Both DNA methylation and histone modification are involved in establishing patterns of gene repression during development. Certain forms of histone methylation cause local formation of heterochromatin, which is readily reversible, whereas DNA methylation leads to stable long-term repression. It has recently become apparent that DNA methylation and histone modification pathways can be dependent on one another, and that this crosstalk can be mediated by biochemical interactions between SET domain histone methyltransferases and DNA methyltransferases. Relationships between DNA methylation and histone modification have implications for understanding normal development as well as somatic cell reprogramming and tumorigenesis.
Many genes associated with CpG islands undergo de novo methylation in cancer. Studies have suggested that the pattern of this modification may be partially determined by an instructive mechanism that recognizes specifically marked regions of the genome. Using chromatin immunoprecipitation analysis, here we show that genes methylated in cancer cells are specifically packaged with nucleosomes containing histone H3 trimethylated on Lys27. This chromatin mark is established on these unmethylated CpG island genes early in development and then maintained in differentiated cell types by the presence of an EZH2-containing Polycomb complex. In cancer cells, as opposed to normal cells, the presence of this complex brings about the recruitment of DNA methyl transferases, leading to de novo methylation. These results suggest that tumor-specific targeting of de novo methylation is pre-programmed by an established epigenetic system that normally has a role in marking embryonic genes for repression.
Oct-3/4 is a POU domain homeobox gene that is expressed during gametogenesis and in early embryonic cells, where it has been shown to be important for maintaining pluripotency. Following implantation, this gene undergoes a novel multi-step programme of inactivation. Transcriptional repression is followed by a pronounced increase in histone H3 methylation on Lys 9 that is mediated by the SET-containing protein, G9a. This step sets the stage for local heterochromatinization via the binding of HP1 and is required for subsequent de novo methylation at the promoter by the enzymes Dnmt3a/3b. Genetic studies show that these epigenetic changes actually have an important role in the inhibition of Oct-3/4 re-expression, thereby preventing reprogramming.
Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.
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