Distribution and DNA methylation of a repetitive promoter sequence cloned from mouse embryonal carcinoma cells

A 203 bp HpaII fragment (X9) cloned from F9 embryonal carcinoma cell DNA showed multiple bands on Southern hybridization towards mouse DNA digested with various restriction enzymes. No tandem repeats were evident. A 200 bp MspI band corresponded to several copies of the cloned fragment and many elements appeared each associated with a 12-kb PvuII fragment. X9 is therefore a member of a moderately repetitive family probably dispersed in the mouse genome. X9 displayed polymorphisms between different Mus species, whereas multiple hybridizing bands were detected neither in DNA from rodent species outside the Mus genus nor in DNA from man. Therefore, X9 provides a useful probe for studies of murine evolution. X9 contains a TATA box and a CCAAT box as well as several homologies to known transcription factor binding sites. When cloned in front of a reporter gene, it acted as a strong promoter in several different cell types. Most genomic sequences detected by X9 were highly methylated. A single copy appeared hypomethylated in adult mouse tissues, whereas several copies were hypomethylated in F9 and embryonic stem cells. It is speculated that X9 might represent the promoter of a murine gene family whose activity is regulated by DNA methylation.