Factors affecting de novo methylation of foreign DNA in mouse embryonic stem cells

Integration of foreign DNA into an established host genome can lead to changes in methylation in both the inserted DNA and in host sequences and potentially alters transgene and cellular transcription patterns. This work addresses the questions of what factors influence de novo methylation, and whether the integration site or inserted DNA can affect de novo methylation. Homologous recombination was used to integrate foreign DNA into a specific gene, B lymphocyte kinase (BLK), in mouse embryonic stem (ES) cells. Two plasmids were chosen for integration; one contained the adenovirus type 2 E2AL promoter upstream of the luciferase reporter gene, and the second carried the early SV40 promoter. The methylation patterns were analyzed using HpaII and MspI restriction endonucleases for both homologously recombined and randomly integrated foreign DNA in the ES cell clones. Upon homologous reinsertion of the BLK gene into the genome of mouse ES cells, methylation patterns in this gene were reestablished. In DNA segments adjoined to the BLK gene, the de novo patterns of DNA methylation depended on the viral sequences in these clones and on the locations of the inserts, i.e. on whether the insertions resulted from homologously recombined or randomly integrated foreign DNA. In homologously recombined DNA, sequences carrying the adenovirus type 2 promoter were heavily methylated, and those with an SV40 promoter and an SV40 enhancer element remained unmethylated or hypomethylated. Upon removal of the enhancer element, these inserted constructs also became heavily methylated. In addition, all randomly integrated constructs were heavily methylated independently of the promoter and enhancer element present in the construct. These results indicate that modes and sites of integration as well as the inserted nucleotide sequence, possibly promoter strength, are factors affecting de novo methylation.