Proc. Natl. Acad. Sci. USA Vol. 86, pp. 4195-4199, June 1989 Immunology Chromatin configuration of the human CD2 gene locus during T-cell development (gene regulation/ differentiation/DNase I hypersensitivity/DNA methylation) D. WOTTON, B. F. FLANAGAN, AND M. J. OWEN Imperial Cancer Research Fund, St. Bartholomew's Hospital, Dominion House, Bartholomew Close, London, EC1A 7BE, United Kingdom Communicated by Walter Bodmer, February 3, 1989 (receivedfor review December 21, 1988)

ABSTRACT To investigate the molecular basis for the tion-independent, and copy number-dependent expression of tissue-specific expression ofthe human CD2 gene, its chromatin the human CD2 gene in transgenic mice after introduction of configuration was assessed by determin*ig DNase I hypersen- a 28.5-kilobase (kb) genomic fragment suggests that most, if sitivity and the degree of methylation during T-cell lineage not all, ofthe CD2 regulatory sequences are contained within commitment and development. Tissue-specific DNase I-hyper- this region of DNA (15). The introduction into the mouse sensitive sites were found within the 5' promoter region and a germ line of a CD2 gene lacking different regions has defined region 3' of the gene essential for . DNase I a 3' region that is absolutely required for tissue-specific hypersensitivity of the 5' region correlated strictly with tran- expression (17). scriptional activity, whereas hypersensitivity of the 3' region In this study we have determined variations in DNase I correlated with T-cell progenitor activity or lineage commit- hypersensitivity and methylation patterns associated with the ment but not necessarily with transcription. Hha I and Hpa II human CD2 gene during T-cell lineage commitment and sites around the 5' and 3' regions were undermethylated in development. The results define areas both 5' and 3' of the CD2-expressing T cells but were more extensively methylated gene that are likely to control its expression. Furthermore, in other cell types. These results derme likely regulatory differences in hypersensitivity and methylation during devel- elements both upstream and downstream of the CD2 gene that opment suggest that the CD2 gene possesses an open chro- control its tissue-specific expression. Further, they show that matin configuration and is poised for transcription prior to the 3' regulatory region adopts an open chromatin configura- T-cell lineage commitment and subsequent expression. tion prior to lineage commitment and during early stages of T-cell development before the CD2 gene is transcribed. MATERIALS AND METHODS Cell Culture. All cell lines were grown in RPMI 1640 T- development occurs entirely within the thy- medium supplemented with L-glutamine and 10% fetal bovine mus and is characterized by the expression of a variety of serum in 5% CO2 at 370C except for KG1, KGla, and KG1b, tissue-specific genes in a defined temporal sequence (1). The where the serum concentration was increased to 20%. In- tissue-specific and temporal expression of these genes is duction of KG1 cells toward the macrophage lineage was likely to be controlled, by analogy with other systems studied achieved by incubation for 4 days in medium containing (2-4), by the initial local unfolding of the chromatin regions ionomycin (1.6 ttg/ml) and phorbol 12,13-dibutyrate (0.1 followed by the interaction of trans-acting factors (some of AM). which are cell type-specific) with cis-acting regulatory ele- DNase I Hypersensitivity. Cells (2-5 x 108) were swollen on ments to form a functional transcription complex. ice for 30 min by incubation in hypotonic buffer (IB: 10 mM A moleculargenetic description ofthe steps involved inT-cell Tris'HCl, pH 7.5/10 mM NaCl/5 mM MgCl2), lysed with lineage commitment and development within the thymus re- Nonidet P-40, and immediately disrupted by Dounce homog- quires a knowledge of the regulatory elements that control enization with a loose pestle. The nuclei were centrifuged tissue-specific and temporal expression ofT-cell-specific genes. through an 8.5% (wt/vol) sucrose cushion and resuspended One frequent indication of the position of such regulatory sites in 1 ml of IB per digestion. Nuclei were then digested with is the existence ofDNase I-hypersensitive areas and regions of various amounts of pancreatic DNase I (0, 0.6, 0.8, 1.0, 1.5, undermethylation within genes that are actively transcribed (5- 2.0, 2.5, 3.0, 3.5, 4.5, and 5.5 pg). After a 5-min incubation 7). These regions have been shown to be correlated with binding at 37°C, 1 ml of 1% SDS in STE (100 mM NaCl/50 mM sites for trans-acting factors (8-10). Thus, an analysis ofDNase Tris HCl, pH 8.0/1 mM EDTA) was added to terminate the I hypersensitivity and methylation patterns of a T-cell-specific reaction and lyse the nuclei. Twenty microliters of each gene should define regions controlling its expression. sample was electrophoresed overnight in a 0.8% agarose gel. The CD2 gene is expressed almost exclusively in hemato- DNA from the untreated nuclei and from five samples that poietic cells ofthe T lineage, transcription first being detected ranged from >20 kb to a median of -10 kb was digested with shortly after entry of progenitor cells into the thymus and proteinase K overnight at 37°C, extracted with phenol/ being maintained throughout later stages of T-cell develop- chloroform, and precipitated with ethanol. Twenty micro- ment (11). The CD2 may function within the thymus grams of each sample was digested overnight with a 5-fold to provide proliferation signals associated with the acquisi- excess of EcoRI or BamHI. tion of major histocompatibility complex (MHC) restriction CpG Methylation. DNA was extracted from 1-5 x 107 cells and tolerance and in the peripheral compartment to amplify prior to digestion of 20 Ag with Hpa II, Msp I, or Hha I in the signal transduced by the T-cell antigen receptor (12-14). conjunction with BamHI, EcoRI, or Stu I. The organization of the human and murine CD2 genes has Southern Blotting Analysis. Restriction enzyme-digested recently been elucidated (15, 16). The tissue-specific, posi- DNA (20 ,g per digestion) was electrophoresed in 0.8% agarose, and the separated fragments were transferred to The publication costs of this article were defrayed in part by page charge GeneScreenPlus nylon membrane (NEN-Du Pont) and hy- payment. This article must therefore be hereby marked "advertisement" bridized overnight at 650C with 32P-labeled probes (specific in accordance with 18 U.S.C. §1734 solely to indicate this fact. activity, >108 dpm/,g) in 1 M NaCI/1% SDS/10% (wt/vol) 4195 Downloaded by guest on September 28, 2021 4196 Immunology: Wotton et al. Proc. Natl. Acad. Sci. USA 86 (1989)

EcoRI SspI Ndel Stul EcoRI corresponding to the genomic fragment containing the first and second exons (see Fig. 6), was detected in BamHI- I______5, digested DNA from untreated nuclei (Fig. 2). Two additional bands of 2.3 and 0.8 kb hybridized in DNA isolated from 0 1000 1500 DNase I-treated nuclei from CD2-expressing cell lines. These bp I bands corresponded to DNase I-hypersensitive sites situated AlI I B 1.5 kb and 50 bp 5' to the transcriptional initiation site of the ClIi CD2 gene. These hypersensitive sites were detected only in CD2-expressing cells (Fig. 2). FIG. 1. Summary of probes used (A-C). A scheme for the Probe B (Fig. 1) was used to assay for DNase I- full-length CD2 cDNA clone is shown (solid bar depicts coding hypersensitive areas at the 3' end of the CD2 gene. Hybrid- region). Subfragments, and the restriction enzyme sites used to a generate them, are shown. Arrows indicate exon boundaries. bp, ization of EcoRI-digested DNA with this probe revealed Base pairs. 20-kb fragment in DNA from untreated nuclei and an addi- tional 3.6-kb subfragment, which identified a hypersensitive dextran sulfate/50 mM Tris HCl, pH 7.5/1 mM EDTA con- site about 0.5 kb 3' to the polyadenylylation site of the gene, taining salmon sperm DNA at >100 ug/ml. Blots were in DNA from DNase I-treated nuclei (Fig. 3). This site was washed once at room temperature with 2x SSC (lx is 0.15 present in all T cells analyzed and in KG1, KGla, and KGlb M NaCl/0.015 M sodium citrate, pH 7), twice at 65°C with 2x cells. In contrast, this 3' DNase I-hypersensitive site was SSC/1% SDS/1 mM EDTA, and once at room temperature undetectable in KG1 cells that had been induced to differ- with O.1x SSC. Autoradiography was carried out at -70°C entiate along the macrophage lineage (Fig. 3). The 3' site was with Kodak XAR-5 film. also undetectable in B-cell and HeLa DNA. The probes used were subfragments of a CD2 cDNA clone No additional DNase I-hypersensitive sites were detected (Fig. 1 and ref. 18), isolated from agarose gels and labeled by with the probes described in Fig. 1. the random primer method (19). Degree of Methylation of the CD2 Gene. The extent of Northern Blotting Analysis. RNA was extracted from 1-5 x methylation around the 5' and 3' regions ofthe CD2 gene was 107 cells by the guanidinium isothiocyanate method (20), determined by using the methylation-sensitive enzymes Hpa electrophoresed (10 ,ug per lane) through a 1% agarose gel in II and Hha I. For Hpa II the methylation-insensitive iso- 50% formamide, and blotted onto Hybond-N nylon mem- schizomer Msp I was used as a control. brane (Amersham). Hybridization and washing were per- Methylation around the 5' region of the CD2 gene was formed as described (18). analyzed with probe A (Fig. 1). Four Hha I sites (A1-A4; see Fig. 6), two of which (A3 and A4) overlapped, were detected RESULTS 5' to the first CD2 exon, and a fifth site (A5) was present 3' to exon 2. Sites A3-A5 were unmethylated in CD2-express- Mapping of DNase I-Hypersensitive Sites. The T cells used ing T cells (Fig. 4). Because of complete digestion at A3/A4 in this study (Table 1) were human T-lineage tumor cell lines it was not possible to analyze Al and A2 with the probe used. with surface phenotypes characteristic of different stages of In contrast, CD2-nonexpressing cells, whether T or non-T in thymocyte differentiation (21). Cell lines representative of origin, exhibited at least 70% methylation at each Hha I site immature (HSB-2, CEM), relatively mature (MOLT-4), and (Fig. 4). Probe A was also used to investigate the methylation mature (J6, HPB-ALL) , and peripheral T cells status of the two Hpa II sites 5' to the start of transcription (HuT 78) were analyzed. The putative lymphomyeloid pro- (H1 and H2; see Fig. 6). No differences were found at these genitor cell line KG1 was also analyzed, together with its sites between any of the cell lines (Fig. 4). sublines KGla and KG1b, which express immature T-cell When genomic DNA was digested with EcoRI followed by characteristics (22, 23). either Hpa II or Msp I and hybridized with probe C (Fig. 1), DNA was prepared from DNase I-digested nuclei, digested four Hpa II sites in the final intron and one in the fifth exon with an appropriate restriction enzyme, and analyzed by (H3-H7; see Fig. 6) showed varying methylation patterns. Southern blotting analysis to identify DNase I-hypersensitive All five sites showed some degree of methylation in both sites. With the 5' probe (probe A, Fig. 1) a 9-kb band, CD2-expressing and nonexpressing T cells, although meth- Table 1. Summary of cell lines and expression of DNase I-hypersensitive sites CD2 RNA DHSt Methylation sites§ Cell line Cell type phenotype* expressions 1 2 3 A1+2 A3+4 A5 H1+2 H3-7 H8 H9 J6 T + + + + + ? - - + + - HPB-ALL T + + + + + ? - - + + - 9 CEM T - - -- + ± + + + + + - MOLT-4 T - - -- + ± + + + + + - HSB-2 T - - - - + ± + + + + + - HuT 78 T - - -- + ± + + + + + - ICRFT1¶ T - - -- + ± + + + + + - BR18 B - - - - - + + + - Daudi B - - -- - + + + KG1 Lymphomyeloid - - -- + ± + + + + + KGla T? - - -- + ± + + + + + KG1b T? - - - - + ± - + + + + - HeLa Epithelial - - - - - + - + + - *Determiined by flow cytometry; +, >50%o cells positive. tDetermined by Northern blotting analysis of total RNA. tDNase I-hypersensitive sites 1-3; see Fig. 6. §A, Hha I; H, Hpa II (see Fig. 6). Degree of methylation: +, >90%o; +, 50-90o; +, 10-50%o; -, <10o; ?, not determined. 1A cell line of unknown origin that expresses the y8 T-cell antigen receptor. Downloaded by guest on September 28, 2021 Immunology: Wotton et A Proc. Natl. Acad. Sci. USA 86 (1989) 4197

HPBALL HPBALL or EcoRI/Hpa TI-digested DNA (Fig. 5). Site H8 was un- methylated in CD2-expressing T cells and 30-50% s4b' DNAsel i5b'DNAsel methyl- Q O. 0 --. ated in all CD2-nonexpressing cells. Site H9 was completely unmethylated in CD2-nonexpressing cells. The degree of 23- methylation of site H9 in CD2-expressing cells could not be 23- determined with the probe used because H8 is digested 9- *#4 t w 9- * 0 6- 6- completely by Hpa II. 4- DISCUSSION 2.3- The positions of the three DNase I-hypersensitive sites 4- identified within the human CD2 gene are summarized in 2-3- 203-

J6 CEM Molt 4 BRI8 0-DNseCg DNAse1 v~~~~~~ CS DNAse1 oDNAse

23- w w W 23- 23- 23- 9-0 4 9-s El, g AN W 6- 6- 6- . . ,. 6- 4- 4- 4- 4- - DHS3 .cDHS3 * c DHS3 2 3 - 2 3- 2 0 - 2 0- 2-3 - 2-3- 2 0 - 2 0-

He La KG1 KG1b KG1S IV t DNA sel 0' DNAseI 0DNAseI DNAsel O 0. 0 &

23- _ w v 23- w , P q * * 23- 23- 9- 9- 6- 9- * * 6- 6- 6- 4- 4- A t 4 - -cDHS3 4- o -a DHS3 23- it 2 0- 23 - 23 - 2 3- I 2 0- 2-0- 20- ..:i 2 I FIG. 3. Hypersensitivity 3' to the CD2 gene. Procedures were as for Fig. 2 (0.6-5.5 pg of DNase) except that EcoRI was used. DNA was hybridized with probe B except for J6, which was probed with the full-length cDNA. The hypersensitive band is indicated as DHS 3. KG1S refers to KG1 cells stimulated with phorbol ester plus ionomycin. The band at 7 kb in the KG1S panel results from "star" activity ofthe restriction enzyme. J6 Molt 4 BRI8 HeLa KG1 KG1b J6 Molt 4 BRI8

BA B A B A B A B A B A B H M B H M BH M *~~ 23 23- 6-

6 4-

2.3- 2 0- 2-3- 2 0-

0.5- 0 0*5-

FIG. 4. Methylation around the 5' end of the CD2 gene. DNA was digested with various combinations of restriction enzymes: B, BamHI; A, BamHI plus Hha I; H, BamHI plus Hpa II; M, BamHI plus Msp I. Blots were hybridized with probe A. Positions of molecular size markers (kb) are shown. Downloaded by guest on September 28, 2021 Immunology: Wotton et aL Proc. Natl. Acad. Sci. USA 86 (1989) 4199

J 6 CEM Molt4 HSB2 BRI8 HeLa KG1a KG1b

R H M RH M R H M R H M R HM R H M R HM RH M

23-f 9 '. 6- S 4- D#

2.3- 2.0- p

9 . * 9 . * -9

0.5-

FIG. 5. Methylation around the 3' end of the CD2 gene. Procedures were as for Fig. 4, except that EcoRI was used in place of BamHI. R, EcoRI; H, EcoRI plus Hpa II; M, EcoRI plus Msp I. Blots were hybridized with probe C.

DHSI DHS2 DHS3 v v V 8 K SB B B N R St . K 5' i I _ 3, I * I VI 1 I A A2HiItIA3/A4A * HH4 HH" H2 HS8H 0 5 10 15 20 25 kb L- 20~~~~~~~~~~~ 30 35

FIG. 6. Summary ofDNase I hypersensitivity and methylation around the 5' and 3' regions ofthe CD2 gene. The positions ofthe three DNase I-hypersensitive sites (DHS 1-3) are indicated. The Hpa II (HI-H9) and the Hha I (Al-AS) sites, whose methylation status was studied, are shown. Black boxes represent exons. B, BamHI; K, Kpn I; N, Nde I; R, EcoRP; S, Ssp I; St, Stu I. The maps of Nde I, Ssp I, and Stu I shown are incomplete. functional enhancers located at the 3' end of the CD3 6-chain Mason, D. Y., Crumpton, M. J. & Kioussis, D. (1988) EMBO J. 7, and T-cell receptor a- and p-chain genes (27, 28). This may be 1675-1682. related to the common evolutionary origin for these genes, all 16. Diamond, D. J., Clayton, L. K., Sayre, P. H. & Reinherz, E. L. (1988) Proc. Nati. Acad. Sci. USA 85, 1615-1619. ofwhich are members ofthe immunoglobulin superfamily (29). 17. Greaves, D. R., Wilson, F. D., Lang, G. & Kioussis, D. (1989) Cell We thank Dimitris Kioussis for much helpful discussion and 56, 979-986. communication of unpublished results. 18. Sewell, W. A., Brown, M. H., Dunne, M. J., Owen, M. J. & Crumpton, M. J. (1986) Proc. Natl. Acad. Sci. USA 83, 8718-8722. 1. Von Boehmer, H. (1988) Annu. Rev. Immunol. 6, 309-326. 19. Feinberg, A. 0. & Vogelstein, B. (1984) Anal. Biochem. 137, 266- 2. Maniatis, T., Goodbourn, S. & Fisher, J. A. (1987) Science 236, 267. 1237-1245. 20. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, 3. Schleif, R. (1988) Science 241, 1182-1187. W. J. (1979) Biochemistry 18, 5294-5299. 4. Gerster, T., Matthias, P., Thali, M., Jiricny, J. & Schaffner, W. 21. Greaves, M. F., Rao, J., Hariri, G., Verbi, W., Katovsky, D., (1987) EMBO J. 6, 1323-1330. Kung, P. & Goldstein, G. (1981) Leuk. Res. 5, 281-299. 5. Grosveld, F., Blom van Assendelft, G., Greaves, D. R. & Kollias, 22. Furley, A. J., Reeves, B. R., Mizutani, S., Altass, L. J., Watt, G. (1987) Cell 51, 975-985. S. M., Jacob, M. C., van den Elsen, P., Terhorst, C. & Greaves, 6. Keshet, I., Lieman-Hurwitz, J. & Cedar, H. (1986) Cel44, 535-543. M. F. (1986) Blood 68, 1101-1107. 7. Cedar, H. (1988) Cell 53, 3-4. 23. Watt, S. M., Karhi, K., Gatter, K., Furley, A. J. W., Katz, F. E., 8. Weintraub, H. & Groudine, M. (1976) Science 193, 848-856. Healy, L. E., Altas, L. J., Bradley, N. J., Sutherland, D. R., 9. Mills, F. C., Fisher, L. M., Kuroda, R., Ford, A. M. & Gould, Levinsky, R. & Greaves, M. F. (1987) Leukaemia 1, 417-426. H. J. (1983) Nature (London) 306, 809-812. 24. Busslinger, M., de Boer, E., Wright, S., Grosveld, F. G. & Flavell, 10. Church, G. M., Ephrussi, A., Gilbert, W. & Tonegawa, S. (1985) R. A. (1983) Nucleic Acids. Res. 11, 3559-3569. Nature (London) 313, 798-801. 25. Keshet, E. & Cedar, H. (1983) Nucleic Acids Res. 11, 3571-3580. 11. Kamoun, M., Martin, P. J., Hansen, J. A., Brown, M. A., Siadak, 26. Ford, A. M., Watt, S. M., Furley, A. J. W., Molgaard, H. V. & A. W. & Nowinski, R. C. (1981) J. Exp. Med. 153, 207-212. Greaves, M. F. (1988) EMBO J. 7, 2393-2399. 12. Reinherz, E. L. (1985) Immunol. Today 6, 75-79. 27. Georgopoulos, K., van den Elsen, P., Bier, E., Maxam, A. & 13. Yang, S. Y., Chouaib, S. & Dupont, B. (1986) J. Immunol. 137, Terhorst, C. (1988) EMBO J. 7, 2401-2407. 1097-1102. 28. Krimpenfort, P., de Jong, R., Uematsu, Y., Dembic, Z., Ryser, S., 14. Cantrell, D. A., Verbi, W., Davies, A., Parker, P. & Crumpton, von Boehmer, H., Steinmetz, M. & Berns, A. (1988) EMBO J. 7, M. J. (1988) Eur. J. Immunol. 18, 1391-13%. 745-750. 15. Lang, G., Wotton, D., Owen, M. J., Sewell, W. A., Brown, M. H., 29. Williams, A. F. (1987) Immunol. Today 8, 298-303. Downloaded by guest on September 28, 2021