Allele-specific demethylation at an imprinted mammalian promoter

A screen for imprinted genes on mouse Chromosome 7 recently identified Inpp5f_v2, a paternally expressed retrogene lying within an intron of Inpp5f. Here, we identify a novel paternally expressed variant of the Inpp5f gene (Inpp5f_v3) that shows a number of unusual features. Inpp5f_v3 initiates from a CpG-rich repeat region adjoining two B1 elements, despite previous reports that SINEs are generally excluded from imprinted promoters. Accordingly, we find that the Inpp5f_v3 promoter acquires methylation around the time of implantation, when many repeat families undergo de novo epigenetic silencing. Methylation is then lost specifically on the paternally derived allele during the latter stages of embryonic development, resulting in imprinted transcriptional activation on the demethylated allele. Methylation analyses in embryos lacking maternal methylation imprints suggest that the primary imprinting mark resides within an intronic CpG island ∼1 kb downstream of the Inpp5f_v3 transcriptional start site. These data support the hypothesis that SINEs can influence gene expression by attracting de novo methylation during development, a property likely to explain their exclusion from other imprinted promoters

Widely variable endogenous retroviral methylation levels in human placenta

It is generally assumed that transposable elements, including endogenous retroviruses (ERVs), are silenced by DNA methylation/chromatin structure in mammalian cells. However, there have been very few experimental studies to examine the methylation status of human ERVs. In this study, we determined and compared the methylation status of the 5′ long terminal repeats (LTRs) of different copies of the human endogenous retrovirus (HERV) family HERV-E, which are inserted in various genomic contexts. We found that three HERV-E LTRs which function as alternative gene promoters in placenta are unmethylated in that tissue but heavily methylated in blood cells, where these LTRs are not active promoters. This difference is not solely due to global hypomethylation in placenta, since two general measures of methylation levels of HERV-E and HERV-K LTRs suggest only 10–15% lower overall HERV methylation in placenta compared to blood. Comparisons between methylation levels of the LTR-derived gene promoters and six random HERV-E LTRs in placenta showed that the former display significantly lower methylation levels than random LTRs. Moreover, the differences in methylation between LTRs cannot always be explained by their genomic environment, since methylation of flanking sequences can be very different from methylation of the LTR itself

High-throughput sequence-based epigenomic analysis of Alu repeats in human cerebellum

DNA methylation, the only known covalent modification of mammalian DNA, occurs primarily in CpG dinucleotides. 51% of CpGs in the human genome reside within repeats, and 25% within Alu elements. Despite that, no method has been reported for large-scale ascertainment of CpG methylation in repeats. Here we describe a sequencing-based strategy for parallel determination of the CpG-methylation status of thousands of Alu repeats, and a computation algorithm to design primers that enable their specific amplification from bisulfite converted genomic DNA. Using a single primer pair, we generated amplicons of high sequence complexity, and derived CpG-methylation data from 31 178 Alu elements and their 5′ flanking sequences, altogether representing over 4 Mb of a human cerebellum epigenome. The analysis of the Alu methylome revealed that the methylation level of Alu elements is high in the intronic and intergenic regions, but low in the regions close to transcription start sites. Several hypomethylated Alu elements were identified and their hypomethylated status verified by pyrosequencing. Interestingly, some Alu elements exhibited a strikingly tissue-specific pattern of methylation. We anticipate the amplicons herein described to prove invaluable as epigenome representations, to monitor epigenomic alterations during normal development, in aging and in diseases such as cancer

Inactive X chromosome-specific reduction in placental DNA methylation

Genome-wide levels of DNA methylation vary between tissues, and compared with other tissues, the placenta has been reported to demonstrate a global decrease in methylation as well as decreased methylation of X-linked promoters. Methylation is one of many features that differentiate the active and inactive X, and it is well established that CpG island promoters on the inactive X are hypermethylated. We now report a detailed analysis of methylation at different regions across the X in male and female placenta and blood. A significant (P < 0.001) placental hypomethylation of LINE1 elements was observed in both males and females. Relative to blood placental promoter hypomethylation was only observed for X-linked, not autosomal promoters, and was significant for females (P < 0.0001) not males (P = 0.9266). In blood, X-linked CpG island promoters were shown to have moderate female methylation (66% across 70 assays) and low (23%) methylation in males. A similar methylation pattern in blood was observed for ∼20% of non-island promoters as well as 50% of the intergenic or intragenic CpG islands, the latter is likely due to the presence of unannotated promoters. Both intragenic and intergenic regions showed similarly high methylation levels in male and female blood (68 and 66%) while placental methylation of these regions was lower, particularly in females. Thus placental hypomethylation relative to blood is observed globally at repetitive elements as well as across the X. The decrease in X-linked placental methylation is consistently greater in females than males and implicates an inactive X specific loss of methylation in the placenta

Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells

Methylation of the cytosine is the most frequent epigenetic modification of DNA in mammalian cells. In humans, most of the methylated cytosines are found in CpG-rich sequences within tandem and interspersed repeats that make up to 45% of the human genome, being Alu repeats the most common family. Demethylation of Alu elements occurs in aging and cancer processes and has been associated with gene reactivation and genomic instability. By targeting the unmethylated SmaI site within the Alu sequence as a surrogate marker, we have quantified and identified unmethylated Alu elements on the genomic scale. Normal colon epithelial cells contain in average 25 486 ± 10 157 unmethylated Alu's per haploid genome, while in tumor cells this figure is 41 995 ± 17 187 (P = 0.004). There is an inverse relationship in Alu families with respect to their age and methylation status: the youngest elements exhibit the highest prevalence of the SmaI site (AluY: 42%; AluS: 18%, AluJ: 5%) but the lower rates of unmethylation (AluY: 1.65%; AluS: 3.1%, AluJ: 12%). Data are consistent with a stronger silencing pressure on the youngest repetitive elements, which are closer to genes. Further insights into the functional implications of atypical unmethylation states in Alu elements will surely contribute to decipher genomic organization and gene regulation in complex organisms

Exploring the functional role of the CHRM2 gene in human cognition: results from a dense genotyping and brain expression study

<p>Abstract</p> <p>Background</p> <p>The <it>CHRM2 </it>gene, located on the long arm of chromosome 7 (7q31-35), is involved in neuronal excitability, synaptic plasticity and feedback regulation of acetylcholine release, and has been implicated in higher cognitive processing. The aim of this study is the identification of functional (non)coding variants underlying cognitive phenotypic variation.</p> <p>Methods</p> <p>We previously reported an association between polymorphisms in the 5'UTR regions of the <it>CHRM2 </it>gene and intelligence.. However, no functional variants within this area have currently been identified. In order to identify the relevant functional variant(s), we conducted a denser coverage of SNPs, using two independent Dutch cohorts, consisting of a children's sample (N = 371 ss; mean age 12.4) and an adult sample (N= 391 ss; mean age 37.6). For all individuals standardized intelligence measures were available. Subsequently, we investigated genotype-dependent <it>CHRM2 </it>gene expression levels in the brain, to explore putative enhancer/inhibition activity exerted by variants within the muscarinic acetylcholinergic receptor.</p> <p>Results</p> <p>Using a test of within-family association two of the previously reported variants – rs2061174, and rs324650 – were again strongly associated with intelligence (<it>P </it>< 0.01). A new SNP (rs2350780) showed a trend towards significance. SNP rs324650, is located within a short interspersed repeat (SINE). Although the function of short interspersed repeats remains contentious, recent research revealed potential functionality of SINE repeats in a gene-regulatory context. Gene-expression levels in post-mortem brain material, however were not dependent on rs324650 genotype.</p> <p>Conclusion</p> <p>Using a denser coverage of SNPs in the <it>CHRM2 </it>gene, we confirmed the 5'UTR regions to be most interesting in the context of intelligence, and ruled out other regions of this gene. Although no correlation between genomic variants and gene expression was found, it would be interesting to examine allele-specific effects on CHRM2 transcripts expression in much more detail, for example in relation to transcripts specific halve-life and their relation to LTP and memory.</p

Genomic Imprinting in Mammals: Emerging Themes and Established Theories

The epigenetic events that occur during the development of the mammalian embryo are essential for correct gene expression and cell-lineage determination. Imprinted genes are expressed from only one parental allele due to differential epigenetic marks that are established during gametogenesis. Several theories have been proposed to explain the role that genomic imprinting has played over the course of mammalian evolution, but at present it is not clear if a single hypothesis can fully account for the diversity of roles that imprinted genes play. In this review, we discuss efforts to define the extent of imprinting in the mouse genome, and suggest that different imprinted loci may have been wrought by distinct evolutionary forces. We focus on a group of small imprinted domains, which consist of paternally expressed genes embedded within introns of multiexonic transcripts, to discuss the evolution of imprinting at these loci

Reduction of DNA damage by interaction s of dietary compounds with DNA.

Plant-derived dietary compounds and supplements are potential sources of chemicals that reduce cancer risk. The purpose of this study was to examine the effect of genistein, a soy-derived isoflavone, on DNA adduct formation. Cisplatin, a DNA damaging agent used in anti-cancer treatment, was selected as a model for assaying genotoxicity in vitro. Since DNA damage frequently occurs at hotspot sequences, we chose the human p53 gene (exon 7 - 9) as hotspot target DNA. Target and excess of carrier DNA were incubated with cisplatin in the presence of genistein or control chemicals overnight at 37°C in buffered saline solution. The extent of DNA damage was detected by a polymerase chain reaction (PCR) and agarose gel electrophoresis assay developed for this purpose. Reduction in the amount of a p53-specific PCR product was taken as evidence of DNA damage. Genistein was found to negatively affect cisplatin-induced DNA damage. A significant retention of band intensity was observed at 2 mM genistein. Quercetin, another plant-derived flavone had a similar effect at the same concentration. The intercalator ethidium bromide also restored band intensity, indicating intercalation could be a mechanism involved in DNA damage reduction by flavones and isoflavones

Reduction of DNA damage by interaction s of dietary compounds with DNA.

Plant-derived dietary compounds and supplements are potential sources of chemicals that reduce cancer risk. The purpose of this study was to examine the effect of genistein, a soy-derived isoflavone, on DNA adduct formation. Cisplatin, a DNA damaging agent used in anti-cancer treatment, was selected as a model for assaying genotoxicity in vitro. Since DNA damage frequently occurs at hotspot sequences, we chose the human p53 gene (exon 7 - 9) as hotspot target DNA. Target and excess of carrier DNA were incubated with cisplatin in the presence of genistein or control chemicals overnight at 37°C in buffered saline solution. The extent of DNA damage was detected by a polymerase chain reaction (PCR) and agarose gel electrophoresis assay developed for this purpose. Reduction in the amount of a p53-specific PCR product was taken as evidence of DNA damage. Genistein was found to negatively affect cisplatin-induced DNA damage. A significant retention of band intensity was observed at 2 mM genistein. Quercetin, another plant-derived flavone had a similar effect at the same concentration. The intercalator ethidium bromide also restored band intensity, indicating intercalation could be a mechanism involved in DNA damage reduction by flavones and isoflavones