DNA methylation is a major epigenetic mechanism that has been suggested to 
control developmental gene regulation during embryogenesis, but its regulatory 
mechanisms remain unclear. In this report, we show that CpG islands associated 
with the X-linked homeobox gene cluster Rhox, which is highly expressed in the 
extraembryonic trophectoderm, are differentially methylated in a stage- and 
lineage-specific manner during the post-implantation development of mice. 
Inactivation of both Dnmt3a and Dnmt3b, DNA methyltransferases essential for the 
initiation of de novo DNA methylation, abolished the establishment of DNA 
methylation and the silencing of Rhox cluster genes in the embryo proper. The 
Dnmt3-dependent CpG-island methylation at the Rhox locus extended for a large 
genomic region ( approximately 1 Mb) containing the Rhox cluster and surrounding 
genes. Complementation experiments using embryonic stem (ES) cells deficient in 
the DNA methyltransferases suggested that the CpG-island methylation by Dnmt3a 
and Dnmt3b was restricted within this large genomic region, and did not affect 
the neighboring genes outside it, implicating the existence of region-specific 
boundaries. Our results suggest that DNA methylation plays important roles in 
both long-range gene silencing and lineage-specific silencing in embryogenesis.