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 ␤ (TCR␤) 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 enhancerbased chromatin alterations that are observed during activation of the TCR␤ locus for transcription and recombination. The H19-ICR insulator abrogated enhancer-promoter contact-dependent chromatin alterations and additionally prevented E␤-mediated histone modifications that have been suggested to be independent of enhancer-promoter interaction. Observed enhancerpromoter-insulator interactions, in conjunction with the chromatin structure of the E␤-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 TCR␤ 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.T ranscriptional insulators regulate the enhancer-promoter communication that orchestrates the epigenetic landscape of specific loci to activate or repress genes in metazoan genomes. Enhancers can regulate their cognate promoters by diverse mechanisms (1, 2). These may involve direct contact with the promoter by looping and/or alteration of the epigenetic landscape of large domains that render them "open," i.e., associated with chromatin modifications that make them accessible to trans-acting factors. Alteration of the large domains could be due to "tracking" of some proteins initially bound at the enhancer or, as proposed during "facilitated tracking," the movement of enhancer itself along the chromatin. Insulators in the vertebrate genomes have been proposed to modulate these interactions by binding to CTCF, a multiZn-finger protein (3). Genome-wide analysis of CTCF demonstrates its crucial role in higher-order chromatin organization that can facilitate enhancer-promoter interactions as well as curtail them depending on the relative positions of these regulatory elements (4). Importantly, insulators block enhancer-promoter communication only when present between them. Several models have been proposed for enhancer blocking, keeping in view the varied mechanisms underlying enhancer activity (5). It has been postulated that the CTCF-bound insulator partitions the enhancer and promoter to topologically distinct chromatin loops and thus prevents their interaction. The i...

Chromatin Domain Organization of the TCRb Locus and Its Perturbation by Ectopic CTCF Binding

“…The excluded PDb1-DJCb1 region is likely to escape Eb-mediated chromatin changes. Consistent with this, our 3C-qPCR analysis indicated reduced Eb-PDb1 interaction, reduced PDb1-DJCb1 chromatin accessibility (37), and a marked reduction in DJCb1 recombination (30). As predicted, Eb-based activation of PDb2 and the linked DJCb2 cluster was unaltered.…”

“…This suggested that in addition to proximity, the precise relative configuration of the chromatin loop encompassing the RC and the V segments in TCR-ins was influenced by the altered CBS interactions and impacted RSS-mediated recombination. Based on functional analysis, we have earlier proposed that CTCF can both facilitate and inhibit RSS-mediated recombination (30,37). Our 3C-qPCR-based analysis of the chromatin interactions in wild-type and mutant TCRb alleles supports this contention.…”

“…This was evidenced by a severe reduction in Eb-PDb1 looping interaction, as well as by a lack of chromatin accessibility and an enormous decrease in the usage of the DJCb1 cluster in VDJ recombination (30,37). The competitive advantage of CBS-ecto could be due to the convergent orientation of the CBS-ecto with respect to CBS-E augmented by the clustering of four CBS on H19-ICR.…”

“…These mutations abrogate CTCF binding in vitro and in vivo (39). TCR-mut behaved like the wild-type allele during previous analyses of chromatin organization, transcription, and VDJ recombination (30,37).…”

“…In each case, the paternal allele was TCR-cas (37), which had the wild-type TCRb locus of Mus castaneus castaneus origin and hence had several single nucleotide differences compared to the maternal alleles (wild type, TCR-ins, or TCR-mut) of Mus musculus domesticus origin and were useful in designing various allele-specific PCR-based assays. The activity of the ectopic insulator in TCR-ins might render the HindIII site proximal to CBS-D inaccessible to HindIII digestion and interfere in 3C-qPCR-based interpretation.…”