ACTIVATION OF AN IMPRINTED IGF 2 GENE IN MOUSE SOMATIC-CELL CULTURES

The mouse insulin-like growth factor II gene (Igf2), located on distal chromosome 7, is parentally imprinted such that the paternal allele i s expressed while the maternal allele is transcriptionally silent. We derived a cell line from a mouse embryo maternally disomic and paterna lly deficient for distal chromosome 7 (MatDi7) to determine the stabil ity of gene repression in culture. MatDi7 cells maintained Igf 2 in a repressed state even after immortalization, except for one randomly pi cked clone which spontaneously expressed the gene. Igf 2 was expressed in a cell culture derived from a normal littermate; this expression w as growth regulated, with Igf 2 mRNA levels increasing in the stationa ry phase of growth. Analysis of the methylation status of 28 sites dis tributed over 10 kb of the gene did not show consistent differences as sociated with expression level in the normal and MatDi7 cell lines, an d the CpG island in the Igf 2 promoter remained unmethylated in all of the cell lines. Only with an oncogenically transformed cell line did the promoter become extensively methylated. We attempted to derepress the imprinted gene in MatDi7 cells by treatments known to alter gene e xpression. Expression of the Igf 2 allele in MatDi7 cells was increase d in a dose-dependent manner by treatment with 5-aza-2'-deoxycytidine or bromodeoxyuridine, agents known to change DNA methylation patterns or chromatin conformation. Treatment of the cells with 1-beta-D-arabin ofuranosylcytosine, 2'-deoxycytidine, calcium ionophore, heat shock, c old shock, or sodium butyrate did not result in increases in the level s of Igf 2 expression. It seems likely that the mechanism of the Igf 2 imprint involves subtle changes in the methylation or chromatin confo rmation of the gene which are affected by 5-aza-2'-deoxycytidine and b romodeoxyuridine.