ENHANCEMENT OF REPORTER GENE DE-NOVO METHYLATION BY DNA FRAGMENTS FROM THE ALPHA-FETOPROTEIN CONTROL REGION

The 5'-upstream region of the rat a-fetoprotein (AFP) gene strongly in creased de novo methylation of an adjacent chloramphenicol acetyltrans ferase (CAT) gene upon transfection into F9 mouse embryonal carcinoma cells. The same effect was exerted by a distal 775-base pair (bp) frag ment and by 300- and 1-kb fragments preceding the transcriptional star t site, but not by other parts of the control region. Further division of the larger, strongly active fragments resulted in a gradual decrea se of methylation and clonal variation in the methylation patterns. Th e effect of the 775-bp fragment did not depend on its orientation. It was ablated by insertion of the mouse metallothionein I promoter betwe en the AFP gene fragment and the CAT gene, but not by its insertion up stream of the AFP gene fragment. Two fragments from the AFP control re gion increasing methylation contained B1 and B2 small interspersed rep etitive elements, respectively. B1 and B2 sequences of different origi n also acted strongly to increase methylation. These findings support the idea that mammalian genes contain specific sequences involved in r egulating their methylation. The effects of these sequences appear to be exerted in cis, to be dependent on proximity, but not on orientatio n, and to require an optimal size of 500-700 bp. Small retrotransposon sequences within such elements may be particularly effective in attra cting de novo methylation.