CDCP1, a novel stem cell marker, is expressed in hematopoietic cell line K562 but not in Jurkat. When CDCP1 promoter was transfected exogenously, Jurkat showed comparable promoter activity with K562, suggesting that the factor to enhance transcription was present but interfered to function in Jurkat. The reporter assay and si-RNA-mediated knockdown experiment revealed that zfp67, a zinc-finger protein, enhanced CDCP1 transcription. Amount of zfp67 in Jurkat was comparable with K562, but chromatin immunoprecipitation showed that zfp67 bound to CDCP1 promoter in K562 but not in Jurkat. There are CpG sequences around the promoter of CDCP1, which were heavily methylated in Jurkat but not in K562. Addition of demethylating reagent to Jurkat induced CDCP1 expression, and increased the zfp67 binding to CDCP1 promoter. Among normal hematopoietic cells such as CD34+CD38− cells, lymphocytes and granulocytes, inverse correlation between proportion of methylated CpG sequences and CDCP1 expression level was found. Demethylation of CpG sequences in lymphocytes, in which CpG sequences were heavily methylated, induced CDCP1 expression and its expression level further increased through zfp67 overexpression. The methylation of DNA appeared to regulate the cell-type-specific expression of CDCP1 through the control of interaction between chromatin DNA and transcription factors.
CDCP1 is a transmembrane protein containing three extracellular CUB domains and an intracellular tyrosine kinase domain.1, 2, 3, 4, 5 This molecule was originally identified as an epithelial tumor antigen through comparison of lung cancer cell lines and normal lung tissues.1 Very recently, it was demonstrated that the transplantation of human CDCP1-positive bone marrow cells into non-obese diabetic/severe-combined immunodeficient mice gives rise to chimeric hematopoiesis, indicating the CDCP1 to be a novel stem cell marker.3, 4 In hematopoietic system, CDCP1 is expressed in CD34-positive stem cells but not in differentiated cells. To date, K562 is the only CDCP1-positive hematopoietic cell line, which is derived from the blast crisis of chronic myeloid leukemia (CML).1, 2
Recently, the correlation between stem cell marker expression in tumor cells and their malignant grade has been reported in brain tumor.6 However, the regulatory mechanism for the expression of stem cell markers remained to be elucidated. Here, by comparing two hematopoietic cell lines, the role of DNA methylation for cell-type-specific CDCP1 expression was examined.
Materials and methods
Human cell lines used in this study (K562 and Jurkat) were obtained from the American Type Culture Collection (Rockville, MD, USA), and cultured in RPMI1640 (Sigma, St Louis, MO, USA) supplemented with 10% fetal calf serum (FCS, Nippon Bio-supp Center, Tokyo, Japan). K562 are derived from CML in blast crisis and Jurkat are from T-cell leukemia. To determine the effect of methylation on CDCP1 expression, 2 μ M of demethylating reagent 5-aza deoxycytidine (5-aza dC) (Sigma) was added. Peripheral blood cells from healthy volunteers and umbilical cord blood from normal full-term deliveries were collected after informed consent. The present study was performed under the authors' institutional guidelines and was approved by the institutional review boards (No. 535). CD34+CD38− cells were purified from umbilical cord blood using FACSAria (BD, Franklin Lakes, NJ, USA). Granulocytes and lymphocytes were purified with Polymorphprep (Axis-Shield, Oslo, Norway) and lymphocyte separation medium (Cappel, Aurora, OH, USA), respectively. Monocytes were purified using the magnetic-activated cell sorter (Miltenyi Biotech, Bergish Gladbach, Germany) according to the manufacturer's guidelines.
Semiquantitative reverse transcription (RT)-PCR analysis
Total RNAs were extracted with RNeasy kit (Qiagen, Valencia, CA, USA) with DNase I treatment. Two micrograms of total RNA were subjected to reverse transcription by Superscript III (Invitrogen, Carlsbad, CA, USA), and the single-strand cDNAs were obtained. One microliter, 0.1 μl or 0.01 μl of the reverse-transcribed product was added to 25 μl of polymerase chain reaction (PCR) mixture containing 1.25 U of Taq DNA polymerase (Roche Diagnostics GmbH, Mannheim, Germany) and 25 pmol of each of the primers. The sequence of primers was as follows: 5′-IndexTermGGGCGCGCATTCATGATCATCCAGG-3′ corresponding to the sequence of exon 7 and 5′-IndexTermCCTCTGGCTGCAGGAAGGAGCCGCTGGA-3′ corresponding to the sequence of exon 9 of CDCP1 gene. The sequence of primers for β-actin was 5′-IndexTermGCCGAGCGGGAAATCGTGCG-3′ and 5′-IndexTermACGATGGAGGGGCCGGACTC-3′. PCR conditions were 30 s at 95°C, 30 s at 55°C and 1 min at 72°C for 35 or 40 cycles in CDCP1 and for 25 cycles in β-actin.
Quantification of mRNA levels by real-time RT-PCR
RNA was extracted using an RNeasy kit (Qiagen) with DNase I treatment. The mRNA levels for CDCP1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were verified using TaqMan gene expression assays from Applied Biosystems (ABI, Foster City, CA, USA) as recommended by the manufacturer. The amount of CDCP1 mRNA was normalized to that of GAPDH mRNA.
Transient transfection and luciferase assay
The PCR-amplified fragment containing promoter and a part of exon1 (nucleotide (nt) −150 to +140, +1 means a transcription initiation site) was cloned into pSP-Luc vector containing luciferase gene as reported previously.7 The reporter plasmid containing the deleted CDCP1 promoter (nt −130 to +140, nt −110 to +140 and nt −60 to +140), and the reporter plasmid mutated at the binding site for zfp67, a zinc-finger type of transcription factor,8 were also constructed by PCR. The sequence of CDCP1 fragment was verified by ABI 3100 sequencer. After the DNA isolation, 5 μg of the reporter plasmid was transiently transfected into K562 and Jurkat, together with 1 μg of the β-galactosidase control vector by using electroporation (Bio-Rad Laboratories, Richmond, CA, USA). Forty-eight hours after the transfection, a luciferase activity was measured and was normalized to β-galactosidase activity as described previously.7 The normalized value of each reporter plasmid was divided by that of reporter plasmid starting from nt −60, and the resultant value was shown as relative luciferase activity.
Small interfering RNA transfection and immunoblot
The predesigned small interfering RNA (siRNA) against zfp67 was purchased from Ambion Inc. (Austin, TX, USA). The siRNA (150 pmol) was transfected into 2 × 106 of K562 by using Amaxa cell line nucleofector kit (Amaxa Inc., Gaithersburg, MD, USA). After 72 h, cells were harvested, and lysed in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride. The resulting lysates were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred to Immobilon (Millipore, Bedford, MA, USA) and reacted with anti-zfp67 (Abcam, Cambridge, UK) or anti-actin (Sigma). After washing, the blots were incubated with an appropriate peroxidase-labeled secondary antibody, and then reacted with Renaissance reagents (NEN, Boston, MA, USA) before exposure.
Chromatin immunoprecipitation (ChIP) assay was carried out with ChIP assay kit (Upstate, Lake Placid, NY, USA) according to the manufacturer's instruction. Briefly, K562 and Jurkat were fixed with 1% formaldehyde for 10 min, washed twice with ice-cold phosphate buffer solution (PBS) and sonicated 15 times for 10 s each at power setting ‘3’ with the ultrasonicator (Tomy Seiko, Tokyo, Japan). Soluble chromatin solution was cleared by centrifugation and adjusted to 0.1% SDS, 1% Triton X-100 and 140 mM NaCl. Immunoprecipitation reactions were performed overnight with salmon-sperm DNA/protein A agarose and anti-zfp67, anti-acetyl histone H3 (Upstate) or normal rabbit IgG (for negative control, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The immunoprecipitates were washed, and reverse-crosslinked at 65°C for 6 h. DNA purified from immunoprecipitates was subjected to PCR analysis. In some experiments, the real-time PCR using Sybr-green system (ABI) was carried out. The sequence of primers for amplifying CDCP1 promoter (−248 to +214) was as follows: 5′-IndexTermCAGACTTGGGAAGGAAGACTAAGC-3′ and 5′-IndexTermAGCAGAACCCCCTAGCAGTGCGATA-3′. DNA directly purified from soluble chromatin solution was used as a positive control (input).
Bisulfite modification and DNA sequencing analysis
One microgram of genomic DNA was modified by sodium bisulfite using the CpGenome DNA modification kit (Chemicon, Temecula, CA, USA). PCR was carried out with the modified DNA as a template. The PCR reaction mixture contained 1 μl of DNA in 25 μl of total volume, which also included 2.5 μl of 10 × PCR buffer II, 5 mM MgCl2, 250 μ M deoxynucleoside triphosphate, 1 μ M of each primer and 1.25 U Taq gold. The PCR buffer II, MgCl2 and Taq gold are parts of the AmpliTaq Gold with Gene Amp kit from ABI. The sequence of the primers was as follows: 5′-IndexTermTAGATTTGGGAAGGAAGATTAAGT-3′ and 5′-IndexTermAACAACAAAACCCCTAACAATA-3′. The PCR was carried out at 95°C for 10 min, followed by 45 cycles at 95°C for 1 min, 50°C for 1 min and 72°C for 1 min. In this condition, we confirmed that both methylated and unmethylated fragments were amplified at a comparable level. The amplified PCR fragment was purified with QIAquick gel extraction kit (Qiagen), and was subcloned into pGEM-T Easy vector (Promega, Madison, WI, USA). The sequence of the amplified fragment was analyzed using an ABI 3100 sequencer (Applied Biosystems). Ten clones were analyzed for each condition.
In vitro methylation
The luciferase construct that contained promoter region of CDCP1 starting from nt −150 was incubated with SssI methylase (New England Biolabs, Beverly, MA, USA) in the presence (methylated) or absence (mock) of S-adenosylmethionine, as recommended by the manufacturer. After DNA isolation, 1 μg of the methylated or mock luciferase construct was transiently transfected into K562, together with 0.2 μg of the β-galactosidase control vector using Transfast transfection reagent (Promega). Forty-eight hours after transfection, luciferase activity was measured and normalized to the β-galactosidase activity.
Overexpression of zfp67 in lymphocytes
The coding region of zfp67 was amplified by PCR, and cloned into pIRES2-AcGFP1 vector (Clontech Laboratories, Mountain View, CA, USA). The sequence was verified with an ABI 3100 sequencer. Overexpression of zfp67 in lymphocytes was induced with Human T-cell nucleofector kit (Amaxa) according to the protocol of manufacturer's for unstimulated T cells. Thirty-two hours after transfection, cells were harvested, and RNA was extracted. A part of harvested cells was used for immunoblot to confirm the overexpression of zfp67.
The values are shown as the mean±s.e. of at least three experiments. In bisulfite sequencing, 10 clones were analyzed for each cell type, and the proportion of methylated CpG sequences is shown as the mean±s.e. of these 10 clones. Statistical comparisons were carried out using Student's t-tests.
Semiquantitative RT-PCR demonstrated that K562 expressed CDCP1 but Jurkat did not (Figure 1a). The real-time quantitative RT-PCR revealed the amount of CDCP1 mRNA in K562 to be approximately 10 000 times higher than that of Jurkat (Figure 1b), indicating that K562 was a CDCP1-positive, whereas Jurkat was a CDCP1-negative cell line. Then, these two cell lines were used to investigate the regulatory mechanism of CDCP1 expression.
First, the cis-acting motif(s) mediating CDCP1 transcription was searched using reporter assay. K562 and Jurkat were transfected with the reporter plasmid containing various length of CDCP1 promoter (Figure 2). In K562, the remarkable luciferase activity was detected when the CDCP1 promoter started from nt −150, whereas the luciferase activity decreased when the CDCP1 promoter was deleted to nt −130, −110 and −60 (Figure 2). The luciferase activity was also decreased in Jurkat by deleting the region between nt −150 and −130 (Figure 2). These findings indicated that K562 and Jurkat possessed the trans-acting factor(s) to express CDCP1, which activated the exogenously transfected CDCP1 promoter via the motif locating between nt −150 and −130.
Putative binding motifs for transcription factor were searched in the CDCP1 promoter region between nt −150 and −130 using Genomatix software (http://www.genomatix.de/index.html). One binding motif for zinc-finger transcription factor zfp67 (also called c-Krox or Th-POK)8, 9 was found between −139 and −133 (Figure 3a; consensus sequence for zfp67 is GGGAGGG). The mutation at zfp67 binding motif (GGGAGGG to GTAAGGG; the mutated nucleotides were underlined) decreased the luciferase activity in K562 and Jurkat, suggesting that zfp67 transactivated the exogenously transfected CDCP1 promoter (Figure 3b). To examine whether zfp67 mediated CDCP1 transcription in vivo, zfp67 was knocked down in K562 by siRNA (Figure 3c). The zfp67-knocked down K562 showed the lower expression of CDCP1 than the original K562 (Figure 3d), indicating that zfp67 transactivate CDCP1 promoter in K562.
The in vivo binding of zfp67 to CDCP1 promoter was examined with ChIP assay using antibody specific for zfp67. The CDCP1 promoter was enriched for zfp67 in K562 but not in Jurkat (Figure 4a). Immunoblot analysis revealed the amount of zfp67 in Jurkat to be comparable with K562 (Figure 4b), indicating that zfp67 was present in Jurkat but was interfered to bind with the CDCP1 promoter.
There are many CpG sequences around the transcription initiation site of CDCP1 gene. As the methylation at CpG sequences is known to inhibit binding of transcription factor by packing chromatin structure,10 the effect of methylation on CDCP1 expression was examined by demethylating reagent 5-aza dC. The addition of 5-aza dC for 4 days increased the expression of CDCP1 mRNA approximately 100 times in Jurkat (Figure 5a). Then, the methylation status of CpG sequences was examined by bisulfite sequencing in K562, Jurkat and Jurkat treated with 5-aza dC. K562 showed a low frequency of methylation, whereas Jurkat showed a high frequency (Figure 5b). The addition of 5-aza dC decreased the methylation frequency in Jurkat (94.1–20.9%). These findings suggest that zfp67 might be interfered to bind with CDCP1 promoter through packing of chromatin structure as a result of methylation at CpG sequences. To confirm this, the histone acetylation status, which reflects the chromatin decondensation,10 was examined. ChIP assay revealed CDCP1 promoter to be enriched for the acetylated histone H3 in K562 but not in Jurkat (Figure 6). The addition of 5-aza dC induced the enrichment of CDCP1 promoter for acetylated histone H3 in Jurkat, indicating that 5-aza dC caused the chromatin decondensation. The binding of zfp67 to CDCP1 promoter was also induced in Jurkat by the addition of 5-aza dC (Figure 6).
To confirm the inhibitory effect of CpG methylation on CDCP1 expression, in vitro methylation of CDCP1 promoter was performed. The luciferase construct containing CDCP1 promoter was methylated in vitro using SssI methylase. The methylated or mock-methylated promoter construct was transiently transfected into K562, and luciferase activity was measured. The methylated promoter construct, but not the mock-methylated one, was resistant to digestion with methylation-sensitive restriction enzyme HpaII (Figure 7a), which confirmed that the CpG sequences of methylated construct were completely methylated in vitro. When transfected into K562, luciferase activities for the mock-methylated CDCP1 promoter were higher than those for methylated one (Figure 7b).
The relationship between methylation status of CpG sequences and CDCP1 expression level was examined in normal hematopoietic cells. The real-time quantitative RT-PCR revealed that the amount of CDCP1 mRNA in CD34+CD38− cells was approximately 100 times higher than that of granulocytes, lymphocytes and monocytes (Figure 8a). Proportion of methylated CpG was low in CD34+CD38− cells, whereas that in granulocytes, lymphocytes, and monocytes was high (Figure 8b). The addition of 5-aza dC to lymphocytes induced the CDCP1 expression, which was further increased through the overexpression of zfp67 (Figure 9a and b).
CDCP1 was expressed in K562 but not in Jurkat, showing a cell-type-specific expression in hematopoietic cell lines. K562, derived from the blast crisis of CML, can differentiate into erythrocytes or megakaryocytes by stimulation with hemin11 or phorbol ester.12 Jurkat express T-cell lineage markers, and thus appears to be more differentiated cell line than K562. Therefore, present results were consistent with the report by Buhring et al.4 that CDCP1 is expressed only in the undifferentiated state of hematopoietic cells.
The exogenously transfected CDCP1 promoter was activated in Jurkat and K562, but CDCP1 was expressed in K562 but not in Jurkat. This might be owing to failure of trans-activating factor to activate CDCP1 transcription in Jurkat. By reporter assay and siRNA experiment, the trans-activating factor to express CDCP1 was demonstrated to be zfp67. As expected, zfp67 existed in Jurkat. ChIP assay revealed that zfp67 bound to CDCP1 promoter in K562 but not in Jurkat. It is well known that the packing of chromatin structure inhibits the binding of transcription factors, and the packing of chromatin structure is enhanced by methylation of CpG sequences and deacetylation of histone.10 The proportion of methylated CpG around CDCP1 promoter was high in Jurkat but low in K562. In addition, the histone around CDCP1 promoter was highly acetylated in K562 but not in Jurkat. These findings indicate that the chromatin was packed in Jurkat, which inhibits the binding of factor to enhance CDCP1 transcription. In fact, demethylation of CpG sequences induced the binding of zfp67 and increased the expression level of CDCP1 in Jurkat.
Role of methylation on CDCP1 expression was examined in normal hematopoietic cells. As reported by Buhring et al.,4 CD34+CD38− cells, possible human hematopoietic stem cells, abundantly expressed CDCP1. In contrast, mature hematopoietic cells such as granulocytes, lymphocytes and monocytes hardly expressed CDCP1. This was also consistent with the previous report.4 The proportion of methylated CpG around CDCP1 promoter was high in mature hematopoietic cells but low in stem cells, showing the inverse correlation between CDCP1 expression level and proportion of methylated CpG sequences. Demethylation of CpG sequences in lymphocytes by 5-aza dC treatment induced CDCP1 expression, and its level increased through zfp67 overexpression. These findings indicated the similar regulatory mechanism of CDCP1 expression in hematopoietic cells to that in K562 and Jurkat.
Buhring et al.4 demonstrated that CDCP1 is expressed on cells phenotypically identical to mesenchymal stem/progenitor cells and neural progenitor cells. Thus, CDCP1 is not only a novel marker for immature hematopoietic progenitor cell subsets but also for cells with phenotypes reminiscent of stem/progenitor cells with non-hematopoietic lineages. Besides CDCP1, other stem cell markers of both hematopoietic and non-hematopoietic lineage cells, such as Nanog and Oct-4, possess CpG sequences around the transcription initiation site.13 Although the relationship between transcription factors and the CpG methylation status in Nanog and Oct-4 gene has not been demonstrated as yet, Deb-Rinker et al.13 recently reported that their CpG sequences were sequentially methylated as differentiation proceeds in human embryonal carcinoma cell line, resulting in the decrease of expression. As demonstrated in the present study, the epigenetic regulation of stem cell marker expression might be a common mechanism for gene silencing in the process of differentiation of hematopoietic cells.
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We thank Mr M Kohara, Ms M Sugano and Ms T Sawamura (Osaka University) for technical supports. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, and from the Osaka Cancer Research Foundation.
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Kimura, H., Morii, E., Ikeda, J. et al. Role of DNA methylation for expression of novel stem cell marker CDCP1 in hematopoietic cells. Leukemia 20, 1551–1556 (2006) doi:10.1038/sj.leu.2404312
- transcriptional regulation
- stem cell marker
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