Trans-chromosomal methylation

The epigenome plays a vital role in helping to maintain and regulate cell functions in all organisms. Alleles with differing epigenetic marks in the same nucleus do not function in isolation but can interact in trans to modify the epigenetic state of one or both alleles. This is particularly evident when two divergent epigenomes come together in a hybrid resulting in thousands of alterations to the methylome. These changes mainly involve the methylation patterns at one allele being changed to resemble the methylation patterns of the other allele, in processes we have termed trans-chromosomal methylation (TCM) and trans-chromosomal demethylation (TCdM). These processes are primarily modulated by siRNAs and the RNA directed DNA methylation pathway. Drawing from other examples of trans-allelic interactions, we describe the process of TCM and TCdM and the effect such changes can have on genome activity. Trans-allelic epigenetic interactions may be a common occurrence in many biological systems.     

Trans-chromosomal methylation

The epigenome plays a vital role in helping to maintain and regulate cell functions in all organisms. Alleles with differing epigenetic marks in the same nucleus do not function in isolation but can interact in trans to modify the epigenetic state of one or both alleles. This is particularly evident when two divergent epigenomes come together in a hybrid resulting in thousands of alterations to the methylome. These changes mainly involve the methylation patterns at one allele being changed to resemble the methylation patterns of the other allele, in processes we have termed trans-chromosomal methylation (TCM) and trans-chromosomal demethylation (TCdM). These processes are primarily modulated by siRNAs and the RNA directed DNA methylation pathway. Drawing from other examples of trans-allelic interactions, we describe the process of TCM and TCdM and the effect such changes can have on genome activity. Trans-allelic epigenetic interactions may be a common occurrence in many biological systems.     

Microwave-accelerated methylation of starch

A novel microwave-accelerated method for methylating soluble starch is described. Soluble starch could be fully methylated in 72% yield within 4.66 min using iodomethane and 30% potassium hydroxide under microwave irradiation. The completely methylated starch thus obtained was hydrolyzed with 60% HCO(2)H for 1.5 min under 80% MW power, followed by 0.05 M H(2)SO(4) for 2.0 min under 100% MW power. The partially methylated monosaccharides were separated by preparative paper chromatography and identified by their melting points and optical rotations.     

Inhibitors of histone methylation

Two classes of inhibitors of histone methyltransferase I from calf thymus are reported. High concentrations ($\text{≧ 10}\text{m}\text{M}$) of various alkyl or aralkyl amines and polyamines were inhibitory to the enzyme. Spermine and spermidine were among the most potent compounds in this group. The best monoamine inhibitor was 2-phenylethylamine, which gave 47% inhibition at 10 mM.The substituted phenanthridinium compound ethidium bromide was also an inhibitor of the enzyme. A number of analogs of ethidium bromide were tested, and the most potent compound (17) gave 50% inhibition at 0.125 mM. $\text{S-}\text{Adenosyl}\text{-}\text{l}\text{-}\text{ethionine}$ (SAM) showed competitive inhibition of the enzyme as determined from a Lineweaver-Burke plot, while ethidium bromide was noncompetitive.     

Avoidance of DNA methylation

Theocr ⁺ gene of bacterial virus T7 codes for the first protein recognized to inhibit a specific group of DNA methylases. The recognition sequences of several other DNA methylases, not susceptible to Ocr inhibition, are significantly suppressed in the virus genome. The bacterial virus T3 encodes an Ado-Met hydrolase, destroying the methyl donor and causing T3 DNA to be totally unmethylated. These observations could stimulate analogous investigations into the regulation of DNA methylation patterns of eukaryotic viruses and cells. For instance, an underrepresentation of methylation sites (5′-CG) is also true for animal DNA viruses. Moreover, we were able to disclose some novel properties of DNA restriction-modification enzymes concerning the protection of DNA recognition sequences in which only one strand can be methylated (e.g., type III enzymeEcoP15) and the primary resistance of (unmethylated) DNA recognition sites towards type II restriction endonucleaseEcoRII.     

Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers

Background

Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve neuroblastoma patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers.

Results

To identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight neuroblastoma cell lines. Specifically, we used re-expression profiling upon 5-aza-2''-deoxycytidine (DAC) treatment and massively parallel sequencing after capturing with a methyl-CpG-binding domain (MBD-seq). Putative methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR on 20 primary neuroblastoma tumors, as well as through MBD- seq in combination with publicly available neuroblastoma tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput methylation-specific PCR on an independent cohort of 89 primary neuroblastoma tumors that had been selected for risk classification and survival. Based on this analysis, methylation of KRT19, FAS, PRPH, CNR1, QPCT, HIST1H3C, ACSS3 and GRB10 was found to be associated with at least one of the classical risk factors, namely age, stage or MYCN status. Importantly, HIST1H3C and GNAS methylation was associated with overall and/or event-free survival.

Conclusions

This study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in neuroblastoma performed thus far. We identified several novel prognostic DNA methylation markers and provide a basis for the development of a DNA methylation-based prognostic classifier in neuroblastoma.     

Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers

ChIP-seq is a powerful method for obtaining genome-wide maps of protein-DNA interactions and epigenetic modifications. CHANCE (CHip-seq ANalytics and Confidence Estimation) is a standalone package for ChIP-seq quality control and protocol optimization. Our user-friendly graphical software quickly estimates the strength and quality of immunoprecipitations, identifies biases, compares the user's data with ENCODE's large collection of published datasets, performs multi-sample normalization, checks against quantitative PCR-validated control regions, and produces informative graphical reports. CHANCE is available at https://github.com/songlab/chance.