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Gene During In Vitro Differentiation of Human Peripheral Blood T Cells into a Th2 Population
Lymphocyte Cell Biology Section, Laboratory of Immunology, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224
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, resulting in a cellular response that helps to eliminate infected cells. In contrast, Th2 cells produce IL-4, IL-5, IL-6, and IL-10, stimulating an Ab response that attacks extracellular pathogens, thereby preventing the cells from becoming infected. To elucidate the mechanisms of differential regulation of cytokine genes by these two different subsets of T cells, we established an in vitro differentiation model of freshly isolated human peripheral blood T cells in which IFN- was used as an index gene to study the transcriptional regulation. The data presented here demonstrate that the IFN- promoter undergoes differential methylation during in vitro differentiation: the promoter becomes hypermethylated in Th2 cells, whereas it is hypomethylated in Th1 cells. Hypermethylation in Th2 cells results in chromatin condensation and exclusion of CREB proteins from the IFN- promoter. Treatment with 5-azacytidine, a demethylating agent, causes Th2 cells to reverse histone condensation and enables CREB recruitment to the hypomethylated promoter. This results in the increased production of IFN-. These data indicate the importance of promoter methylation in the regulation of the IFN- gene during differentiation. |
Introduction |
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, IL-2, and lymphotoxin, and are responsible for cellular immunity; and Th2 cells, which produce IL-4, 5, 6, and 10, and are responsible for humoral immunity (1). The development of these subsets of T cells is dictated by the cytokines present in the environment during differentiation: IL-12 and IFN- dictate Th1 differentiation, whereas IL-4 dictates Th2 differentiation (2). IL-12, largely produced by APCs, activates the transcription factor Stat4 and, accordingly, Stat4-deficient mice have impaired Th1 differentiation (3, 4, 5, 6). T-bet, a member of the T box transcription factor family, is expressed specifically in Th1 cells and is considered to be the master regulator of both IFN- production and Th1 differentiation (7, 8, 9). On the other hand, IL-4 promotes Th2 development by activating Stat6 and Th2-specific factors GATA-3 and c-Maf (10, 11, 12, 13, 14, 15).
The production of IFN- is restricted to T cells and NK cells. It is well established that methylation of DNA is an epigenetic mechanism for the modulation of gene expression in mammalian cells (16). DNA methylation changes chromatin structure and possibly impedes the recruitment of transcription factors to the target genes (17). In fact, certain transcription factors (i.e., CREB) bind with a low affinity to the methylated recognition elements, and this reduction in affinity has been correlated to the low promoter activity (18). We have previously shown that in long-term murine clones, the promoter region of IFN- was hypomethylated in Th1 clones, whereas the promoter was hypermethylated in Th2 clones (19). We and others have shown that the methylation-sensitive promoter region is recognized by CREB/activating transcription factor, AP-1, and octamer families of transcription factors (20, 21). In vitro methylation of this site markedly reduced the binding of these transcription factors (19, 21).
In the present study, we have addressed the role of methylation in the regulation of the IFN- gene during in vitro differentiation of human peripheral blood T cells. Although the methylation status of the IFN- gene has been shown to correlate with its expression (22, 23, 24, 25), no attempt has been made to investigate the effect of methylation on the chromatin condensation and the recruitment of transcription factors to the promoter. We have examined the acetylation status of histone H3 and H4 as an indicator of transcriptionally active chromatin, as well as the recruitment of two important transcription factors, CREB and c-Jun, in the context of genomic DNA by the chromatin immunoprecipitation assay (ChIP) during in vitro differentiation. The data presented here demonstrate that promoter methylation plays an important role in IFN- gene expression during in vitro differentiation of a Th2 population.
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Materials and Methods |
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PBMC (collected from healthy National Institutes of Health Blood Bank donors) were isolated by Ficoll-Hypaque density gradient centrifugation. Total resting T cells or CD4+ T cells were purified from PBMC with a Human T Cell Enrichment Column kit or Human CD4 Subset Column kit (R&D Systems, Minneapolis, MN). T cells were >95% CD3+ cells.
Differentiation of pTh cells into Th1 and Th2 cells
Culture flasks were prepared for differentiation by coating with 20 µg/ml rabbit anti-mouse IgG solution (Zymed Laboratories, South San Francisco, CA), 200 ng/ml anti-CD3, and 1 µg/ml anti-CD28 (BD PharMingen, San Diego, CA). T cells were cultured in clone medium: complete RPMI 1640 medium with 10% FCS, 2% human serum, 50 µM 2-ME, 1 mM sodium pyruvate, 2 mM glutamine, 1x nonessential amino acids, 100 µg/ml gentamicin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 20 mM HEPES. pTh cells were seeded at a density of 2.5 x 106 cells/ml into the culture flask supplemented with 1 µg/ml anti-IL-4 (clone 34019.111, IgG2b; R&D Systems) and 1 ng/ml IL-12 (R&D Systems) to differentiate into Th1 cells, and 1 µg/ml anti-IFN- (clone 25718.11, IgG2b; R&D Systems) and 20 ng/ml IL-4 (R&D Systems) to differentiate into Th2 cells. The Th cells were split once (1/5) with the clone medium supplemented with 10 U/ml rIL-2 (Teceleukin; Hoffmann-LaRoche, Nutley, NJ).
Cytokine measurement
Th cells were harvested at day 5 (D5) and day 7 (D7) after initial activation. The cells were then washed with complete RPMI 1640 and were cultured either with medium alone (control) or with PMA (10 ng/ml) and ionomycin (1 µg/ml). After 24 h, supernatants were collected and analyzed for cytokine production using a Cytometric Bead Array kit (BD Biosciences, San Diego, CA) according to the manufacturer’s instructions.
DNA isolation and Southern blot analysis
Genomic DNA was extracted from human T cells using a Qiagen DNA Blood Midi kit (Qiagen, Valencia, CA). Extracted DNA (10 µg) was digested overnight with 50 U of PvuII and 25 U of SnaBI at 37 °C. The digested DNA was subjected to 1% agarose gel electrophoresis and transferred to a nylon membrane (NEN Research Products, Boston, MA). After UV cross-linking, the membrane was baked for 1 h at 80 °C. A DNA probe consisting of the first exon of the IFN- gene was prepared from a PCR product amplified with the primers: sense, 5'-TATAAATACCAGCAGCCAGAGGAG-3' and antisense, 5'-TCACAACTGAATGAGTTCCC-3'. The PCR fragment was labeled with [32P]dCTP by random oligonucleotide priming. The membrane was hybridized with the probe using Church’s hybridization buffer (National Institutes of Health Media Unit, Bethesda, MD). To determine the effect of a demethylating agent on Th2 cells, 2 µM 5-azacytidine was added to the culture at the beginning of the differentiation. Cells were harvested after 72 h and were restimulated with PMA and ionomycin (P/I) for 24 h for the IFN- measurement.
ChIP assay
The recruitment of transcription factors to the IFN- gene was examined by ChIP assay. The ChIP assay was performed using the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY). The cells were stimulated with or without P/I for 4 h and were then washed with complete RPMI 1640. Cells (1 x 106) were cross-linked with 1% formaldehyde for 10 min at 37 °C. The cross-linking reaction was terminated by the addition of glycine to a final concentration of 125 mM for 5 min. The cells were washed twice with ice-cold PBS and were lysed with 200 µl of warm SDS lysis buffer (Upstate Biotechnology catalogue no. 20-163). The samples were sonicated using a Cell Disruptor 350 (Sonifier) on ice for a total of 10 cycles per sample to generate DNA fragments ranging from 200 to 1000 bp in length. The supernatant from the sonicated samples was diluted 10-fold in ChIP dilution buffer (Upstate Biotechnology catalogue no. 20-153). An aliquot from each sample was collected for an input positive control. After incubating the supernatants with salmon sperm DNA/protein A agarose slurry (Upstate Biotechnology catalogue no. 16-157) for 30 min at 4°C, the following Abs were added for immunoprecipitation: anti-c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA; catalogue no. sc: 44), anti-CREB (Santa Cruz Biotechnology; catalogue no. sc: 186), anti-phospho-CREB (Ser133) (Upstate Biotechnology catalogue no. 06-519), anti-acetyl-histone H3 (Lys9 and Lys14) (Upstate Biotechnology catalogue nos. 07-352 and 07-353), and anti-acetyl-histone H4 (Upstate Biotechnology catalogue no. 06-866). The immunoprecipitation was done at 4°C overnight with rotation. The immune complexes were collected and the beads were washed five times with the wash buffer. The immune complexes were eluted with freshly prepared elution buffer (1% SDS and 0.1 M NaHCO3). The cross-linking was reversed by heating at 65°C for 4 h in 500 µl of elution buffer containing 20 µl of 5 M NaCl. Proteins were degraded by adding 10 µl of 0.5 M EDTA, 20 µl of 1 M Tris-HCl, and 2 µl of 10 mg/ml proteinase K followed by incubation for 1 h at 45°C. DNA was recovered by phenol/chloroform extraction and ethanol precipitation in the presence of 20 µg of glycogen. Pellets were resuspended in 20 µl of TE (10 mm Tris and 1 mM EDTA). PCR were performed for 35 cycles with 2 µl of DNA sample. PCR cycles were optimized to ensure a linear range of amplification. The DNA samples were amplified with the following primer pairs: sense, 5'-TGCCTCAAAGAATCCCACC-3' (nt 105–123) and antisense, 5'-CAGTAACAGCCAAGAGAACC-3' (nt 542–523). The amplified reaction was electrophoresed on a 1% agarose gel and stained with ethidium bromide.
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Results |
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To examine the regulation of IFN- gene expression during the differentiation of pTh cells into Th1 and Th2 cells, we established an in vitro differentiation system using freshly isolated human peripheral blood T cells. As shown in Fig. 1a, successful differentiation resulted in polarized T cell populations: Th1 cells produced much more IFN- than did Th2 cells on both days 5 and 7, whereas these cells produced much less IL-4, IL-5, and IL-10 compared with Th2 cells. With regard to IL-4 production, the difference between Th1 and Th2 cells was much more pronounced at day 7 than at day 5. This could be due to the fact that the IL-4-producing cells require a higher number of cell divisions compared with the IFN--producing cells (6). Similar results were also obtained from the isolated CD4+ T cells (Fig. 1b).
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gene in differentiated Th cells
Using established murine clones, we have previously shown that the methylation status of a CpG dinucleotide contained within a TATA proximal regulatory element of the IFN- promoter correlated with the transcription of the gene (19). Th1 clones were hypomethylated at the promoter regions of the IFN- gene, whereas Th2 clones were hypermethylated at the same regions (19). We wanted to know whether freshly differentiated Th cells follow the same methylation pattern. To determine this, we took advantage of the presence of a methylation-sensitive restriction cut site of the enzyme SnaBI. As shown in the schematic of Fig. 2a, the digestion of the chromosomal DNA by the combination of PvuII and SnaBI will produce a DNA fragment of 6.7 kb in Southern blot analysis, if the SnaBI site is methylated. On the other hand, if the SnaBI site is unmethylated, the same digestion will produce a DNA fragment of 2.6 kb. As shown in Fig. 2a, the pTh cells produced both 6.7- and 2.6-kb fragments. To determine the degree of hypo- or hypermethylation, we measured the intensity of the bands obtained in the Southern blot analysis and expressed the results as a percentage of hypomethylation using the following formula: percent hypomethylation = density of 2.6-kb fragment/(densities of 6.7- plus 2.6-kb fragment). As shown in Fig. 2b, pTh cells had 65% of the promoter hypomethylated (Fig. 2b). At day 5, DNA from Th1 cells had 67% hypomethylation, whereas 23% of the DNA was hypomethylated in Th2 cells. The high degree of hypomethylation in pTh cells and an absence of inducible hypomethylation in Th1 cells could be due to the fact that total resting peripheral blood T cells instead of naive T cells were used in these experiments. A similar difference in the degree of hypomethylation was also obtained at day 7: DNA from Th1 cells had 57% hypomethylation compared with 34% hypomethylation in Th2 cells. To correlate the status of promoter hypomethylation to IFN- gene transcription, we treated Th2 cells with a demethylating agent, 5-azacytidine, during the differentiation process. As shown in Fig. 2c, Th2 cells treated with 5-azacytidine produced a higher amount of IFN- compared with untreated Th2 cells. Interestingly, 5-azacytidine treatment caused a 2-fold increase in the degree of hypomethylation (Fig. 2, d and e), boosting hypomethylation to approximately the same degree as IFN- production. These data suggest that the methylation status of the promoter correlates with IFN- gene transcription during T cell differentiation.
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Next, we wanted to identify the transcription factors that were differentially recruited to the methylation-sensitive promoter region of the IFN- gene during T cell differentiation. It has been shown that both CREB and c-Jun proteins recognized the methylation-sensitive promoter region by the EMSA (20, 21). Based on these results, we decided to assess the recruitment of CREB and c-Jun to the IFN- promoter region by ChIP assay. Besides CREB and c-Jun, we also examined the status of acetylated histones H3 and H4 to determine the activation status of the chromatin, since the acetylation of histones H3 and H4 has been correlated to the transcriptionally active chromatin (17, 26). T cells were differentiated as before for different periods of time and, at each time point, cells were restimulated and subjected to the ChIP assay as described in Materials and Methods. As shown in Fig. 3a, the degree of acetylation of histones H3 and H4 was greater in Th1 than in pTh and Th2 cells at day 3, indicating that the methylation-sensitive IFN- promoter was more active transcriptionally in Th1 than in pTh and Th2 cells. Interestingly, 5-azacytidine treatment of Th2 cells caused more acetylation of histone H4, indicating that the methylation status of the promoter might be connected to the transcriptional activity of the IFN- gene (Fig. 3b). Input controls demonstrated that equivalent amounts of materials were added to each immunoprecipitation (Fig. 3). In the case of CREB proteins, the recruitment to the promoter region was significantly higher in Th1 cells as compared with pTh and Th2 cells (Fig. 3c). This was also true for phospho-CREB; the degree of recruitment was higher in Th1 cells as compared with pTh and Th2 cells. Again 5-azacytidine treatment of Th2 cells caused more recruitment of phospho-CREB to the promoter, indicating the positive role of phospho-CREB in the differential regulation of IFN- gene expression in Th1 cells. In repeated experiments, we observed that 5-azacytidine treatment of Th2 cells did not cause any increased recruitment of total CREB protein to the promoter. Whether the 5-azacytidine treatment interfered with the immunoprecipitation with the anti-CREB Ab is currently under investigation. However, the recruitment of c-Jun was found to be the same in both Th1 and Th2 cells, and 5-azacytidine treatment of Th2 cells did not affect the level of c-Jun recruitment (Fig. 3b). These findings suggest that the in vitro differentiation of pTh cells to Th1 cells results in a higher degree of acetylation of histones H3 and H4 as well as increased recruitment of CREB to the methylation-sensitive IFN- promoter. In contrast, both acetylation of histones H3 and H4 and CREB recruitment to the promoter are suppressed during in vitro differentiation of pTh cells to Th2 cells.
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Discussion |
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gene during in vitro differentiation of human peripheral blood T cells to a Th2 population. The promoter became hypermethylated during differentiation to Th2 cells, whereas the promoter remained hypomethylated during Th1 differentiation. The degree of promoter methylation correlated with the production of IFN- as demonstrated by the fact that treatment of Th2 cells with a demethylating agent, 5-azacytidine, caused an increase in promoter hypomethylation and a subsequent increase in IFN- production. The inhibitory effect of the promoter methylation on the transcription of the IFN- gene was also demonstrated by the ChIP assay where acetylation of histones H3 and H4 was substantially lower in Th2 cells as compared with Th1 cells, and phospho-CREB was excluded from the IFN- promoter in Th2 conditions. Interestingly, treatment with the demethylating agent increased the degree of acetylation of histone H4 and increased the recruitment of phospho-CREB to the promoter in Th2 cells. Acetylation of histones plays a crucial role in chromatin remodeling and gene transcription (17, 27). N-terminal acetylation of histones decreases its affinity for DNA, which in turn might allow the termini to be dislodged from the nucleosome, rendering nucleosomes open in conformation and allowing access to transcription factors (28). DNA methylation could interfere with transcription by multiple mechanisms (29). One direct mechanism may be blocking the binding of both the basal transcription machinery and ubiquitous transcription factors to promoters. As an example, the transcription factor CREB binds with low affinity to the methylated recognition elements and this reduced binding affinity has subsequently been shown to be linked to low promoter activity (18).
Differential methylation of the IFN- gene has been closely associated with IFN- expression (22, 23, 24, 25). Katamura et al. (25) have shown that the inhibitory effect of prostaglandin E2 and IL-4 on the production of IFN- by in vitro-differentiated CD4+ cells was due to the promoter methylation of the IFN- gene. The low level of IFN- production by naive CD4+ T cells from human cord blood has also been shown to be due to the hypermethylation of both CpG and non-CpG sites within and adjacent to the IFN- promoter (24). Mikovits et al. (23) have demonstrated that promoter methylation is responsible for the dysregulation of IFN- gene expression following HIV-1 infection of human peripheral blood T cells. Using long-term murine clones, we have previously shown that the differences in IFN- expression in Th1 and Th2 clones have been associated with the differential CpG methylation in this gene (19). Even with all of this information, the mechanism by which CpG methylation controls gene transcription is not well understood. In this report, we investigated whether the differential methylation of the IFN- promoter was closely associated with chromatin condensation and the transcription factor exclusion of the promoter during in vitro differentiation of human peripheral blood T cells to Th1 and Th2 populations. The data presented here clearly demonstrated that the IFN- promoter underwent differential methylation during in vitro differentiation to a Th2 population. Interestingly, the promoter became hypermethylated during differentiation of pTh cells to Th2 cells and the inhibition of this inducible methylation by 5-azacytidine allowed Th2 cells to produce more IFN- compared with the untreated cells. In contrast, the hypomethylation status of the IFN- promoter remained the same during differentiation to Th1 cells. The high degree of hypomethylation in pTh cells and the lack of increase in hypomethylation during differentiation to Th1 cells could be due to the fact that total resting peripheral blood T cells instead of naive T cells were used in these experiments. The degree of acetylation of histones H3 and H4 was also found to be differentially regulated during the in vitro differentiation process. Since the status of histone acetylation is an accepted indicator for transcriptionally active chromatin (17, 26), data presented here suggest that the IFN- promoter is transcriptionally less active in pTh and Th2 cells as compared with Th1 cells. The decreased activity of the IFN- promoter in Th2 cells could be due to the hypermethylation of the promoter, since treatment with the demethylating agent increased the degree of histone H4 acetylation.
The consequences of promoter methylation were also observed in the recruitment of transcription factors to the responsive regions. Among several transcription factors that have been shown to recognize the methylation-sensitive promoter region of the IFN- gene (20, 21), we examined the status of CREB and c-Jun by ChIP assay. The recruitments of phospho-CREB and CREB to the promoter were significantly lower in Th2 cells as compared with Th1 cells (Fig. 3c). Of much interest was that demethylation of the promoter by 5-azacytidine treatment of Th2 cells caused increased recruitment of phospho-CREB to the promoter. The lack of CREB recruitment to pTh cells despite its hypomethylated promoter is intriguing. One possibility is that CREB undergoes modification necessary for its DNA recognition during the differentiation process. Although we cannot explain the CREB exclusion from the IFN- promoter under pTh conditions, the CREB exclusion under Th2 conditions is in agreement with the fact that CREB proteins bind less well to methylated recognition elements. These data also suggest a positive role of CREB in the regulation of IFN- gene transcription. The role of CREB in the transcriptional regulation of the IFN- gene remains controversial. Some reports have indicated a negative role of CREB in IFN- transcription (28, 29), whereas some other reports have indicated a positive role (20, 30). However, some of these studies have been conducted either in the Jurkat T cell line or in transgenic mice; their relevance to the human system is uncertain. During in vitro differentiation of human peripheral blood T cells, the higher levels of phospho-CREB recruitment to the IFN- promoter of Th1 cells in the context of genomic DNA coupled with increased IFN- production strongly suggests a positive role for CREB in IFN- transcription.
The role of c-Jun in the regulation of the IFN- gene has been proposed to be a positive one (20, 21). The methylation-sensitive promoter region of IFN- has been shown to be recognized by c-Jun as a c-Jun/activating transcription factor 2 heterodimeric complex (21). In transient transfection assays, the expression of c-Jun plasmids containing either a DNA-binding domain mutation or activation domain mutations inhibited IFN- promoter function (20, 21). These data suggest a positive role for c-Jun in IFN- gene transcription through its binding to the promoter region. Interestingly, in the ChIP assay, the extent of c-Jun recruitment to the promoter region was found to be the same in the Th1 and Th2 populations. These data indicated that the recruitment of c-Jun was insensitive to promoter methylation, which was in agreement with the fact that 5-azacytidine treatment of Th2 cells did not affect the level of recruited c-Jun (Fig. 3b).
In summary, our data demonstrate that the methylation of the promoter plays an important role in the regulation of the IFN- gene during in vitro differentiation of human peripheral blood T cells to Th2 populations. The methylation of the promoter might prevent the recruitment of acetylase to the local chromatin, thereby inhibiting acquisition of the open conformation suitable for the recruitment of transcription factors. During the preparation of this manuscript, Messi et al. (31) have described the role of histone acetylation in the regulation of polarized cytokine gene expression during priming of CD4+ T cells under Th1 and Th2 conditions. The present data also indicate a positive role of CREB for IFN- transcription, which is in agreement with the recently published report describing the reduced levels of IFN- production in patients infected with Mycobacterium tuberculosis due to the reduced expression of CREB proteins (30). In contrast, c-Jun was also capable of binding the promoter but no differential binding was noted in high-producing Th1 cells or low-producing Th2 cells. Furthermore, manipulations that enhanced IFN- production from Th2 cells (5-azacytidine) did not affect c-Jun binding. Thus, c-Jun may be required for IFN- gene transcription but it is not involved in modulating the level of expression.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: pTh, precursor T cells; ChIP, chromatin immunoprecipitation; P/I, PMA and ionomycin.
Received for publication December 10, 2002. Accepted for publication July 2, 2003.
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References |
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loci accompany Th1/Th2 differentiation. J. Immunol. 169:647. promoter function in disease due to an intracellular pathogen. J. Immunol. 168:3520.This article has been cited by other articles:
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) and Th2 (
) cells from pTh cells. a, T cells were harvested at day 5 (D5) and day 7 (D7) after in vitro differentiation as described in Materials and Methods. Cells were then cultured either with medium alone (control) or with P/I for 24 h, and culture supernatants were collected and analyzed for IFN-
