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 event s'. In the mouse blastula, most DNA is unmethylated, but after implantation a wave of de novo methylation modifies most of the genome, excluding the m ajority 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 gen e 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 nor mal development Cis-acting regulatory elements and trans-acting factors app ear to be involved in both stage- and tissue-specific demethylation process es(10,11). Spl-like elements have a key role in protecting the CpG island o f Aprt (encoding adenine phosphoribosyl transferase) from de novo methylati on, and when these elements are specifically mutated, the Aprf CpG island b ecomes methylated genome, in transgenic mice(12,13) . We have now character ized 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 elemen t on a removable cassette adjacent to a human HBB (encoding P-globin) repor ter and generated a transgene whose methylation pattern can be switched in vivo. Analysis of globin transcription in this system showed that methylati on in cis inhibits gt,ne expression in a variety of tissues, indicating tha t DNA modification may serve as a global genomic repressor.