Epigenetic Control of Mammalian Development : Studies on an imprinting control region

Abstract: A subset of autosomal genes is preferentially or exclusively expressed from one of the parental alleles. This phenomenon, termed genomic imprinting, is highlighted by the neighboring Igf2 and H19 genes, which are monoallelically expressed on opposite parental chromosomes. These features are governed by a 2.2 kb differentially methylated domain, hereafter termed imprinting control region (ICR) in the 5'-flank of the H19 gene. It was shown that the H19 ICR displays unique chromatin conformation features with nuclease hypersensitive sites in linker elements flanked by positioned nucleosomes on the maternally derived allele. Moreover, it was demonstrated that the unmethylated ICR functions as a unidirectional chromatin insulator, which involves the chromatin insulator protein CTCF.The methylated and unmethylated states of the paternal and maternal H19 ICR alleles are known to be stably propagated in the soma throughout development. During in vitro organogenesis, however, the stability of the H19 ICR was demonstrated to be disturbed due to presence of environmental cues. The methylation plasticity of the H19 ICR was nevertheless tolerated without affecting the imprinted status of either Igf2 or H19 genes. It was also observed that some human cancer cell lines possess strong de novo methylation activities. Following transfection of an episomal construct, which contains the H19 miningene with the core H19 ICR and its human counterpart, the H19 reporter gene became rapidly do novo methylated and eventually silenced in choriocarcinoma cells (JEG-3), but not in heptoma cells (HEP3B). Although the H19 ICR was initially protected from being methylated by JEG3 cells, progressive waves of de novo methylation generated a heavily methylated H19 ICR in later passages, with concomitant loss of its insulator function. It is generally accepted that parental marks undergo erasure and reestablishment during gametogenesis. It was shown that CTCF and its paralogue, BORIS, are expressed in reciprocal patterns during adult male germline development. By means of laser-dissection and bisulfite genomic sequencing, it was observed that de novo methylation of CTCF target sites occurred in BORIS-expressing spermatocytes that exhibit repression of CTCF gene. It was also shown, by chromatin immunopurification analyses of adult mouse testes, that CTCF and BORIS were associated with H19 ICR. A model is proposed to explain the acquisition of differential methylation marks in molecular terms.