Low-dose Cd induces hepatic gene hypermethylation, along with the persistent reduction of cell death and increase of cell proliferation in rats and mice

BACKGROUND: Cadmium (Cd) is classified as a human carcinogen probably associated with epigenetic changes. DNA methylation is one of epigenetic mechanisms by which cells control gene expression. Therefore, the present study genome-widely screened the methylation-altered genes in the liver of rats previously exposed to low-dose Cd. METHODOLOGY PRINCIPAL FINDINGS: Rats were exposed to Cd at 20 nmol/kg every other day for 4 weeks and gene methylation was analyzed at the 48(th) week with methylated DNA immunoprecipitation-CpG island microarray. Among the 1629 altered genes, there were 675 genes whose promoter CpG islands (CGIs) were hypermethylated, 899 genes whose promoter CGIs were hypomethylated, and 55 genes whose promoter CGIs were mixed with hyper- and hypo-methylation. Caspase-8 gene promoter CGIs and TNF gene promoter CGIs were hypermethylated and hypomethylated, respectively, along with a low apoptosis rate in Cd-treated rat livers. To link the aberrant methylation of caspase-8 and TNF genes to the low apoptosis induced by low-dose Cd, mice were given chronic exposure to low-dose Cd with and without methylation inhibitor (5-aza-2'-deoxyctidene, 5-aza). At the 48(th) week after Cd exposure, livers from Cd-treated mice displayed the increased caspase-8 CGI methylation and decreased caspase-8 protein expression, along with significant increases in cell proliferation and overexpression of TGF-β1 and cytokeratin 8/18 (the latter is a new marker of mouse liver preneoplastic lesions), all which were prevented by 5-aza treatment. CONCLUSION/SIGNIFICANCE: These results suggest that Cd-induced global gene hypermethylation, most likely caspase-8 gene promoter hypermethylation that down-regulated its expression, leading to the decreased hepatic apoptosis and increased preneoplastic lesions.     

Fetal exposure to cocaine causes programming of Prkce gene repression in the left ventricle of adult rat offspring

Previous studies demonstrated that maternal cocaine administration caused a significant decrease in protein kinase C epsilon (PRKCE) abundance in the left ventricle and an increase in susceptibility of the heart to ischemic injury in adult male offspring. The present study tested the hypothesis that epigenetic modification has a key role in cocaine-mediated programming of cardiac Prkce gene repression. Pregnant Sprague-Dawley rats were administered saline or cocaine (30 mg/kg/day i.p.) from Days 15 to 21 of gestational age, and hearts of 3-mo-old adult offspring were studied. Cocaine exposure significantly decreased Prkce mRNA levels in the left ventricle of male but not female offspring. CpG dinucleotides identified in Bhlhb2, Pparg, E2f, and Egr1 binding sites at the Prkce gene promoter were densely methylated in males and females and were unaffected by cocaine exposure. In contrast, methylation of CpGs in the two Sp1 binding sites (-346 and -268) was low and was significantly increased by cocaine exposure in male offspring. In females, methylation of the Sp1 binding site at -268 but not -346 was increased. Reporter gene assays showed that both Sp1 binding sites had a strong stimulatory role in Prkce gene activity. Methylation of the Sp1 binding sites significantly decreased SP1 binding to the Prkce promoter. Cocaine exposure did not affect nuclear SP1 protein levels but decreased the SP1 binding affinity to its binding site at -268. The results demonstrate an epigenetic mechanism of DNA methylation in programming of cardiac Prkce gene repression, linking fetal cocaine exposure and pathophysiological consequences in the heart of adult male offspring in a gender-dependent manner.     

Exposure to diethylhexyl phthalate (DEHP) results in a heritable modification of imprint genes DNA methylation in mouse oocytes

Diethylhexyl phthalate (DEHP) is an estrogen-like compound widely used as a plasticizer in commercial products and is present in medical devices, and common household items. It is considered an endocrine disruptor since studies on experimental animals clearly show that exposure to DEHP can alter epigenetics of germ cells. This study was designed to assess the effects of DEHP on DNA methylation of imprinting genes in germ cells from fetal and adult mouse. Pregnant mice were treated with DEHP at doses of 0 and 40 μg DEHP/kg body weight/day from 0.5 to 18.5 day post coitum. The data revealed DEHP exposure significantly reduced the percentage of methylated CpG sites in Igf2r and Peg3 differentially methylated regions (DMRs) in primordial germ cells from female and male fetal mouse, particularly, in the oocytes of 21 dpp mice (F1), which were produced by the pregnant micetreated with DEHP. More surprisingly, the modification of the DNA methylation of imprinted genes in F1 mouse oocytes was heritable to F2 offspring which exhibit lower percentages of methylated CpG sites in imprinted genes DMRs. In conclusion, DEHP exposure can affect the DNA methylation of imprinting genes not only in fetal mouse germ cells and growing oocytes, but also in offspring’s oocytes.     

Identifying differentially methylated genes using mixed effect and generalized least square models

Background  

DNA methylation plays an important role in the process of tumorigenesis. Identifying differentially methylated genes or CpG islands (CGIs) associated with genes between two tumor subtypes is thus an important biological question. The methylation status of all CGIs in the whole genome can be assayed with differential methylation hybridization (DMH) microarrays. However, patient samples or cell lines are heterogeneous, so their methylation pattern may be very different. In addition, neighboring probes at each CGI are correlated. How these factors affect the analysis of DMH data is unknown.     

Modeling genomic data with type attributes, balancing stability and maintainability

Background

The computational prediction of DNA methylation has become an important topic in the recent years due to its role in the epigenetic control of normal and cancer-related processes. While previous prediction approaches focused merely on differences between methylated and unmethylated DNA sequences, recent experimental results have shown the presence of much more complex patterns of methylation across tissues and time in the human genome. These patterns are only partially described by a binary model of DNA methylation. In this work we propose a novel approach, based on profile analysis of tissue-specific methylation that uncovers significant differences in the sequences of CpG islands (CGIs) that predispose them to a tissue- specific methylation pattern.

Results

We defined CGI methylation profiles that separate not only between constitutively methylated and unmethylated CGIs, but also identify CGIs showing a differential degree of methylation across tissues and cell-types or a lack of methylation exclusively in sperm. These profiles are clearly distinguished by a number of CGI attributes including their evolutionary conservation, their significance, as well as the evolutionary evidence of prior methylation. Additionally, we assess profile functionality with respect to the different compartments of protein coding genes and their possible use in the prediction of DNA methylation.

Conclusion

Our approach provides new insights into the biological features that determine if a CGI has a functional role in the epigenetic control of gene expression and the features associated with CGI methylation susceptibility. Moreover, we show that the ability to predict CGI methylation is based primarily on the quality of the biological information used and the relationships uncovered between different sources of knowledge. The strategy presented here is able to predict, besides the constitutively methylated and unmethylated classes, two more tissue specific methylation classes conserving the accuracy provided by leading binary methylation classification methods.     

Methylation changes of H19 gene in sperms of X-irradiated mouse and maintenance in offspring

The nature of imprinting is just differential methylation of imprinted genes. Unlike the non-imprinted genes, the methylation pattern of imprinted genes established during the period of gametogenesis remains unchangeable after fertilization and during embryo development. It implies that gametogenesis is the key stage for methylation pattern of imprinted genes. The imprinting interfered by exogenous factors during this stage could be inherited to offspring and cause genetic effect. Now many studies have proved that ionizing irradiation could disturb DNA methylation. Here we choose BALB/c mice as a research model and X-ray as interfering source to further clarify it. We discovered that the whole-body irradiation of X-ray to male BALB/c mice could influence the methylation pattern of H19 gene in sperms, which resulted in some cytosines of partial CpG islands in the imprinting control region could not transform to methylated cytosines. Furthermore, by copulating the interfered male mice with normal female, we analyzed the promoter methylation pattern of H19 in offspring fetal liver and compared the same to the pattern of male parent in sperms. We found that the majority of methylation changes in offspring liver were related to the ones in their parent sperms. Our data proved that the changes of the H19 gene methylation pattern interfered by X-ray irradiation could be transmitted and maintained in the first-generation offspring.     

Methylation status of putative differentially methylated regions of porcine IGF2 and H19

This study was designed to identify the putative differentially methylated regions (DMRs) of the porcine imprinted genes insulin-like growth factor 2 and H19 (IGF2-H19), and to assess the genomic imprinting status of IGF2-H19 by identifying the methylation patterns of these regions in germ cells, and in tissues from porcine fetuses, an adult pig, as well as cloned offspring produced by somatic cell nuclear transfer (SCNT). Porcine IGF2-H19 DMRs exhibit a normal monoallelic methylation pattern (i.e., either the paternally- or the maternally derived allele is methylated) similar to the pattern observed for the same genes in the human and mice genomes. Examination of the methylation patterns of the IGF2-H19 DMRs revealed that the zinc finger protein binding sites CTCF1 and 2 did not exhibit differential methylation in both control and cloned offspring. In contrast, the CTCF3 and DMR2 loci of the IGF2 gene showed abnormal methylation in cloned offspring, but a normal differential or moderate methylation pattern in tissues from control offspring and an adult pig. Our data thus suggest that regulation of genomic imprinting at the porcine IGF2-H19 loci is conserved among species, and that the abnormal methylation pattern in the regulatory elements of imprinted genes may lead to an alteration in the coordinated expression of genes required for successful reprogramming, which, in consequence, may contribute to the low efficiency of porcine genome reprogramming induced by nuclear transfer.     

A primary role of TET proteins in establishment and maintenance of De Novo bivalency at CpG islands

Ten Eleven Translocation (TET) protein-catalyzed 5mC oxidation not only creates novel DNA modifications, such as 5hmC, but also initiates active or passive DNA demethylation. TETs’ role in the crosstalk with specific histone modifications, however, is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domains underlying methylated DNA CpG islands (CGIs). Overexpression of wild type (WT), but not catalytic inactive mutant (Mut), TET2 in low-TET-expressing cells results in an increase in the level of 5hmC with accompanying DNA demethylation at a subset of CGIs. Most importantly, this alteration is sufficient in making de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of essential developmental gene promoters, which are located within CGIs and are previously silenced due to DNA methylation. On the other hand, deletion of Tet1 and Tet2 in mouse embryonic stem (ES) cells results in an apparent loss of H3K27me3 at bivalent domains, which are associated with a particular set of key developmental gene promoters. Collectively, this study demonstrates the critical role of TET proteins in regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at CGIs associated with developmental genes.