Global 5-hydroxymethylcytosine content is significantly reduced in tissue stem/progenitor cell compartments and in human cancers

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.

Figure 1. Specificity of immuohistochemical detection of 5hmC

To assess the specificity of 5hmC immuno-labeling of formalin fixed paraffin embedded cells, HEK293 cells were transfected with expression plasmids encoding for myc-tagged TET2 or control. Cell pellets were fixed and embedded in paraffin. Sections of the resulting paraffin block were co-immuno-labeled with anti-myc and anti-5hmC specific antibodies and visualized using fluorophore conjugated secondary antibodies (A, B, C). Note that only cells that express high levels of TET2 (indicated by arrowheads) showed strong staining for 5hmC. (E, F) To show that 5hmC can be specifically detected using a chromogenic immunohistochemistry method, HEK293 cells overexpressing TET2-myc and HEK293 control cells were stained with 5hmC specific antibodies and immunocomplexes were visualized using HRP conjugated secondary antibodies with DAB as a chromogen.

Figure 2. 5hmC is abundant in embryonal and adult tissues, with differential abundance in basal vs. luminal cell compartments of stratified epithelia

Micrographs show 5hmC staining in the intestine (A) and skin (B) of a 17 day old mouse embryo. Note the reduced staining of 5hmC staining in the basal cell compartment (indicated by arrowheads) compared to the luminal/apical epithelial cells (indicated by arrows). (C) Normal human adult colonic mucosa exhibits strong staining for 5hmC in apical epithelial cells (indicated by arrows); epithelial cells in the base of the crypt (indicated by arrowheads) show greatly reduced staining intensities. Note the strong 5hmC staining of associated stromal nuclei. (D) Hierarchical distribution of 5hmC staining in murine cervix. Asterisk (*) indicates apical/luminal surface.

Figure 3. Quantitative analysis of the hierarchical distribution of 5hmC in stratified epithelia

(A-C) Representative micrographs of normal prostate epithelia co-immunolabeled for 5hmC (red) and basal cell specific cytokeratin 903 (green). Nuclei were counterstained with DAPI (blue). (D) Box-plots show the distribution of 5hmC fluorescence intensities in basal (903+) and luminal (903-) cells, normalized to DAPI. (E-G) Representative micrographs of normal esophageal mucosa co-immunolabeled for 5hmC (red) and basal cell specific CK15 (green). (H) Distribution of 5hmC staining intensities in basal (CK15+) and luminal (CK15-) cells, normalized to DAPI. Arrowheads indicate basal cells, arrows indicate luminal cells, Asterisks (*) indicate lumen.

Figure 4. Quantitative analysis of the hierarchical distribution of 5hmC in hematopoietic cells

Ficoll-Paque enriched, CD34-depleted bone marrow, or FACS sorted CD34+;CD38-;ALDH-high hematopoietic stem cells, or CD34+;CD38+ progenitor cells were spotted on glass slides, stained with 5hmC specific antibodies and visualized using immunofluorescence microscopy. Signal intensities were determined by quantitative image analysis. (A) Distribution of 5hmC signal intensities in the stem cell (CD34 positive ALDH high), progenitor cell (CD34, CD38 positive) and differentiated cell (CD34 negative) compartments. (B) Representative micrographs of each enriched fraction.

Figure 5. Significant reduction in 5hmC levels in cancers

Micrographs of representative 5hmC staining in normal human prostate (A) and prostate adenocarcinoma (B), normal breast (D) and ductal breast cancer (E) and normal colon mucosa and adenocarcinoma of the colon (H). (C, F, I) show distributions of semi-quantitative intensities scores in normal and tumor cells in box-and-whisker plots. Note that (A) contains a small focus of cancerous glands (indicated by arrowheas) infiltrating normal prostatic epithelium. Arrows indicate normal epithelial cells; arrowheads show tumor cell nuclei with reduced 5hmC staining.