Analysis of the H19ICR insulator

Abstract

Transcriptional insulators are specialized cis-acting elements that protect promoters from inappropriate activation by distal enhancers. The $H19 Imprinting Control Region (ICR)$ functions as a CTCF-dependent, methylation-sensitive transcriptional insulator. Several insertional mutations and knock-out mouse were examined to demonstrate the H19ICR functions as a methylation-regulated maternal-specific insulator in novel chromosomal contexts. To investigate the configuration of cis-acting elements at several insertion sites, Chromosome Conformation Capture (3C) and Chromatin immunoprecipitation (ChIP) assays were used. By comparing maternal and paternal organizations on wild type and mutant chromosomes, mechanisms for ICR/insulator function were identified. The results indicate that promoter and enhancer element invariably associate to form DNA loop domains at transcriptionally active loci. Conversely, active insulators always prevent these promoter-enhancer interactions. Instead the ICR/insulator forms novel loop domains by associating with the blocked promoters and enhancers. The results suggest that these associations are fundamental to insulator function.