Abstract
DNA methylation at the 5-position of cytosines (5 mC) represents an important epigenetic modification involved in tissue differentiation and is frequently altered in cancer. Recent evidence suggests that 5 mC can be converted to 5-hydroxymethylcytosine (5 hmC) in an enzymatic process involving members of the TET protein family. Such 5 hmC modifications are known to be prevalent in DNA of embryonic stem cells and in the brain, but the distribution of 5 hmC in the majority of embryonic and adult tissues has not been rigorously explored. Here, we describe an immunohistochemical detection method for 5 hmC and the application of this technique to study the distribution of 5 hmC in a large set of mouse and human tissues. We found that 5 hmC was abundant in the majority of embryonic and adult tissues. Additionally, the level of 5 hmC closely tracked with the differentiation state of cells in hierarchically organized tissues. The highest 5 hmC levels were observed in terminally differentiated cells, while less differentiated tissue stem/progenitor cell compartments had very low 5 hmC levels. Furthermore, 5 hmC levels were profoundly reduced in carcinoma of the prostate, breast and colon compared to normal tissues. Our findings suggest a distinct role for 5 hmC in tissue differentiation, and provide evidence for its large-scale loss in cancers.
Highlights
Epigenetic modifications play a crucial role in cellular differentiation and have been implicated in numerous disease states including cancer [1,2,3,4]
The recent discovery of a group of enzymes of the ten-eleven translocated (TET) family that can modify these DNA methylation marks by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) has added another dimension of complexity to our understanding of DNA methylation [7]. It has been well established for decades that certain bacteriophages contain 5-hydroxymethylcytosine rather than cytosine in their genome to protect themselves from host-controlled nucleases [8]
TET2 expressing and control HEK293 cells were incubated with 5hmC antibodies; immunocomplexes were visualized using HRP conjugated secondary antibodies with diaminobenzidine tetrahydrochloride (DAB) as a chromogen
Summary
Epigenetic modifications play a crucial role in cellular differentiation and have been implicated in numerous disease states including cancer [1,2,3,4]. The recent discovery of a group of enzymes of the ten-eleven translocated (TET) family that can modify these DNA methylation marks by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) has added another dimension of complexity to our understanding of DNA methylation [7]. It has been well established for decades that certain bacteriophages contain 5-hydroxymethylcytosine rather than cytosine in their genome to protect themselves from host-controlled nucleases [8]. The tissue specific cellular distribution of 5hmC in normal adult tissues and neoplasia, has far not been welldocumented
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