Epigenetics, which scientists define as heritable changes in gene expression that do not involve alterations in the DNA sequence, is drawing the increasing attention of investigators all around the globe. In addition to its role in helping researchers better understand organismal development, epigenetics has emerged as an important facet of disease pathogenesis.
The creation of new experimental strategies represents a fundamental component of the efforts to gain deeper insights into the epigenetic modulation of gene expression. For example, at Keystone Symposia’s “Epigenomics” conference last month, Peter A. Jones, Ph.D., distinguished professor of urology and biochemistry at the University of Southern California, talked about a new method that investigators in his lab developed to simultaneously map the presence of nucleosomes and DNA methylation on individual DNA molecules.
“The idea is that we place a chemical marker on the DNA molecule, which allows us to determine which DNA areas are open and which ones are wrapped around nucleosomes, and we can relate this to DNA methylation,” explained Dr. Jones.
For the past 30 years, nucleosomes were mapped by limited digestion with DNA-cleaving enzymes such as DNAse I or micrococcal nuclease, and the location of DNA methylation sites was revealed by parallel experiments.
The nucleosome occupancy methylome-sequencing, or NOMe-seq, single-molecule high-resolution nucleosome positioning assay that Dr. Jones and colleagues developed is based on the accessibility of the DNA to the M.CviPI GpC methyltransferase that recognizes GpC dinucleotides not associated with nucleosomes or transcription factors.
This technique allows the simultaneous visualization, within the same DNA molecule, of the endogenous DNA methylation and nucleosome occupancy distribution, irrespective of the density or methylation status of CpG sites.
When used to examine the well-characterized enhancer/promoter pair of the MYOD1 gene in somatic cells, the NOMe-seq method revealed that a nucleosome-depleted region exists at enhancers in opposing transcriptional states, and Dr. Jones and collaborators showed that this architecture may be associated both with active and repressed promoter states.
The presence of epigenetic marks correlated with both activation and repression mirrors previous findings from embryonic stem cells, where similar bivalent marks were characterized and are thought to keep genes poised for induction. The presence of permissive enhancers associated with certain promoters that are repressed by the polycomb-repressive complex allows these loci to retain their permissiveness and ensures epigenetic plasticity during reprogramming in somatic cells.
Dr. Jones and colleagues previously revealed that DNA methylation acts as a lock to silence transcriptionally inactive chromosomal domains.
The scientists showed that three nucleosomes, which in normal cells are almost completely absent from the start sites on MLH1, a gene implicated in colorectal and breast cancer, exist in methylated and in silenced promoters, suggesting that the stable placement of nucleosomes into previously vacant positions could provide a mechanism for epigenetic silencing.
“The way this works is that the nucleosome is a substrate for de novo DNA methylation, and in order for genes to become silenced, the first thing that happens is that the nucleosome is placed into the region, followed by de novo DNA methylation,” noted Dr. Jones.