DNA sequencing has had the power of technology behind it as novel platforms to produce more sequencing faster and at lower cost have been introduced. But the tools to unravel the epigenetic control mechanisms that influence how cells control access of transcriptional proteins to DNA are just beginning to emerge.
Among these mechanisms, DNA methylation, or the enzymatically mediated addition of a methyl group to cytosine or adenine dinucleotides, serves as an inherited epigenetic modification that stably modifies gene expression in dividing cells. The unique methylomes are largely maintained in differentiated cell types, making them critical to understanding the differentiation potential of the cell.
In the DNA methylation process, cytosine residues in the genome are enzymatically modified to 5-methylcytosine, which participates in transcriptional repression of genes during development and disease progression. 5-methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. DNA methylation affects gene transcription by physically interfering with the binding of proteins involved in gene transcription.
Methylated DNA may be bound by methyl-CpG-binding domain proteins (MBDs) that can then recruit additional proteins. Some of these include histone deacetylases and other chromatin remodeling proteins that modify histones, thereby forming compact, inactive chromatin, or heterochromatin. While DNA methylation doesn’t change the genetic code, it influences chromosomal stability and gene expression.
Epigenetics and Cancer Biomarkers
And because of the increasing recognition that DNA methylation changes are involved in human cancers, scientists have suggested that these epigenetic markers may provide biological markers for cancer cells, and eventually point toward new diagnostic and therapeutic targets. Cancer cell genomes display genome-wide abnormalities in DNA methylation patterns, some of which are oncogenic and contribute to genome instability. In particular, de novo methylation of tumor suppressor gene promoters occurs frequently in cancers, thereby silencing them and promoting transformation.
Cytosine hydroxymethylation (5-hydroxymethylcytosine, or 5hmC), the aforementioned DNA modification resulting from the enzymatic conversion of 5mC into 5-hydroxymethylcytosine by the TET family of oxygenases, has been identified as another key epigenetic modification marking genes important for pluripotency in embryonic stem cells (ES), as well as in cancer cells.
The base 5-hydroxymethylcytosine was recently identified as an oxidation product of 5-methylcytosine in mammalian DNA. In 2011, using sensitive and quantitative methods to assess levels of 5-hydroxymethyl-2'-deoxycytidine (5hmdC) and 5-methyl-2'-deoxycytidine (5mdC) in genomic DNA, scientists at the Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California investigated whether levels of 5hmC can distinguish normal tissue from tumor tissue. They showed that in squamous cell lung cancers, levels of 5hmdC showed up to five-fold reduction compared with normal lung tissue. In brain tumors, 5hmdC showed an even more drastic reduction with levels up to more than 30-fold lower than in normal brain, but 5hmdC levels were independent of mutations in isocitrate dehydrogenase-1, the enzyme that converts 5hmC to 5hmdC.
Immunohistochemical analysis indicated that 5hmC is “remarkably depleted” in many types of human cancer. Importantly, they said, an inverse relationship between 5hmC levels and cell proliferation was observed with lack of 5hmC in proliferating cells. Their data, they say, suggest that 5hmdC is strongly depleted in human malignant tumors, a finding that adds another layer of complexity to the aberrant epigenome found in cancer tissue. In addition, a lack of 5hmC may become a useful biomarker for cancer diagnosis.
But according to New England Biolabs’ Sriharsa Pradhan, Ph.D., methods for distinguishing 5mC from 5hmC and analyzing and quantitating the cell’s entire “methylome” and “hydroxymethylome” remain less than optimal.
The protocol for bisulphite conversion to detect methylation remains the “gold standard” for DNA methylation analysis. This method is generally followed by PCR analysis for single nucleotide resolution to determine methylation across the DNA molecule. According to Dr. Pradhan, “It’s a pretty good method but the DNA gets pretty badly battered. The polymer can’t be copied by conventional polymerase easily and the strands are of a different sequence. Furthermore, bisulphite conversion does not distinguish 5mC and 5hmC,” which may hamper the analysis.
Several years ago, he explained, “We started looking at enzymes that can detect 5-methylcytosine. Recently we found an enzyme, a unique DNA modification-dependent restriction endonuclease, AbaSI, and this unique microbial enzyme can decode the hydryoxmethylome of the mammalian genome. You easily can find out where the hydroxymethyl regions are.”
AbaSI, recognizes 5-glucosylatedmethylcytosine (5gmC) with high specificity when compared to 5mC and 5hmC, and cleaves at narrow range of distances away from the recognized modified cytosine. By mapping the cleaved ends, the exact 5hmC location can, the investigators reported, be determined.
Dr. Pradhan and his colleagues at NEB; the Department of Biochemistry, Emory University School of Medicine, Atlanta; and the New England Biolabs Shanghai R&D Center described use of this technique in a paper published in Cell Reports this month, in which they described high-resolution enzymatic mapping of genomic hydroxymethylcytosine in mouse ES cells.
In the current report, the authors used the enzyme technology for the genome-wide high-resolution hydroxymethylome, describing simple library construction even with a low amount of input DNA (50 ng) and the ability to readily detect 5hmC sites with low occupancy.
As a result of their studies, they propose that factors affecting the local 5mC accessibility to TET enzymes play important roles in the 5hmC deposition including include chromatin compaction, nucleosome positioning, or TF binding. The most striking example, they said, is the regularly oscillating 5hmC profile around the CTCF-binding sites, suggesting 5hmC ‘‘writers’’ may be sensitive to the nucleosomal environment. They further proposed that some transiently stable 5hmCs may indicate a poised epigenetic state or demethylation intermediate, whereas others may suggest a locally accessible chromosomal environment for the TET enzymatic apparatus.
“We were able to do complete mapping in mouse embryonic cells and are pleased about what this enzyme can do and how it works,” Dr. Pradhan said.
And the availability of novel tools that make analysis of the methylome and hypomethylome more accessible will move the field of epigenetic analysis forward and potentially novel biomarkers for cellular development, differentiation, and disease.