectively. The CpG-island margins were hypermethylated and a low number of genes were active in the normal stomach compared with other normal tissues. In gastric cancers, the CpG-island margins and non-island-CpG sites were hypomethylated in association with high-level chromosomal losses, and the number of active genes increased. Colon, breast, and nasopharyngeal cancers showed no significant association between the chromosomal losses and methylation changes. These findings suggest that chromosomal losses in gastric cancers are associated with the hypomethylation of the gene-control regions and the increased number of active genes

Relationship Between the Extent of Chromosomal Losses and the Pattern of CpG Methylation in Gastric Carcinomas

The extent of unilateral chromosomal losses and the presence of microsatellite instability (MSI) have been classified into high-risk (high- and baseline-level loss) and low-risk (low-level loss and MSI) stem-line genotypes in gastric carcinomas. A unilateral genome-dosage reduction might stimulate compensation mechanism, which maintains the genomic dosage via CpG hypomethylation. A total of 120 tumor sites from 40 gastric carcinomas were examined by chromosomal loss analysis using 40 microsatellite markers on 8 chromosomes and methylation analysis in the 13 CpG (island/non-island) regions near the 10 genes using the bisulfite-modified DNAs. The high-level-loss tumor (four or more losses) showed a tendency toward unmethylation in the Maspin, CAGE, MAGE-A2 and RABGEF1 genes, and the other microsatellite-genotype (three or fewer losses and MSI) toward methylation in the p16, hMLH1, RASSF1A, and Cyclin D2 genes (p<0.05). The non-island CpGs of the p16 and hMLH1 genes were hypomethylated in the high-level-loss and hypermethylated in the non-high-level-loss sites (p<0.05). Consequently, hypomethylation changes were related to a high-level loss, whereas the hypermethylation changes were accompanied by a baseline-level loss, a low-level loss, or a MSI. This indicates that hypomethylation compensates the chromosomal losses in the process of tumor progression

DNA Methylation and Expression Patterns of Key Tissue-specific Genes in Adult Stem Cells and Stomach Tissues

CpG-island margins and non-island-CpG sites round the transcription start sites of CpG-island-positive and -negative genes are methylated to various degrees in a tissue-specific manner. These methylation-variable CpG sites were analyzed to delineate a relationship between the methylation and transcription of the tissue-specific genes. The level of tissue-specific transcription was estimated by counting the number of the total transcripts in the SAGE (serial analysis of gene expression) database. The methylation status of 12 CpG-island margins and 21 non-island CpG sites near the key tissue-specific genes was examined in pluripotent stromal cells obtained from fat and bone marrow samples as well as in lineage-committed cells from marrow bulk, stomach, colon, breast, and thyroid samples. Of the 33 CpG sites examined, 10 non-island-CpG sites, but none of the CpG-island margins were undermethylated concurrent with tissue-specific expression of their nearby genes. The net methylation of the 33 CpG sites and the net amount of non-island-CpG gene transcripts were high in stomach tissues and low in stromal cells. The present findings suggest that the methylation of the non-island-CpG sites is inversely associated with the expression of the nearby genes, and the concert effect of transitional-CpG methylation is linearly associated with the stomach-specific genes lacking CpG-islands

DNA Methylation Patterns of Ulcer-Healing Genes Associated with the Normal Gastric Mucosa of Gastric Cancers

Recent evidence suggests that gastric mucosal injury induces adaptive changes in DNA methylation. In this study, the methylation status of the key tissue-specific genes in normal gastric mucosa of healthy individuals and cancer patients was evaluated. The methylation-variable sites of 14 genes, including ulcer-healing genes (TFF1, TFF2, CDH1, and PPARG), were chosen from the CpG-island margins or non-island CpGs near the transcription start sites. The healthy individuals as well as the normal gastric mucosa of 23 ulcer, 21 non-invasive cancer, and 53 cancer patients were examined by semiquantitative methylation-specific polymerase chain reaction (PCR) analysis. The ulcer-healing genes were concurrently methylated with other genes depending on the presence or absence of CpG-islands in the normal mucosa of healthy individuals. Both the TFF2 and PPARG genes were frequently undermethylated in ulcer patients. The over- or intermediate-methylated TFF2 and undermethylated PPARG genes was more common in stage-1 cancer patients (71%) than in healthy individuals (10%; odds ratio [OR], 21.9) and non-invasive cancer patients (21%; OR, 8.9). The TFF2-PPARG methylation pattern of cancer patients was stronger in the older-age group (≥55 yr; OR, 43.6). These results suggest that the combined methylation pattern of ulcer-healing genes serves as a sensitive marker for predicting cancer-prone gastric mucosa

The gene-reduction effect of chromosomal losses detected in gastric cancers

<p>Abstract</p> <p>Background</p> <p>The level of loss of heterozygosity (LOH) that reduces a gene dose and exerts a cell-adverse effect is known to be a parameter for the genetic staging of gastric cancers. This study investigated if the cell-adverse effect induced with the gene reduction was a rate-limiting factor for the LOH events in two distinct histologic types of gastric cancers, the diffuse- and intestinal-types.</p> <p>Methods</p> <p>The pathologic specimens obtained from 145 gastric cancer patients were examined for the level of LOH using 40 microsatellite markers on eight cancer-associated chromosomes (3p, 4p, 5q, 8p, 9p, 13q, 17p and 18q).</p> <p>Results</p> <p>Most of the cancer-associated chromosomes were found to belong to the gene-poor chromosomes and to contain a few stomach-specific genes that were highly expressed. A baseline-level LOH involving one or no chromosome was frequent in diffuse-type gastric cancers. The chromosome 17 containing a relatively high density of genes was commonly lost in intestinal-type cancers but not in diffuse-type cancers. A high-level LOH involving four or more chromosomes tended to be frequent in the gastric cancers with intestinal and mixed differentiation. Disease relapse was common for gastric cancers with high-level LOH through both the hematogenous (38%) and non-hematogenous (36%) routes, and for the baseline-level LOH cases through the non-hematogenous route (67%).</p> <p>Conclusions</p> <p>The cell-adverse effect of gene reduction is more tolerated in intestinal-type gastric cancers than in diffuse-type cancers, and the loss of high-dose genes is associated with hematogenous metastasis.</p

The overmethylated genes in Helicobacter pylori-infected gastric mucosa are demethylated in gastric cancers

<p>Abstract</p> <p>Background</p> <p>The transitional-CpG sites between weakly methylated genes and densely methylated retroelements are overmethylated in the gastric mucosa infected with <it>Helicobacter pylori </it>(<it>H. pylori</it>) and they are undermethylated in the gastric cancers depending on the level of loss of heterozygosity (LOH) events. This study delineated the transitional-CpG methylation patterns of CpG-island-containing and -lacking genes in view of the retroelements.</p> <p>Methods</p> <p>The transitional-CpG sites of eight CpG-island-containing genes and six CpG-island-lacking genes were semi-quantitatively examined by performing radioisotope-labelling methylation-specific PCR under stringent conditions. The level of LOH in the gastric cancers was estimated using the 40 microsatellite markers on eight cancer-associated chromosomes. Each gene was scored as overmethylated or undermethylated based on an intermediate level of transitional-CpG methylation common in the <it>H. pylori</it>-negative gastric mucosa.</p> <p>Results</p> <p>The eight CpG-island genes examined were overmethylated depending on the proximity to the nearest retroelement in the <it>H. pylori</it>-positive gastric mucosa. The six CpG-island-lacking genes were similarly methylated in the <it>H. pylori</it>-positive and -negative gastric mucosa. In the gastric cancers, long transitional-CpG segments of the CpG-island genes distant from the retroelements remained overmethylated, whereas the overmethylation of short transitional-CpG segments close to the retroelements was not significant. Both the CpG-island-containing and -lacking genes tended to be decreasingly methylated in a LOH-level-dependent manner.</p> <p>Conclusions</p> <p>The overmethylated genes under the influence of retroelement methylation in the <it>H. pylori</it>-infected stomach are demethylated in the gastric cancers influenced by LOH.</p