In it’s third year, the Epigenetic World Congress continued to deliver a great experience and create new fans. See what our volunteer conference reporter, Sadie Marjani had to say about all the epigenetic knowledge getting dropped at this event. The Epigenetics World Congress was held on April 26-27, 2011 in conjunction with the RNAi & miRNA World Congress, Advances in qPCR and the Next-Gen Sequencing Congress. The conference attracted a good mix of academic scientists and industry professionals, which no doubt resulted in many fruitful interactions.
Plasma DNA Methyaltion as Cancer Biomarkers
Victor Levenson, Rush University Medical Center
Everyone has cell-free circulating DNA in his or her plasma. Typically, the concentration is around 5 ng/ml; however, this is oftentimes increased in patients with diseases such as cancer and multiple sclerosis (MS). Dr. Levenson described the potential for the methylation of this DNA as a biomarker and specifically discussed a method that involved methylation sensitive restriction enzyme digestion, PCR amplification and profiling with a candidate gene CpG-island microarray.
Groups of genes represented on MethDet-56 microarray were used to distinguish between patients in MS remission or exacerbation with high sensitivity and specificity. Levenson went on to show the ability of this tool to distinguish between malignant or benign masses, and the affect of specific cancer drugs on DNA methylation. The power of this assay is its minimally invasive nature, which makes it a desirable diagnostic tool.
Evolution of Eukaryotic DNA Methylation
Daniel Zilberman, University of California, Berkeley
Dr. Zilberman used MethylC-seq to assess the genome-wide methylation levels of seventeen eukaryotic species. RNA-seq was also performed to profile transcription. Arabidopsis, rice, algae (Chlorella) and puffer fish all have extensive CG methylation in their gene bodies, moderately transcribed genes were the most likely to be methylated, and transposable elements (TE) were highly methylated. The sea squirt had a similar pattern; however, in contrast to the above species, it had hypomethylated TEs.
This reduced TE methylation was also seen in honeybees, silk moths and anemones. In fungi, methylation is concentrated at transcriptionally silent, repetitive loci (i.e., TEs) and not present in active genes. Zilberman concluded that methylation of gene bodies is highly conserved and very ancient as it is observed in plants, invertebrates and vertebrates. Based on an analysis of DNA methyltransferases in plants and fungi, he suggested that TE methylation/silencing is ancient and that it is lost in early animal evolution and then re-established in vertebrates.
Zilberman postulated that this is possibly due to the fact that TE aggressiveness depends on sexual out-crossing and thus asexual reproducing species would have less need to methylate and thereby silence TEs in their genomes.
Epigenome-Wide Association Studies
John Greally, Albert Einstein College of Medicine
Dr. Greally discussed the parameters necessary for determining non-random associations of epigenetic characteristics with disease state. Factors such as cell type, which epigenetic modification to study, which assay to use and sample size are critical. Greally focused on the analysis of global DNA methylation and showed data that demonstrated the superiority of massively parallel sequencing (MPS) techniques over microarray based techniques due to a reduction in noise. He advocated the combination of HELP-tagging (HpaII tiny fragment Enrichment by Ligation-mediated PCR) with MPS to interrogate both CG-rich and depleted genomic regions. Additionally, the technique is amendable to multiplexing further reducing sequencing costs.
Using data sets generated by HELP-tagging MPS and directional RNA-seq, Greally showed a 20-30% difference in the methylation levels of intergenic regions in rat intrauterine growth restriction model. This modest change in methylation levels correlated with a drop in gene expression. He concluded that it is necessary to have at least 100 samples to provide enough power for all the methylation changes observed to be informative, and that samples need to be stratified by age, sex and ethnicity.
Next-Gen Sequencing Uncovers Allele-Specific Expression
Sergio Baranzini, University of California, San Francisco
Dr. Baranzini described his cutting edge work where differential allelic expression was analyzed by RNA-seq in monozygotic twins discordant for multiple sclerosis. Single nucleotide polymorphisms (SNPs) were identified in coding regions, RNA reads were counted for each allele, and the deviation from the 50:50 ratio was plotted along the genome. Allelic imbalance, i.e., preference for one allele, was seen in both twins; however, the affected twin had significantly more differential allelic expression.
Allelic imbalance and differential allelic expression between the twins was associated when SNPs were located in transcription factor binding sites, indicating a possible mechanism. When combined with RRBS-sequencing data, a statistically significant number of regions were identified that displayed differential allelic expression and differential CG methylation. Additionally, SNPs that resided in miRNA binding sites were much more likely to exhibit allelic imbalance.
Epigenetic Reprogramming by Early Life Experiences
Douglas Ruden, Wayne State University
Using mDIP with antibodies for 5mC or 5hmC in combination with high-throughput sequencing or Illumina Infinium assays, Dr. Ruden examined methylation patterns in killer bees and human neural precursor cells (NPCs). Killer bee brains have 5hmC and there was 90% overlap between regions that were pulled down by either 5mC or 5hmC antibodies. MethylC-seq identified CpG methylation in the exons and CHH methylation in the introns of Killer bees. In human NPCs, hundreds of genes dramatically increased the 5hmC/5mC ratio in CpG islands (assessed by mDIP-Infinium) when grown under 20% oxygen conditions compared to 5% oxygen.
Dr. Ruden postulated that 5hmC might be a marker of oxidative stress. He further speculated that because human IVF is routinely done under 20% oxygen, an increase in 5hmC could be responsible for the higher incidence of imprinting disorders in children produced by IVF. More specifically, a switch from 5mC to 5hmC would lead to a loss of methylation during replication because 5hmC is not recognized by DNMT1.
**EpiGenie would like to thank Sadie Marjani, PhD who is a Postdoctoral Fellow in the Department of Genetics at the Yale University School of Medicine, for providing this conference coverage.