DNA methylation alters chromatin structure and regulates Thy-1 expression in EL-4 T cells.
Thy-1 exhibits marked differences in expression in various tissues in many species; therefore, it is of interest to define possible mechanisms that may regulate Thy-1 expression. We produced Thy-1 negative variants of the murine T cell lymphoma EL-4 by mutagenesis with ethylmethanesulfonate (EMS), negative selection with anti-Thy-1 monoclonal antibodies (mAb) plus complement, and fluorescence-activated cell sorting (FACS). Thy-1 surface negative (Thy-1-) mutants produced in this manner were shown to produce no detectable Thy-1 mRNA, but contained an intact Thy-1 gene as determined by Southern blotting. 5' CG sequences, which had been demethylated in the parent EL-4 clone, were completely methylated in the EMS-induced Thy-1-variant. In addition, a DNase I hypersensitive site that mapped to the 5' end of the Thy-1 gene in EL-4 was absent in the Thy-1- variant. Treatment of this Thy-1- clone with 5-azadeoxycytidine (5-dAZA) resulted in re-expression of surface Thy-1, demethylation of the 5' CG sequences, and regeneration of the DNase I hypersensitive site. These studies indicate that methylation of certain critical DNA sequences in the 5' region of the Thy-1 gene can alter local chromatin structure and regulate expression of this gene.
D044503:5-Methylcytosine; D000818:Animals; D000954:Antigens, Surface / Q000096:biosynthesis*; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D002843:Chromatin / Q000648:ultrastructure*; D003596:Cytosine / Q000031:analogs & derivatives / Q000032:analysis; D004273:DNA, Neoplasm / Q000032:analysis*; D000077209:Decitabine; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D008223:Lymphoma; D008745:Methylation; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000096:biosynthesis; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D018800:Thy-1 Antigens
D000954:Antigens, Surface; D002843:Chromatin; D004273:DNA, Neoplasm; D012333:RNA, Messenger; D018800:Thy-1 Antigens; D044503:5-Methylcytosine; D000077209:Decitabine; D003596:Cytosine; D005020:Ethyl Methanesulfonate; D001374:Azacitidine