Based on what we’ve seen so far in journals, conferences and interviews, 2012 could be a banner year for epigenetic analysis methods. New twists on old tricks, and futuristic technology advances have converged to create some really exciting new approaches in the field. Here are just a few that you’ll want to keep track of in the coming months.
NoME-Seq: Nucleosome Occupancy and Methylome Sequencing
Nucleosome occupancy and methylome sequencing (NoME-Seq) was actually published late last year (PNAS, August 2011), but began making the rounds on the conference circuit in early 2012 when USC’s Peter Jones presented at the Keystone Epigenomics Meeting, and again when co-author Teresa Kelly spoke at the CHI X-Gen Congress.
NoMe-Seq is a high-resolution, single molecule technique that looks at nucleosome positions and DNA methylation all at once. The assay is based on the accessibility of the DNA to the M.CviPI GpC methyltransferase that scopes out GpC dinucleotides not associated with nucleosomes or transcription factors. The idea is that DNA molecules are tagged with a chemical that lets the researcher determine which DNA areas are open and which ones are wrapped around nucleosomes, that info is then compared to DNA methylation status.
Dr. Jones and his team have already put NoME-Seq to good use by proving that movement of nucleosomes into empty locations can be a mechanism for epigenetic silencing. The scientists showed that three nucleosomes, which are absent in the MLH1 start sites of normal cells are found, in silenced promoters; making the nucleosome a substrate for de novo DNA methylation.
oxBS-Seq: Oxidative Bisulfite Sequencing
As the newest flavor of bisulfite-based DNA methylation analysis methods, oxidative bisulfite sequencing (oxBS-Seq) is poised to make a big splash due mostly to one unique feature: It can distinguish between cytosine (C), methylcytosine (5mC) and hydroxymethylcytosine (5hmC) all at single base resolution.
Wolf Reik and his collaborators at the Babraham Institute and University of Cambridge achieved the ability to effectively sequence 5mC and 5hmC with the addition of an oxidation step prior to bisulfite conversion, which selectively alters 5hmC into formylcytosine (5fC) causing it to appear as a normal C when sequenced. A little careful comparison of standard and oxBS-seq data will quickly show you which locations were 5hmC or 5mC.
There are other ways to discriminate 5hmC from 5mC, but the single base resolution, and genome wide power that the sequencing component provides could really launch a new wave of research into 5-hmC and the epigenetic role of TET proteins.
BisChip-seq/ ChIP BS-Seq: Combined Bisulfite & Chromatin Immunoprecipitation Sequencing
In a case of ‘the sum of the parts being greater than the whole’, two familiar techniques, bisulfite conversion (BS or Bis) and chromatin immunoprecipitation ChIP), were combined to form a technique to address growing interest in the cross talk between DNA methylation and chromatin states. Apparently great minds think alike, because two groups both published very similar combo approaches in the March 2012 edition of Genome Research. The two new methods, BisChIP-Seq and ChIP-BS-Seq use similar steps, just in a different order. Either way, the bisulfite/ChIP combo offers new insights into how DNA methylation and chromatin work together, and represent a big upgrade over the correlation studies used until now.
Nanofluidic Epigenetic Analysis: SCAN (Single Chromatin Analysis at the Nanoscale) & Nanofluidic DNA Methylation Detection
For Paul Soloway and his various partners, working with technology is much more than just closely following the details of the Facebook IPO. Soloway’s lab at Cornell has been involved in creating very cutting edge, nanofluidic devices that can analyze single DNA molecules for various epigenetic marks.
In 2010, their first instrument was designed to assess multiple chromatin marks simultaneously as a single molecule was passed through a nanopore and was called Single Chromatin Analysis at the Nanoscale (SCAN). The SCAN technique used fluorescence microscopy to capture the light that different histone modifications emit when excited by a laser in order to tell them apart. SCAN still had a few things to iron out back then, but the development continues, in fact, Dr. Soloway was spotted presenting the latest updates at last month’s Genomics Research Meeting in Boston.
Another innovation from Soloway and friends has just hit the scene, too. In this month’s PNAS issue a new technique for using nanofluidics to sort and analyze single methylated DNA molecules in real-time was introduced. In this method, DNA is labeled with a red fluorescent marker, while MBD1 (which binds to methylated DNA) is labeled green. Once they are mixed, the device sorts the molecules based on color, sending methylated DNA to one channel and unmethylated into another. So, not only do you get a real-time readout of methylation density, but since the sample is separated and saved, you can go back and look for the exact locations of those methyl marks by qPCR.