Cytosine methylation in the EcoRI site of active and inactive herpesvirus thymidine kinase promoters.

The herpesvirus thymidine kinase (tk) gene integrated in the human cell line, 2.1-a, can be inactivated by limited de novo methylation. All these TK- clones show partial EcoRI digestion of the recognition site (cGAATTCg) in the tk promoter in contrast to complete digestion of this site in the original cell line. Studies on well-defined substrates prepared in vitro showed that methylation of one cytosine in the EcoRI recognition sequence resulted in partial and methylation of both cytosines in severe inhibition of digestion by EcoRI. This characteristic was used to determine whether no, one or both cytosines in the EcoRI site of the tk promoter were methylated in various TK- clones derived from 2.1-a and in TK+ clones re-expressing the gene after 5-azacytidine treatment. A high correlation was found between inactivity of the tk gene and methylation of only one of the two cytosines in the EcoRI recognition site. The results also show that the tk promoter can be active despite the presence of a methylated cytosine.