STRAIN-SPECIFIC TRANSGENE METHYLATION OCCURS EARLY IN MOUSE DEVELOPMENT AND CAN BE RECAPITULATED IN EMBRYONIC STEM-CELLS

A murine transgene, HRD, is methylated only when carried in certain in bred strain backgrounds. A locus on distal chromosome 4, Ssm1 (strain- specific modifier), controls this phenomenon. In order to characterize the activity of Ssm1, we have investigated developmental acquisition of methylation over the transgene. Analysis of postimplantation embryo s revealed that strain-specific methylation is initiated prior to embr yonic day (E) 6.5. Strain-specific transgene methylation is all-or-non e in pattern and occurs exclusively in the primitive ectoderm lineage, A strain-independent pattern of partial methylation occurs in the pri mitive endoderm and trophectoderm lineages, To examine earlier stages, embryonic stem (ES) cells were derived from E3.5 blastocysts and exam ined for transgene methylation before and after differentiation. Thoug h the transgene had already acquired some methylation in undifferentia ted ES cells, differentiation induced further, de novo methylation in a strain-dependent manner, Analysis of methylation in ES cultures sugg ests that the transgene and endogenous genes (such as immunoglobulin g enes) are synchronously methylated during early development. These res ults are interpreted in the context of a model in which Ssm1-like modi fier genes produce alterations in chromatin structure during and/or sh ortly after implantation, thereby marking target loci for de novo meth ylation with the rest of the genome during gastrulation.