SEQUENCE-SPECIFIC METHYLATION OF THE MOUSE H19 GENE IN EMBRYONIC-CELLS DEFICIENT IN THE DNMT-1 GENE

We have used Dnmt(c/c) ES cells that a re homozygous for disruption of the DNA methyltransferase gene io address how de novo methylation is propagated and whether ii is directed io specific sites in the early e mbryo. We examined the imprinted H19 gene and the specific-sequence re gion implicated as an ''imprinting mark'' to determine whether de novo methylation was occurring at a restricted sei of sites. Since the ''i mprinting mark'' was found to be methylated differentially at ail stag es of development, we reasoned that the sequence may still be a target for the de novo methylation activity found in the Dnmt(c/c) cells, ev en though the loss of maintenance methylase activity renders the H19 p romoter active. We used bisulfite genomic sequencing io determine the methylation slate of the imprinted region of the H19 gene and found a low level of DNA methylation at specific single CpG sites in the upstr eam region of the imprinted H19 sequence in the Dnmt(c/c) mutant ES ce lls. Moreover, these CpG sites appeared io be favoured targets for fur ther de novo methylation of neighbouring CpG sites in rescued ES cells , which possess apparently normal maintenance activity. Our data provi de further evidence for a separate methylating activity in ES cells an d indicate that this activity displays sequence specificity. (C) 1998 Wiley-Liss, Inc.