Dynamic DNA methylation change in the CpG island region of p15 during human myeloid development

We examined the kenetics of p15 methylation and expression during myeloid development. We treated human cord blood CD34⁺ cells with either GM-CSF alone or in combination with stem cell factor and followed methylation at this locus using bisulfite genomic sequencing. CD34⁺ cells were found to be either fully methylated or completely unmethylated at 27 CpG dinucleotide sites in exon 1 and at 18 CpG sites in the promoter region of the p15 gene. A time-course study showed that the percentage of the allelic methylation of p15 CpG island increased to approximately 50% to 60% until 7 days after cytokine stimulation, then decreased to less than 10% after 21 days. The methylation was also observed in bone marrow CD34⁺ cells exposed to GM-CSF. p15 expression varied inversely with methylation. Expression was negligible or at low levels until 14 days, after which it increased substantially. The frequency of myeloid colony-forming cells in the progeny decreased and myeloid-specific markers increased in the later stages. Based on our observations on cells grown with GM-CSF and 5-aza-2′-deoxycytidine, DNA methylation of the p15 promoter region CpG island appears to be associated with proliferation rather than differentiation of normal human myeloid progenitors.

p15 (also known as INK4b or MTS2) is a candidate tumor suppressor with structural and functional similarity to p16. The p15 gene is located on chromosome 9p21, and its product is present in lymphocytes, granulocytes, and monocytes (1, 2). Both p15 and p16 specifically inhibit cyclin-dependent kinase 4 (CDK4) and CDK6 (3, 4), key regulators of the progression of eukaryotic cells through the G1 phase of the cell cycle. In T lymphocytes, p15 is downmodulated following stimulation with IL-2 (1). On the other hand, treatment with TGF-β upregulates p15 expression in human keratinocytes, indicating that p15 acts as an effector of TGF-β–mediated cell cycle arrest (3).

DNA methylation is the most common eukaryotic DNA modification and one of many epigenetic phenomena. This reaction occurs at cytosine residues including CpG dinucleotides. With the exception of islands in genes on the inactive X chromosome and some imprinted genes, CpG islands are usually unmethylated in normal somatic cells (5, 6). Recently, it was demonstrated that inactivation of the p15 gene by methylation of its 5′ CpG island and resulting transcription block occur in some gliomas and hematologic malignancies, such as acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS) (7–9). However, it remains unclear whether methylation of the p15 CpG sites occurs during normal hematopoiesis.

In the present study, the kinetics of p15 methylation and expression were examined during the cytokine-dependent differentiation into granulocytes and macrophages from normal hematopoietic progenitors. Here we document, we believe for the first time, a dynamic change in methylation at the p15 CpG island and the inverse expression of p15 during myeloid development.

Selective growth of granulocytes and macrophages from cord blood CD34⁺ cells under stimulation with GM-CSF or GM-CSF + SCF. We compared the ability of 10 ng/ml of GM-CSF and/or 10 ng/ml of SCF to generate granulocytes and macrophages from cord blood CD34⁺ cells. The results are presented in Figure 1. Under stimulation with GM-CSF alone, the number of total viable cells increased to a maximum of 20-fold the input quantity at day 21. The addition of SCF to the culture with GM-CSF caused a marked increment in the numbers of viable cells during the culture period. With both treatments, the cell number decreased at day 28. This may be due in part to apoptotic death, because the frequency of subdiploid nuclei in PI-stained samples of day 28–cultured cells was 20.3% in the presence of GM-CSF and 26.4% in the presence of GM-CSF + SCF (data not shown). When 10 ng/ml of G-CSF was added on day 21 to the culture containing GM-CSF with or without SCF, the number of total viable cells increased by 12% to 15% (data not shown). In the presence of GM-CSF alone, 39% and 11% of day 14–cultured cells were CD14^–CD15⁺ cells and CD14⁺CD15^– cells, respectively. Four percent of them were positive for the two antigens. After 21 days, a large part of the progeny displayed selective expression of CD15 antigen. On the other hand, the two-factor combination hastened myeloid differentiation. A great majority of the day 21– and day 28–cultured cells were positive for CD15, and one-half of them reacted with anti-CD14 mAb. Terminal differentiation into myeloid cell lineage under stimulation with GM-CSF or GM-CSF + SCF was confirmed by differential counting of the cytospin preparations stained with ANB and NASDCA. The cells with other lineage-specific markers (CD2, CD19, CD41, GPA, and c-kit) were at negligible levels after 7 days. SCF alone supported the growth of significant numbers of cells, which was comparable to the level obtained by GM-CSF alone during the culture period. On days 21 and 28, however, only 38% to 55% of the cultured cells expressed myeloid cell antigens, and 12% to 16% of the cultured cells were positive for c-kit (data not shown). From these results, we selected GM-CSF and GM-CSF + SCF as favorable stimuli for the differentiation into the myeloid cell lineage from cord blood hematopoietic progenitors.

Figure 1

Myeloid cell production from cord blood CD34⁺ cells under stimulation with GM-CSF or GM-CSF + SCF. (a) Cord blood CD34⁺ cells (2 × 10⁴) were plated in a well containing 10 ng/ml of GM-CSF with or without 10 ng/ml of SCF. The number of viable cells was serially counted every week. The time course of the relative frequency of cells positive for CD14 and/or CD15 and the relative frequency of ANB⁺ cells and NASDCA⁺ cells were examined. Similar results were obtained in two other experiments. Values are expressed as the mean ± SD. ANB⁺ cells (gray bars) and NASDCA⁺ cells (white bars). (b) Expression of CD14 and CD15 on the cultured cells generated by GM-CSF or GM-CSF + SCF from cord blood CD34⁺ cells. Surface marker expressions were analyzed by a flow cytometer.

To assess the proliferative potential of the individual cultured cells generated by GM-CSF with or without SCF, we performed clonal cell culture experiments. The results are presented in Figure 2a. In response to both types of stimulation, there was a gradual decrease in the frequency of GM precursors with the culture period. The progeny grown after 21 days contained granulocyte-macrophage precursors at negligible or very low levels in the two groups.

Figure 2

Time course of GM precursor number, intracellular p15 expression, and methylation of the p15 CpG island in the cultured cells generated by GM-CSF or GM-CSF + SCF from cord blood CD34⁺ cells. (a) Numbers of GM colony-forming cells (GM-CFCs) in 1,000 cultured cells at various time points. The cultured cells generated by GM-CSF were plated in a dish containing methylcellulose culture medium supplemented with 10 ng/ml of GM-CSF. In the case of the cultured cells generated by GM-CSF + SCF, the incidence of GM precursors was assayed in a semisolid culture medium containing 10 ng/ml of GM-CSF and 10 ng/ml of SCF. *Significant difference between the two groups; NS, no significance. (b) Intracellular p15 expression of the cultured cells determined by flow cytometry. Data are expressed as RFI. (c) The percentage of allelic methylation in the CpG island region of p15. The data are the sum total of the results obtained by the sequence method and by PCR analysis.

p15 expression during myeloid differentiation supported by GM-CSF or GM-CSF + SCF. We examined changes of p15 expression in this differentiation pathway. First, we analyzed the intracellular p15 expression of several leukemic cell lines according to the cytofluorometric analysis. K562 cells were used as a negative control on the basis of homozygous deletion of the 9p21 region containing the p15 and p16. As presented in Figure 3a, there was no significant p15 positivity in K562 cells (the RFI was 1.07). In Raji cells, the RFI was 1.25. On the other hand, HL-60 cells were reactive to anti-p15 mAb (the RFI was 3.24). The RFI of KU812 cells was 3.81 (data not shown). These results were consistent with the evidence obtained previously (22). Cord blood CD34⁺ cells were weakly positive for p15. Under stimulation with GM-CSF or GM-CSF + SCF, p15 was expressed at negligible or low level in the cultured cells up to day 14, as shown in Figures 2b and 3b. Subsequently, intracellular p15 content increased to the level comparable to p15-positive leukemic cell lines. A time-dependent increment in p15 protein levels was confirmed by 15% SDS-PAGE analysis followed by Western blot analysis, as shown in Figure 3c.

Figure 3

Intracellular p15 expression of leukemic cell lines, cord blood CD34⁺ cells, and the cultured cells generated by GM-CSF or GM-CSF + SCF from cord blood CD34⁺ cells. The cells were fixed and subsequently permeabilized. Then, the intracellular p15 expression was determined by flow cytometry. (a) Leukemic cell lines and cord blood CD34⁺ cells. (b) The cultured cells generated by GM-CSF or GM-CSF + SCF from cord blood CD34⁺ cells. Solid lines indicate the cells stained with anti-p15 mAb; the dotted lines indicate cells stained with mouse IgG1. (c) Thirty micrograms of cell lysates from cord blood CD34⁺ cells or cultured cells were subjected to 15% SDS-PAGE, transferred to PVDF membranes, and immunoblotted with anti-p15 mAb. Actin was used as a control.

Allelic methylation status of CpG sites at the p15 locus during myeloid differentiation supported by GM-CSF or GM-CSF + SCF. To elucidate the regulatory mechanism for p15 expression during the differentiation into granulocytes and macrophages, we first studied the methylation status of each CpG site within the region located between positions –12 and +251 relative to the transcriptional starting site of the p15 gene, by bisulfite genomic sequencing. Hypermethylation of this sequence has been demonstrated to be associated with transcriptional silencing in AML and MDS (9, 23, 24). As shown in Figure 4a, the region contains 27 CpG dinucleotides and 60 non-CpG cytosines. Primers were designed in the region without CpG dinucleotides to amplify both methylated and unmethylated alleles. Figure 4, b and c, show the allelic patterns of CpG island methylation in three leukemic cell lines. HL60 cells were nearly devoid of methylated CpG sites, whereas ML1 cells were methylated at almost all CpG sites on every allele sequenced. Most Raji cells had completely methylated alleles. In part, these results are consistent with those described by Cameron et al. (25).

Figure 4

Bisulfite genomic sequencing. (a) Sequence of the CpG island region of the p15 gene (–12 to +251 relative to the transcriptional starting site). The binding sites of the primers used in this experiment are also shown (p15-F and p15-R). (b) Sequence analysis of HL60 cells and ML1 cells. The region encompassing the 9th to 14th CpG site is shown. In HL60 cells, all cytosines, including six CpG dinucleotides, were invariably converted to uracils by the treatment with sodium bisulfite and appeared as thymidines after PCR. On the other hand, methylated cytosines at all of six CpG sites in ML1 cells remained unreactive. (c) The allelic methylation status of the p15 CpG island in various leukemic cell lines. Each row represents an individual cloned and sequenced allele following the bisulfite treatment. Methylated CpG sites are marked as filled circles and unmethylated sites as open circles. Numbers at the top correspond to those of the CpG sites shown in a.

We examined whether methylation occurred within this region during myeloid differentiation. As shown in Figure 5a, approximately 20% of cord blood CD34⁺ cells sorted by flow cytometry were methylated at all of the 27 CpG sites within the target sequence. The cultured cells grown with GM-CSF alone were found to be either fully methylated or completely unmethylated at the p15 CpG island until day 28 of culture. Similar results were obtained in the stimulation with GM-CSF + SCF. For an easy discrimination of methylated and unmethylated p15 CpG islands, we designed two sets of primers (p15-MF and p15-MR; p15UF and p15-UR, shown in Table 1). If these primers mixed together with the individual clones annealed to either the methylated or unmethylated DNA, the expected molecular size would be 147 bp and 99 bp, respectively. As presented in Figure 6a, the result of PCR with a mixture of the four primers (p15-MF, p15-MR, p15-UF, and p15-UR) corresponded to the data obtained by the separated PCR. The accuracy of the PCR analyses was also confirmed from the results of individual alleles characterized by the sequencing, as shown in Figure 6b. The sum total of the results obtained by the sequencing method and those by the PCR analysis showed that the percentage of alleles with methylated CpG islands increased to 50% until day 7 and then decreased in the presence of GM-CSF alone, as shown in Figures 2c and 6c. On stimulation with GM-CSF + SCF, methylation of the p15 CpG island was maintained for up to 14 days at a modestly high level relative to the values obtained from the stimulation with GM-CSF alone. Then, the incidence of methylation declined to less than 10%. Normal bone marrow cells could also be induced to methylate by GM-CSF, as shown in Figure 5b. None of ten alleles derived from marrow CD34⁺ cells were methylated, which was confirmed by the PCR analysis. This result was in agreement with that reported previously (26). The frequency of alleles with methylated p15 CpG islands increased to 50% on day 7 under stimulation with GM-CSF and decreased on day 21. In clonal cell culture experiments of bone marrow CD34⁺ cells with GM-CSF, methylation data emerged in all ten 10-day GM colonies, ranging from 10% to 60% (38% ± 23%).

Figure 5

Allelic patterns of CpG island methylation at the p15 locus during myeloid development from cord blood or bone marrow CD34⁺ cells. Methylation of the p15 CpG island located between positions –12 and +251 relative to the transcriptional starting site was examined by bisulfite genomic sequencing. Methylated CpG sites are marked as filled circles and unmethylated sites as open circles. Numbers at the top correspond to those of the CpG sites shown in Figure 4a. Each row of circles corresponds to one clone. (a) Cord blood CD34⁺ cells were stimulated with GM-CSF or GM-CSF+SCF. (b) Normal bone marrow CD34⁺ cells were stimulated with GM-CSF.

Figure 6

Detection of CpG island methylation in the p15 gene by PCR analysis. To discriminate between methylated and unmethylated CpG island (–12 and +251 relative to the transcriptional starting site) of the p15 gene, we performed PCR analysis. The primers used are p15-MF, p15-MR, p15-UF, and p15-UR. (a) Comparison of PCR methods. U, p15-UF + p15-UR; M, p15-MF + p15-MR; U&M, p15-UF + p15-UR + p15-MF + p15-MR. Numbers of clones from HL60 cells and Raji cells correspond to those of clones shown in Figure 4c. 99-bp products, unmethylated; 147-bp products, methylated. (b) Comparison of methylation status of 12 clones of the day 7–cultured cells generated by GM-CSF or GM-CSF + SCF between the PCR and sequencing method. Individual clones correspond to those shown in Figure 5a. (c) The PCR analysis for the methylation of the p15 CpG island in another 15 clones of the day 7– or day 21–cultured cells.

Next, we attempted to elucidate the methylation status of the CpG dinucleotides in the promoter region (–285 and +12 relative to the transcriptional starting point) of the p15 gene during GM-CSF–dependent myeloid differentiation. The region is known to contain some trans-acting factor binding sites. As presented in Figure 7, there was a dynamic change of methylation in the promoter region examined. All of 18 CpG sites in the promoter region were either methylated or unmethylated in individual alleles. Although we failed to determine whether allelic methylation in the promoter region synchronized with that in exon 1 at the p15 locus because of a technical difficulty, allelic methylation pattern was similar between the two regions.

Figure 7

Methylation status of the p15 promoter region during GM-CSF–dependent myeloid development from cord blood CD34⁺ cells. We examined the allelic methylation status of CpG sites in the promoter region of the p15 gene, using bisulfite genomic sequencing. Ten clones were randomly selected and sequenced at each time point. Each row of circles corresponds to one clone. Methylated CpG sites are marked as filled circles and unmethylated sites as open circles. All of the CpG sites between position –285 and +12 relative to the transcriptional point stand in a line. The binding sites of the primers used in this experiment are also shown (p15-PF and p15-PR).

Physiologic role of p15 CpG island methylation in myeloid development. Finally, we examined a role of an initial increase in CpG island methylation of the p15 gene in normal myeloid development. Forty thousand day 5–cultured cells grown with GM-CSF from cord blood CD34⁺ cells were incubated in wells containing GM-CSF with or without 5-Aza-CdR for 3 days. The frequency of alleles with methylated CpG islands was 12% on day 5 and increased to 44% on day 8 in the presence of GM-CSF, as presented in Table 2. The addition of 5-Aza-CdR at 10^–6 M canceled the GM-CSF–mediated increment of CpG island methylation, associated with an increased expression of the gene. The mean number of total viable cells was significantly lower in the culture containing GM-CSF plus 5-Aza-CdR at 10^–6 M than in the culture containing GM-CSF alone after 3 days. One-fourth of the cells expressed myeloid markers irrespective of treatment with 5-Aza-CdR. CD14^–/CD15^– cells were more immature than CD14⁺/CD15⁺ cells, based on the expression of CD34 and CD33 (Table 3). In addition, CD14^–/CD15^– cells contributed to an initial increase in p15 CpG island methylation to a greater extent than did CD14⁺/CD15⁺ cells. When day 20–cultured cells grown with GM-CSF from cord blood CD34⁺ cells or HL60 cells were target cells, the exposure to 5-Aza-CdR at 10^–6 M for 3 days did not influence the production of progeny (data not shown).

Table 2

Physiologic role of p15 CpG island methylation in myeloid development

Table 3

Surface marker expression and p15 CpG island methylation status of day 8–cultured cells grown with GM-CSF and/or 5-Aza-CdR

To elucidate whether the methylated cells had a growth advantage, we examined the correlation of the size to the methylation status of GM colonies. On day 10, ten GM colonies grown with GM-CSF + SCF from cord blood CD34⁺ cells were individually picked up and then divided into small GM colony groups and large colony groups. Small GM colonies were defined as those consisting of fewer than 1,000 constituent cells (594 ± 283 cells, n = 5) and large colonies as those consisting of more than 2,000 constituent cells (2,936 ± 1,277 cells, n = 5). The incidence of p15 CpG island methylation was significantly greater in large GM colonies (86% ± 5%) than in small colonies (54% ± 8%).

It has been demonstrated that the transcriptional repression of p15 due to cytosine methylation of the CpG island region occurs in several types of hematological malignancies, including AML and MDS (7–9). In peripheral blood MNCs of normal individuals, the p15 CpG island was initially reported to be unmethylated as determined by Southern blot analysis and PCR analysis based on restriction enzyme reaction cleavage (8). On the other hand, bisulfite genomic sequencing showed that lymphocytes of normal subjects are methylated at some CpG sites in most alleles (24, 25). This discrepancy between the two different approaches may be accounted for by an ability or an inability to assess the methylation status of each CpG site within individual alleles. Therefore, we used the latter technique in the present study. On stimulation with GM-CSF or GM-CSF + SCF, a two-phase methylation at the 5′ CpG island in exon 1 of the p15 gene was observed during the differentiation of both cord blood and bone marrow CD34⁺ cells into granulocytes and macrophages. Similar methylation emerged in the p15 promoter region in the culture with cord blood CD34⁺ cells and GM-CSF. By contrast, the p16 promoter region was entirely unmethylated in cord blood CD34⁺ cells and day 7–cultured cells grown with GM-CSF or GM-CSF + SCF (data not shown). These results suggest a close link between p15 CpG island methylation and the cytokine-dependent myeloid development from normal hematopoietic progenitors.

In cord blood CD34⁺ cells cultured with GM-CSF or GM-CSF + SCF, the percentage of the alleles with methylated p15 CpG islands increased to approximately 50% to 60% until day 7, compared with the level obtained at the beginning of culture, after which the percentage declined. On the other hand, the intracellular p15 content of the cultured cells was at negligible or low level up to day 14, but subsequently increased, according to the flow cytometric analysis and Western blot analysis. Treatment of day 5–cultured cells with 5-Aza-CdR at 10^–6 M for 3 days abrogated the ability of GM-CSF to increase the allelic methylation and caused a significant increment in the p15 expression. The mean number of total viable cells was substantially lower in the culture containing GM-CSF plus 5-Aza-CdR at 10^–6 M than in the culture containing GM-CSF alone. However, no significant difference was found in the percentages of CD14⁺/CD15⁺ cells between the two groups. These results imply that methylation of the p15 CpG island regulates negatively the p15 expression, and that the transcriptional silencing of p15 functions as a stimulator for the proliferation rather than differentiation of myeloid lineage cells. Nevertheless, we cannot rule out the involvement of genes other than the p15 gene whose expressions are regulated by promoter DNA methylation in this differentiation pathway.

It is generally held that the p15 promoter region CpG island is not methylated in normal cells and that the presence of this change is a tumor-specific event associated with an aberrant transcriptional silencing of the involved gene and loss of gene function. In the current study, one-half of the early-appearing cultured cells grown from cord blood or bone marrow CD34⁺ cells under stimulation with GM-CSF or GM-CSF + SCF displayed homogeneous methylation at all of the CpG sites in the region located between positions –12 and +251 relative to the transcriptional starting site of the p15 gene. In addition, a small number of cord blood CD34⁺ cells were methylated in the promoter region of the p15 gene at the beginning of culture, whereas normal marrow CD34⁺ cells showed no signs of the CpG island methylation. It is well known that several circulating cytokines are elevated in the neonatal period and that cord blood hematopoietic progenitors exhibit higher proliferative potential in the presence of growth factors compared with marrow progenitors. Taken together, it is possible that endogenously produced hematopoietic factors bind to the surface of cord blood CD34⁺ cells in vivo and lead to methylation of the p15 CpG island. The cytokine stimulation may be able to induce p15 promoter methylation in normal hematopoietic cells, although such methylation is absent in steady-state hematopoietic cells. Cameron et al. (25) demonstrated pronounced intra-allelic heterogeneity of methylation with varying degrees of methylation density at the CpG island within this region of the p15 gene in primary acute leukemia, implying an apparent difference in the methylation pattern of the p15 gene between normal myelopoiesis and hematological malignancies. Considering the existence of hematopoietic progenitors harboring hypermethylated p15 gene alleles, it is speculated that these cells can escape from negative growth regulatory signals emanating from p15, resulting in a proliferative advantage, and perhaps transform into a leukemic clone if combined with other defect(s).

In the mouse, most genes are subject to a process of active demethylation starting promptly after fertilization. Such genome-wide demethylation is followed by programmed de novo methylation. Gene-specific demethylations then form a terminal methylation pattern that is clonally inherited in the soma (27). The present findings suggest that a dynamic DNA methylation change is involved in the expression of the tissue-specific gene during human myeloid development as well as in murine embryo development.

We are deeply indebted to A. Komiyama (Department of Pediatrics, Shinshu University School of Medicine) for helpful comments. We also thank Y. Fukushima (Department of Clinical and Molecular Genetics, Shinshu University School of Medicine) for the helpful advice and S. Ito (Blood Transfusion Service, Shinshu University Hospital) for excellent technical assistance. This work was supported by Grants-in Aid numbers 11670753 and 09041178 from the Ministry of Education of Japan.

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