The differentially methylated 5'-flank of the mouse H19 gene unidirectional
ly regulates the communication between enhancer elements and gene promoters
and presumably represses maternal lgf2 expression in vivo [1-6]. The speci
fic activation of the paternally inherited lgf2 allele has been proposed to
involve methylation-mediated inactivation of the H19 insulator function du
ring male germline development [[1 -4, 6]. Here, we addressed the role of m
ethylation by inserting a methylated fragment of the H19-imprinting control
region (ICR) into a nonmethylated episomal H19 minigene construct, followe
d by the transfection of ligation mixture into Hep3B cells. Individual clon
es were expanded and analyzed for genotype, methylation status, chromatin c
onformation, and insulator function. The results show that the methylated s
tatus of the H19 ICR could be propagated for several passages without sprea
ding into the episomal vector. Moreover, the nuclease hypersensitive sites,
which are typical for the maternally inherited H19 ICR allele [1], were ab
sent on the methylated ICR, underscoring the suggestion that the methylatio
n mark dictates parent of origin-specific chromatin conformations [1] that
involve CTCF [2]. Finally, the insulator function was strongly attenuated i
n stably maintained episomes. Collectively, these results provide the first
experimental support that the H19 insulator function is regulated by CpG m
ethylation.