Differentiation of the T helper phenotypes by analysis of the methylation state of the IFN-gamma gene.
Th1 and Th2 CD4+ T cell clones have been defined by their ability to produce different lymphokines. However, the processes by which CD4+ T cells differentially regulate lymphokine gene expression have not been well defined. In this report, we demonstrate that the methylation status of a CpG dinucleotide contained within a TATA proximal regulatory element of the IFN-gamma promoter correlates with the transcription of the gene. In murine Th1 clones and two human CD4+ Th0 clones, this site is either completely or partially hypomethylated, whereas in murine Th2 clones this site is > 98% methylated. Treatment of murine Th2 clones with 5-azacytidine, an agent that inhibits methylation of the DNA, converts these cells to IFN-gamma producers. Additional targets for methylation outside the transcriptional control regions of the IFN-gamma genetic locus were found to be hypomethylated in Th2 cells but not in Th1 cells. Electrophoretic mobility shift assays (EMSA) revealed at least five distinct protein-DNA complexes that are formed with an oligonucleotide containing the IFN-gamma promoter TATA proximal regulatory element, and in vitro methylation of this site results in a loss of these three complexes. Furthermore, a comparison of nuclear extracts prepared from Th1 and Th2 clones revealed that the EMSA patterns were qualitatively similar but differed quantitatively. In addition, transient transfection of a murine IFN-gamma promoter-chloramphenicol acetyl transferase (CAT) gene construct into both Th1 and Th2 clones produced CAT activity that was not inducible by anti-CD3, indicating that hypomethylation per se of the promoter alone is not sufficient for inducible gene expression.
Young
H A
HA
Laboratory of Experimental Immunology, NCI-FCRDC, MD.
Ghosh
P
P
Ye
J
J
Lederer
J
J
Lichtman
A
A
Gerard
J R
JR
Penix
L
L
Wilson
C B
CB
Melvin
A J
AJ
McGurn
M E
ME
D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D003852:Deoxyribonucleoproteins / Q000737:chemistry; D005786:Gene Expression Regulation / Q000187:drug effects; D005796:Genes; D007371:Interferon-gamma / Q000235:genetics*; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012150:Polymorphism, Restriction Fragment Length; D012045:Regulatory Sequences, Nucleic Acid; D016176:T-Lymphocyte Subsets / Q000502:physiology*; D018417:Th1 Cells / Q000502:physiology*; D018418:Th2 Cells / Q000502:physiology*
D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D009838:Oligodeoxyribonucleotides; D007371:Interferon-gamma; D001374:Azacitidine