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Expression of the T-cell differentiation antigen RT6.2 in T-cell lines is correlated with hypomethylation of the RT6 promoter
Expression of the T-Cell Differentiation Antigen RT6.2 in T-Cell Lines is Correlated With Hypomethylation of the RT6 Promoter S. Rothenburg, F. Koch-Nolte, H.-G. Thiele, and F. Haag
T
HE T-cell differentiation marker RT6 belongs to the family of cell surface mono(ADP-ribosyl)transferases (ARTs), which have been implicated in the regulation of the immune response.1,2 RT6 expression is tissue-specific, being limited to mature peripheral T cells, intraepithelial lymphocytes, and a subpopulation of NK cells.3,4 RT6 is not expressed by thymocytes and recent thymic migrants. This highly regulated RT6 expression led us to investigate whether DNA methylation plays a role in the transcriptional control of RT6. Methylation is thought to be an important mechanism in the control of gene expression. This has been most clearly demonstrated in the case of genomic imprinting, where there is a causal relationship between gene silencing and methylation. Recent data directly link methylation to histone acetylation,5,6 a mechanism long known to be important in altering chromatin structure and thereby influencing gene activity.
RESULTS AND DISCUSSION
We investigated the methylation status of the RT6 promoter in two rat cell lines which differ in RT6 expression (Fig 1a); the RT6 non-expressing thymoma C58NT, and the strongly RT6.2 expressing T-cell hybridoma EpD3. DNA from these cell lines was digested with Msp I or with its methylation-sensitive isoschizomer Hpa II, separated on an 0.8% agarose gel and blotted to a nylon membrane. The membrane was probed with a 300-bp PCR fragment from the main promoter. In digests with methylation-resistant Msp I an approximately 1-kb fragment is detected in both cell lines (Fig 1b, arrow). In contrast, digestion with methylation-sensitive Hpa II reveals striking differences between the cell lines. Hpa II digestion of DNA from C58NT, which does not express RT6, produces almost no 1-kb band. Instead, the RT6 promoter is found in higher molecular weight bands, indicating promoter hypermethylation. The 1-kb band is clearly visible in Hpa II-digested DNA from EpD3 cells, which express RT6 at a high level. This suggests that at least some copies of the promoter are hypomethylated in these cells. However, the intensity of the band is reduced in comparison to the Msp I digest, and higher molecular weight bands can also be seen. This indicates that
Fig 1. RT6 expression correlates with the methylation status of the RT6 promoter. A. RT6 expression of rat T-cell lines C58NT and EpD3. Cells were analyzed for RT6 expression by flow cytometry after incubation with the RT6-specific mAb Gy1/12 and FITC-conjugated anti-rat Ig secondary antibody. B. Methylation status of the RT6 promoter. Genomic DNA (10 mg) of the indicated cell lines was digested with Msp I (M) or Hpa II (H), separated on a 0.8% agarose gel, blotted to a nylon membrane, and probed with a PCR fragment representing 2711 to 2310 relative to the transcriptional start site of the main promoter. The arrow indicates the band generated when both flanking Msp I/Hpa II sites at 215 and 21106 relative to the transcription start site are cut.
part of the EpD3 DNA is hypomethylated while another part is hypermethylated. The EpD3 hybridoma resulted from fusion of DA rat spleen cells with the C58NT thymoma, and carries both From the Institute for Immunology, University Hospital, Hamburg, Germany. This work was supported by DFG Ha2369/1. Address reprint requests to F. Haag, Institute for Immunology, University Hospital, Martinistr 52, D-20246 Hamburg, Germany.
0041-1345/99/$–see front matter PII S0041-1345(99)00062-7
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1624
Transplantation Proceedings, 31, 1624–1625 (1999)
HYPOMETHYLATION OF THE RT6 PROTOMER
parental RT6b copies.7 A likely explanation for the heterogeneity in the level of methylation is that the C58NT allele, which is clearly hypermethylated (see Fig 1b, lanes 1 and 2) remains hypermethylated in EpD3, while the transcribed DA allele is hypomethylated. Our results demonstrate that expression of RT6 is correlated with the methylation status of the RT6 promoter in the investigated cell lines, suggesting that methylation is important in regulating RT6 expression. Further studies to clarify the role of DNA methylation in RT6 expression are in progress.
1625 2. Wang J, Nemoto E, Dennert G: Adv Exp Med Biol 419:191, 1997 3. Thiele HG, Haag F, Koch-Nolte F: Adv Exp Med Biol 419:109, 1997 4. Wonigeit K, Dinkel A, Fangmann J, et al: In Haag F, Koch-Nolte F (eds): ADP-Ribosylation in Animal Tissues: Structure, Function, and Biology of Mono(ADP-Ribosyl)Transferases and Related Enzymes, New York: Plenum Press; 1997, p 229 5. Nan X, Ng HH, Johnson CA, et al: Nature 393:386, 1998
REFERENCES
6. Jones PL, Veenstra GJ, Wade PA, et al: Nature Genet 19:187, 1998
1. Koch-Nolte F, Haag F, Kastelein R, et al: Immunol Today 17:402, 1996
7. Haag F, Rothenburg S, Koch-Nolte F, et al: Transplant Proc 29:1703, 1997
T
HE T-cell differentiation marker RT6 belongs to the family of cell surface mono(ADP-ribosyl)transferases (ARTs), which have been implicated in the regulation of the immune response.1,2 RT6 expression is tissue-specific, being limited to mature peripheral T cells, intraepithelial lymphocytes, and a subpopulation of NK cells.3,4 RT6 is not expressed by thymocytes and recent thymic migrants. This highly regulated RT6 expression led us to investigate whether DNA methylation plays a role in the transcriptional control of RT6. Methylation is thought to be an important mechanism in the control of gene expression. This has been most clearly demonstrated in the case of genomic imprinting, where there is a causal relationship between gene silencing and methylation. Recent data directly link methylation to histone acetylation,5,6 a mechanism long known to be important in altering chromatin structure and thereby influencing gene activity.
RESULTS AND DISCUSSION
We investigated the methylation status of the RT6 promoter in two rat cell lines which differ in RT6 expression (Fig 1a); the RT6 non-expressing thymoma C58NT, and the strongly RT6.2 expressing T-cell hybridoma EpD3. DNA from these cell lines was digested with Msp I or with its methylation-sensitive isoschizomer Hpa II, separated on an 0.8% agarose gel and blotted to a nylon membrane. The membrane was probed with a 300-bp PCR fragment from the main promoter. In digests with methylation-resistant Msp I an approximately 1-kb fragment is detected in both cell lines (Fig 1b, arrow). In contrast, digestion with methylation-sensitive Hpa II reveals striking differences between the cell lines. Hpa II digestion of DNA from C58NT, which does not express RT6, produces almost no 1-kb band. Instead, the RT6 promoter is found in higher molecular weight bands, indicating promoter hypermethylation. The 1-kb band is clearly visible in Hpa II-digested DNA from EpD3 cells, which express RT6 at a high level. This suggests that at least some copies of the promoter are hypomethylated in these cells. However, the intensity of the band is reduced in comparison to the Msp I digest, and higher molecular weight bands can also be seen. This indicates that
Fig 1. RT6 expression correlates with the methylation status of the RT6 promoter. A. RT6 expression of rat T-cell lines C58NT and EpD3. Cells were analyzed for RT6 expression by flow cytometry after incubation with the RT6-specific mAb Gy1/12 and FITC-conjugated anti-rat Ig secondary antibody. B. Methylation status of the RT6 promoter. Genomic DNA (10 mg) of the indicated cell lines was digested with Msp I (M) or Hpa II (H), separated on a 0.8% agarose gel, blotted to a nylon membrane, and probed with a PCR fragment representing 2711 to 2310 relative to the transcriptional start site of the main promoter. The arrow indicates the band generated when both flanking Msp I/Hpa II sites at 215 and 21106 relative to the transcription start site are cut.
part of the EpD3 DNA is hypomethylated while another part is hypermethylated. The EpD3 hybridoma resulted from fusion of DA rat spleen cells with the C58NT thymoma, and carries both From the Institute for Immunology, University Hospital, Hamburg, Germany. This work was supported by DFG Ha2369/1. Address reprint requests to F. Haag, Institute for Immunology, University Hospital, Martinistr 52, D-20246 Hamburg, Germany.
0041-1345/99/$–see front matter PII S0041-1345(99)00062-7
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1624
Transplantation Proceedings, 31, 1624–1625 (1999)
HYPOMETHYLATION OF THE RT6 PROTOMER
parental RT6b copies.7 A likely explanation for the heterogeneity in the level of methylation is that the C58NT allele, which is clearly hypermethylated (see Fig 1b, lanes 1 and 2) remains hypermethylated in EpD3, while the transcribed DA allele is hypomethylated. Our results demonstrate that expression of RT6 is correlated with the methylation status of the RT6 promoter in the investigated cell lines, suggesting that methylation is important in regulating RT6 expression. Further studies to clarify the role of DNA methylation in RT6 expression are in progress.
1625 2. Wang J, Nemoto E, Dennert G: Adv Exp Med Biol 419:191, 1997 3. Thiele HG, Haag F, Koch-Nolte F: Adv Exp Med Biol 419:109, 1997 4. Wonigeit K, Dinkel A, Fangmann J, et al: In Haag F, Koch-Nolte F (eds): ADP-Ribosylation in Animal Tissues: Structure, Function, and Biology of Mono(ADP-Ribosyl)Transferases and Related Enzymes, New York: Plenum Press; 1997, p 229 5. Nan X, Ng HH, Johnson CA, et al: Nature 393:386, 1998
REFERENCES
6. Jones PL, Veenstra GJ, Wade PA, et al: Nature Genet 19:187, 1998
1. Koch-Nolte F, Haag F, Kastelein R, et al: Immunol Today 17:402, 1996
7. Haag F, Rothenburg S, Koch-Nolte F, et al: Transplant Proc 29:1703, 1997