DNA cytosine hydroxymethylation levels are distinct among non-overlapping classes of peripheral blood leukocytes

Abstract

Background: Peripheral blood leukocytes are the most commonly used surrogates to study epigenome-induced risk and epigenomic response to disease-related stress. We considered the hypothesis that the various classes of peripheral leukocytes differentially regulate the synthesis of 5-methylcytosine (5mCG) and its removal via Ten-Eleven Translocation (TET) dioxygenase catalyzed hydroxymethylation to 5-hydroxymethylcytosine (5hmCG), reflecting their responsiveness to environment. Although it is known that reductions in TET1 and/or TET2 activity lead to the over-proliferation of various leukocyte precursors in bone marrow and in development of chronic myelomonocytic leukemia and myeloproliferative neoplasms, the role of 5mCG hydroxymethylation in peripheral blood is less well studied.

Results: We developed simplified protocols to rapidly and reiteratively isolate non-overlapping leukocyte populations from a single small sample of fresh or frozen whole blood. Among peripheral leukocyte types we found extreme variation in the levels of transcripts encoding proteins involved in cytosine methylation (DNMT1, 3A, 3B), the turnover of 5mC by demethylation (TET1, 2, 3), and DNA repair (GADD45A, B, G) and in the global and gene-region-specific levels of DNA 5hmCG (CD4+ T cells≫CD14+ monocytes>CD16+ neutrophils>CD19+ B cells>CD56+ NK cells>Siglec8+ eosinophils>CD8+ T cells).

Conclusions: Our data taken together suggest a potential hierarchy of responsiveness among classes of leukocytes with CD4+, CD8+ T cells and CD14+ monocytes being the most distinctly poised for a rapid methylome response to physiological stress and disease.

Keywords: 5-Hydroxymethylcytosine; Cellular memory; Disease; Epigenetic control; Epigenome-induced risk; Surrogate cells.

Fig. 1

The dynamic modification cycle of DNA cytosine and its impact on gene activity. This model of the turnover of modified cytosine (C) residues emphasizes the central role of DNMTs in the methylation of C to 5-methylcytosine (5mC) and TETs in the rate limiting removal of 5mC by oxidation to 5-hydroxymethylcytosine (5hmC). The dynamic turnover of 5mC appears critical to regulating rapid changes in linked gene expression (Meagher, 2014; Wu and Zhang, 2014). TETs may further oxidize 5hmC to 5-formalcytosine (5fC) and 5-carboxycytosine (5caC). Thymine DNA glycosidase TDG removes the modified 5fC or 5caC bases leaving an abasic nucleotide (–OH). Base excision repair (BER) repairs the single nucleotide gap in double stranded DNA back to a C residue. Enzymes are in square boxes and nucleotide bases are in ovals. The diagram was modified from (Kohli and Zhang, 2013), based on the data in (Chen et al., 2012; Ramon et al., 2012; Dubois-Chevalier et al., 2014; Haseeb et al., 2014; Oger et al., 2014).

Fig. 2

Description of isolation protocols. Graphical outline of the three isolation methods (1, 2, 3) each starting with 5 ml of peripheral blood.

Fig. 3

Nuclear morphologies of isolated cell types. Isolated leukocytes, bound to Dynabeads were stained with: DAPI (upper left blue) and PI (lower left red) and photographed by fluorescence (left) and DIC microscopy (upper right) of each of the seven panels. The three images were merged to yield the image in the lower right. Scale bar = 20 μm.

Fig. 4

Immuno-fluorescent analysis showed a wide distribution of 5hmC levels among various classes of leukocytes. A. Total human leukocyte fraction from fresh peripheral blood (method 2) were labeled with DAPI for DNA (fluorescent green), primary antibody to 5hmC and secondary R-PE (red fluorescence), and then the merged image of DAPI and 5hmC is also presented. The same field of cells is shown in all images. B. Total human leukocyte fraction from frozen peripheral blood (method 3) were labeled with DAPI for DNA (fluorescent green), primary antibody to 5hmC and secondary R-PE (red fluorescence), and then the merged image of DAPI and 5hmC is also presented. The same field of cells is shown in all images. Example cells are labeled based on nuclear morphologies. K: kidney shaped (monocytes or natural killer cells), R: Round (T cells and B cells), M: Multilobed (neutrophils), B: Bilobed (eosinophils). C. 5hmC signal was quantified, and categorized as minimal, low, medium or high for each of the nuclear morphologies in each isolation method (fresh blood: method 2, frozen blood: method 3) and the percent of cells for each nuclear morphology was plotted for each 5hmC signal. Error bars represent standard error of the mean.

Fig. 5

Gene-region-specific 5hmCG levels are distinct among the peripheral leukocyte types and vary by transcript level. A. Map defining the three gene regions assayed (Lister et al., 2013). B. 5hmCG levels were plotted for each of the seven leukocyte types by quintile of transcript expression level. The peak percentage of 5hmCG relative to total CG content for each cell type is estimated at the top of each graph. C. The 5hmCG levels for the quintile of the highest quintile of transcript for each cell type was plotted together. The dots plotted with each line on the graph represent the degree of change from the previous regions level of 5hmCG to the current level. The figure legend to the right of panel C shows the varying levels of significance of this change as determined by NLP. The relative position of each cell type is the same when these data are plotted for the other 4 quintiles of transcript expression (Supplemental Fig. 4).

Fig. 6

Gene-sequence-specific distribution of 5hmCG levels in the peripheral leukocytes for relevant GO term gene lists. The fraction of 5hmCG relative to all CG dinucleotides for seven gene sequence locations in all leukocytes (100 bp upstream of the TSS (UTSS), 100 bp downstream TSS (DTSS), 100 bp upstream of all exons (UEXON), within exons (EXON), 100 bp downstream of exons (DEXON), 100 bp upstream TTS (UTTS), and 100 bp downstream TTS (DTTS)) were plotted for different GO terms related to leukocyte function as box plots with the bar representing the median, and the box extending from the 25th to 75th percentiles. The whiskers represent ±1.5 times the interquartile range. Within each box plot the weighted average of 5hmCG for each leukocyte type was plotted as a colored dot, while the box is the weighted average for all 7 cell types. Similarly plotted data for several other genes grouped by GO terms are presented in Supplemental Fig. 5.

Fig. 7

Expression of transcripts encoding enzymes involved in the establishment and removal of modified DNA cytosine. A–C. qRT-PCR analysis of the relative transcript expression was performed on cDNA prepared from seven leukocyte types. Values are expressed as a scaled Relative Quantity (RQ) of transcript in each cell type using the dCT method. Letters designate significant differences of at least p < 0.05. A. Analysis of transcripts for DNMTs (Fig. 1). The RQ value for each cell type is presented as a scaled value of 10⁴, 10⁵, and 10⁶ times their RQ value for DNMT1, DNMT3A, and DNMT3B, respectively. B. Analysis of transcripts for TETs (Fig. 1). The RQ value for each cell type is presented as a scaled value of 10⁶, 10⁵, and 10⁵ times their RQ value for TET1, TET2, and TET3, respectively. C. Analysis of transcripts for GADD45s (Fig. 1). The RQ value for each cell type is presented as a scaled value of 10⁵, 10⁵, and 10⁶ times their RQ value for GADD45A, GADD45B, and GADD45G, respectively.