DNA methylation represses transcription in vivo

DNA in somatic tissue is characterized by a bimodal pattern of methylation, which is established in the animal through a series of developmental events'. In the mouse blastula, most DNA is unmethylated, but after implantation a wave of de novo methylation modifies most of the genome, excluding the majority of Cpt islands, which are mainly associated with housekeeping genes. This genomic methylation pattern is broadly maintained during the life of the organism by maintenance methylation, and generally correlates with gene expression. Experiments both in vitro(3-5) and in vivo(6-9) indicate that methylation inhibits transcription, It has not yet been possible, however, to determine the role of DNA methylation on specific. sequences during normal development Cis-acting regulatory elements and trans-acting factors appear to be involved in both stage- and tissue-specific demethylation processes(10,11). Spl-like elements have a key role in protecting the CpG island of Aprt (encoding adenine phosphoribosyl transferase) from de novo methylation, and when these elements are specifically mutated, the Aprf CpG island becomes methylated genome, in transgenic mice(12,13) . We have now characterized an embryo-specific element,ment from the CpG island sequence upstream of Aprt that can protect itself from de novo methylation in transgenic mice as well as reduce methylation of flanking sequences. We placed this element on a removable cassette adjacent to a human HBB (encoding P-globin) reporter and generated a transgene whose methylation pattern can be switched in vivo. Analysis of globin transcription in this system showed that methylation in cis inhibits gt,ne expression in a variety of tissues, indicating that DNA modification may serve as a global genomic repressor.