Fig 1.

DNMT3L stimulates de novo methylation by Dnmt3a but not by DNMT3B at the SNRPN IC. DNA harvested from human 293/EBNA1 cells carrying pFC19 or pFC30 (in which a key ≈1-kb region of the SNRPN IC was cloned in either orientation) was digested with an excess of the methylation-sensitive enzyme HpaII. The resulting DNA species were separated by electrophoresis through an agarose gel, transferred to a nylon membrane, and hybridized with a probe corresponding to the SNRPN IC. Two bands (684 and 754 bp for pFC19, and 684 and 221 bp for pFC30) were expected if no methylation was present. CpG methylation prevents cleavage by HpaII and results in higher molecular weight species that can be easily separated. (A) Each target plasmid was introduced into cells alone, together with an expression vector for Dnmt3a, or together with expression vectors for both Dnmt3a and DNMT3L, as indicated. (B) Plasmid pFC19 was introduced in cells either with DNMT3B or with both DNMT3B and DNMT3L expression vectors, as indicated.