Interindividual concordance of methylation profiles in human genes for tumor necrosis factors a and fi

The DNA in mammalian genomes is characterized by complex patterns ofDNA methylation that reflect the states of all genetic activities of that genome. The modified nucleotide 5-methyldeoxycytidine (5mdC) can affect the interactions of specific proteins with DNA sequence motifs. The most extensively studied effect of sequence-specific methylations is that of the long-term silencing of eukaryotic (mammalian) promoters. We have initiated studies on the methylation status of parts of the human genome to view patterns of DNA methylation as indicators for genetic activities. In this report, analyses using both restriction enzyme-Southern blotting and the very precise genomic sequencing technique have been done. The genes for tumor necrosis factors (TNF) a and ,B-in particular, their 5'-upstream and promoter regions-have been investigated in DNA isolated from human lymphocytes, granulocytes, and sperm. The results are characterized by a remarkable interindividual concordance of DNA methylation in specific human cell types. The patterns are identical in the DNA from one cell type for different individuals even of different genetic origins but different in the DNA from different cell types. As an example, in the DNA from human granulocytes of 15 different individuals (ages 20-48 yr, both sexes), 5mdC residues have been localized by the genomic sequencing technique in three identical sequence positions in the 5'upstream region and in one downstream position of the gene encoding TNF-a. The promoter of this gene is free of 5mdC, and TNF-a is expressed in human granulocytes. The TNF-,B promoter is methylated in granulocytes from 9 different individuals, and TNF-fl is not expressed. In human lymphocytes, the main source of TNF-fB the TNF-,B promoter is free of5mdC residues. All 5'-CG-3' sites studied in the TNF-a and -fl genes are methylated in DNA from human sperm. In human cell lines HL-60, Jurkat, and RPMI 1788, the extent ofDNA methylation in TNF-a and -,B genes has also been studied. Sequence-specific promoter methylation can inactivate or strongly inhibit eukaryotic (mammalian) promoters (for reviews, see refs. 1-6). Adenovirus promoters have been used as models to study the mechanism of promoter inactivation (7-15). The development of de novo patterns ofDNA methylation in transformed cell lines is characterized by the ordered, nonrandom spreading of methylation (16-18); By using restriction enzyme and Southern blotting analyses (19), as well as the very exact genomic sequencing procedure (17, 20, 21), we have studied human DNA methylation in segments and in the promoter regions of the tumor necrosis factor (TNF) genes a (cachectin) and 8 (lymphotoxin) (22-29). Depending on the tissue origin of human DNA, highly specific patterns of methylation exist without interindividual variance. In established human hematopoietic cell lines a higher variability in patterns of methylation has been seen. Evidence for inheritance of methylation patterns and for variation in the methylation of allelic sites has been presented (30). MATERIALS AND METHODS Cells, Cell Lines, and Extraction ofDNA. (i) Fresh blood or buffy coats were obtained by venipuncture from healthy human volunteers and diluted with RPMI 1640 medium (31). Subsequently, lymphocytes and granulocytes were fractionated on a Ficoll-Paque gradient. Granulocytes were recovered from the pellet and freed from contaminating erythrocytes by lysing the latter in 0.83% NH4C1. The resulting granulocyte preparation was >95% pure, as determined in a Pappenheimstained (32) cell smear. The lymphocyte preparation was >90o lymphocytes, 5-10%o monocytes, and <3% granulocytes. (ii) Human cell lines HL-60, a human promyelocytic leukemia cell line; Jurkat, a T-cell line; and RPMI 1788, an IgM-secreting B-lymphoblastoid cell line, were propagated in RPMI 1640 medium (GIBCO-BRL) supplemented with 1020%o fetal bovine serum. (iii) Human sperm cells were washed, and the DNA was extracted after dithiothreitol treatment (33). The nuclear DNA from the different cell preparations was extracted by standard protocols (34). Isolation of Cellular RNA. Granulocytes were purified >99%. Granulocytes or the cells from established cell lines were kept in culture for 5 hr in the absence or presence of 8 nM PMA (phorbol 12-myristate 13-acetate). Total cellular RNA was extracted as described (35). Genomic DNA Sequencing. The method of Church and Gilbert (20) was applied (17, 18, 21). For analysis of the TNF-a promoter (Figs. la and 3a), the DNA was cleaved with Stu I and Bgl I. The appropriate promoter fragments were isolated by zone velocity sedimentation in 5-20o sucrose gradients (36, 37) and were subsequently cleaved with BstXI or Sty I. For analysis of the TNF-,B promoter (Figs. lb and 4a), the initial cleavage was with Pvu II or BamHI. After fragment selection, the DNA was cut with Hinfl. The cytidine reaction for DNA sequencing (38) with hydrazine proceeded at 200C (TNF-,B) or at 25°C (TNF-a) for 27 min, the guanosine reaction with dimethyl sulfate was at 20'C for 4 min. Cleavage was effected by 1 M piperidine treatment for 30 min at 90°C. DNA fragments were separated by electrophoresis on 6% polyacrylamide gels containing 8 M urea and were then electrotransferred to a GeneScreenPlus membrane. The TNF-a or -f3 fragments were cloned into M13 (mpl8 or mpl9) DNA. Single-stranded probes were generAbbreviations: 5mdC, 5. -methyldeoxycytidine; TNF, tumor necrosis factor; PMA, phorbol 12-myristate 13-acetate. *Present address: Department of Molecular Biology, Princeton University; Princeton, NJ 08544-1014. tTo whom reprint requests should be addressed at: Institut fur Genetik, Universitat zu Koln, Weyertal 121, D-5000 Koln 41, Federal Republic of Germany. 8830 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 87 (1990) 8831 EcoRI-Sau 3a probe, 812 bp 11 11 I III IIfI I IIl111