Influence of a CTCF-Dependent Insulator on Multiple Aspects of Enhancer-Mediated Chromatin Organization.

ABSTRACT

Developmental stage-specific enhancer-promoter-insulator interactions regulate the chromatin configuration necessary for transcription at various loci and additionally for VDJ recombination at antigen receptor loci that encode immunoglobulins and T-cell receptors. To investigate these regulatory interactions, we analyzed the epigenetic landscape of the murine T-cell receptor ß (TCRß) locus in the presence and absence of an ectopic CTCF-dependent enhancer-blocking insulator, H19-ICR, in genetically manipulated mice. Our analysis demonstrated the ability of the H19-ICR insulator to restrict several aspects of enhancer-based chromatin alterations that are observed during activation of the TCRß locus for transcription and recombination. The H19-ICR insulator abrogated enhancer-promoter contact-dependent chromatin alterations and additionally prevented Eß-mediated histone modifications that have been suggested to be independent of enhancer-promoter interaction. Observed enhancer-promoter-insulator interactions, in conjunction with the chromatin structure of the Eß-regulated domain at the nucleosomal level, provide useful insights regarding the activity of the regulatory elements in addition to supporting the accessibility hypothesis of VDJ recombination. Analysis of H19-ICR in the heterologous context of the developmentally regulated TCRß locus suggests that different mechanisms proposed for CTCF-dependent insulator action might be manifested simultaneously or selectively depending on the genomic context and the nature of enhancer activity being curtailed.