Transcriptional Silencing in the Imprinted Igf2-H19 Loci: The Mystique of Epigenetics

Abstract

Genomic imprinting marks a subset of autosomal loci expressed in parent of origin-dependent monoallelic expression in a non-Mendelian fashion. To restore totipotency and to reset the imprint according to the sex of the individual, the mark must be erased during germline development. The imprinted Igf2-H19 loci located distally on chromosome 7 in mouse and 11p15.5 in human, share common regulatory elements that regulate differential expression. Where the H19 is silenced when paternally inherited, the Igf2 is silenced when maternally inherited. The differentially methylated 5'-flank of H19 gene, termed imprinting control region (ICR), shown to display a unique chromatin organisation harbours hypersensitive sites in linker regions flanked by positioned nucleosomes on the maternal allele. This unique chromatin conformation functions as a methylation-sensitive and unidirectional chromatin insulator, which later was found to depend on the chromatin insulator protein CTCF. The H19 ICR exhibits default-silencing functions in promoter-proximal positions. The maximal distance between the H19 ICR and the promoter of the reporter gene required for this effect was 1.2 ± 0.3kb which can be compared to the 1.9 kb distance between the endogenous H19 ICR and H19 promoter. Results suggest that the H19 ICR adopts a chromatin conformation that must be separated by a minimal distance from pivotal cis-regulatory elements to avoid adverse effects on neighbouring promoters. Poly(ADP-ribosy)lation represents a novel post-translational epigenetic mark that segregates with exclusively the maternal derived H19 ICR and associated with factors that interact with the CTCF target sites. CTCF is itself poly(ADP-ribosy)lated and the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide relieves the insulator function of the H19 ICR. Designed zinc finger proteins were applied to examine if epigenetic marks provided an obstacle for targeted activation and silencing. The zinc finger protein ZFP809 with activator/repressor domain able to efficiently activate/silence the IGF2 target. Murine hybrid cell lines of human chromosome 11, demonstrated that the ZFP809 overcame the epigenetic marks that repressed maternal IGF2 and paternal H19 allele, respectively. Results suggested that imprinted genes are not normally exposed to strong cis-regulatory elements and that the designed ZFPs can be exploited to develop a therapeutic method for rectifying epigenetic lesions