Determining the global DNA methylation status of rat according to the identifier repetitive elements

Abstract

A large portion of the genome represents repetitive elements. Identifier (ID) elements, the major elements of short interspersed repetitive elements, are widespread with about 150 000 copies in the rat genome. Each ID element contains six CpG dinucleotides, which might account for the global methylation status of rat. We validated the CpG methylation of the ID elements by various methods. The methylation of one CpG site (CpG-3) of the ID element was investigated by performing pyrosequencing. The methylation percentage of the CpG-3 site was 53.6% (SD = 2.2) on average from six rat tissues with blood, but 24.6% (SD = 1.0) in rat pheochromocytoma, PC-12, cell line. This CpG-3 methylation was further verified by whole genome amplification (WGA), 5-azacytidine treatment, and proportional mixing of rat WGA genomic DNA (gDNA) with liver gDNA. Methylation-sensitive restriction enzyme PCR method showed that three other CpG sites (CpG-1, CpG-4, and CpG-5) within the ID element were also methylated (about 60%) in rat gDNA, but not in WGA gDNA. The ID elements may be good candidates for routine analysis of the global DNA methylation changes of rat for pharmaceutical treatment and their use can make basic epigenetic research possible with high accuracy.

Low Doses of the Carcinogen Furan Alter Cell Cycle and Apoptosis Gene Expression in Rat Liver Independent of DNA Methylation

“…LINE-1 showed little enrichment, which is consistent with a previous report that LINE-1 was hypomethylated in rat liver (Asada et al 2006). Although the methylation level of one CpG of ID elements has been reported at > 60% (Kim et al 2007), considering the high mutation level and the possible low methylation status of other CpG sites, it is not surprising that few ID elements were enriched in immunoprecipitated DNA.…”

Effect of folate deficiency on placental DNA methylation in hyperhomocysteinemic rats

“…Genomic DNA (gDNA) was isolated from the rat placenta using the PUREGENE Cell and Tissue kit (Gentra, Minneapolis, MN, USA) as we described previously [19]. The bisulfite modification of gDNA was performed using the CpGenome DNA modification kit (CHEMICON, Temecula, CA, USA), after which the ID element was amplified by polymerase chain reaction in a 25-μl volume containing AmpliTaq Gold with 30 cycles of 94°C for 1 min, 37°C for 1 min and 72°C for 1 min using the forward primer 5′-GGGTTGGGGATTTAG-3′ at 0.1 μΜ and the biotinylated reverse primer 5′-AACCCAAAACCTTA-3′.…”

Neurotoxic Doses of Chronic Methamphetamine Trigger Retrotransposition of the Identifier Element in Rat Dorsal Dentate Gyrus

“…SINEs and LINEs are frequently methylated in mouse, rat and human tissues [44,45,46]. The methylation percentage of CpG sites within the ID element in gDNA from six tissues (liver, lung, kidney, spleen, ovary, testis) from two-month-old male and female Sprague-Dawley rats was found to be about 54% at the CpG-1 and about 60% at the CpG-2 [16]. We found a similar methylation percentage at the CpG-1 and about 30% at the CpG-2 in several brain areas in two-month-old male Sprague-Dawley control rats.…”

“…ID elements are about 100 bp long and consists of a core domain containing an internal RNA polymerase III promoter, a poly(A) region and 5′- and 3′-flanking regions [14,15]. Due to their widespread presence in the rat genome, ID elements have been used as determinants of global methylation in rat tissues [16]. Human and mouse counterparts of the rat ID element were demonstrated to be activated by hypomethylation [17]; therefore, ID element transcription is most likely activated by removal of methyl groups within its sequence.…”