Enhancer blocking activity of the insulator at H19-ICR is independent of chromatin barrier establishment.

Transcriptional insulators are cis regulatory elements that organize chromatin into independently regulated domains. At the imprinted murine Igf2/H19 locus, the H19-ICR insulator prevents the activation of the Igf2 promoter on the maternal allele by enhancers that activate H19 on the same chromosome. Given the well-demonstrated role of H19-ICR as an enhancer blocker, we investigated its ability to define a chromatin barrier, as the two activities are coincident on several insulators and may act in concert to define a functional chromatin boundary between adjacent genes with distinct transcriptional profiles. Allele-specific association of posttranslationally modified histones, reflecting the presence of active or inactive chromatin, was analyzed in the region encompassing H19-ICR using chromatin immunoprecipitation. The existence of differential histone modifications upstream and downstream of H19-ICR specifically on the maternal chromosome was observed, which is suggestive of a chromatin barrier formation. However, H19-ICR deletion analysis indicated that distinct chromatin states exist despite the absence of an intervening "barrier." Also, the enhancers can activate the Igf2 promoter despite some parts of the intervening chromatin being in the silent state. Hence, H19-ICR insulator activity is not dependent on preventing the enhancer-mediated alteration of the histone modifications in the region between the Igf2 promoter and the cognate enhancers.