An ectopic CTCF-dependent transcriptional insulator influences the choice of Vβ gene segments for VDJ recombination at TCRβ locus

Abstract

Insulators regulate transcription as they modulate the interactions between enhancers and promoters by organizing the chromatin into distinct domains. To gain better understanding of the nature of chromatin domains defined by insulators, we analyzed the ability of an insulator to interfere in VDJ recombination, a process that is critically dependent on long-range interactions between diverse types of cis-acting DNA elements. A well-established CTCF-dependent transcriptional insulator, H19 imprint control region (H19-ICR), was inserted in the mouse TCRβ locus by genetic manipulation. Analysis of the mutant mice demonstrated that the insulator retains its CTCF and position-dependent enhancer-blocking potential in this heterologous context in vivo. Remarkably, the inserted H19-ICR appears to have the ability to modulate cis-DNA interactions between recombination signal sequence elements of the TCRβ locus leading to a dramatically altered usage of Vβ segments for Vβ-to-DβJβ recombination in the mutant mice. This reveals a novel ability of CTCF to govern long range cis-DNA interactions other than enhancer–promoter interactions and suggests that CTCF-dependent insulators may play a diverse and complex role in genome organization beyond transcriptional control. Our functional analysis of mutated TCRβ locus supports the emerging role of CTCF in governing VDJ recombination.

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

“…established a CTCF-dependent insulator (14). The inserted H19-ICR was able to bind CTCF when maternally inherited, as evidenced by ChIP analysis carried out in thymocytes of TCR-ins/ϩ, H19del13/H19del13 mice (Fig.…”

“…Alternatively, the LM-PCR products were subjected to Snapshot analysis (ABI Snapshot multiplex kit, catalog no. 4323159) that incorporated specific fluorescently labeled dideoxynucleotides by single-nucleotide primer extension based on nucleotide differences between domesticus and castaneus alleles as described previously (14).…”

“…Additionally, insertion of H19-ICR of the imprinted Igf2/H19 locus to the TCR␤ locus ( Fig. 1) led to organization of an ectopic CTCF-dependent insulator that effectively blocked the E␤ activity and led to impaired transcription and recombination patterns in the mutant mice (14).Organization of the ectopic insulator at the TCR␤ locus by H19-ICR (14) provided a very useful system, for several reasons, …”

“…Organization of the ectopic insulator at the TCR␤ locus by H19-ICR (14) provided a very useful system, for several reasons, to evaluate enhancer-promoter-insulator interactions and the impact of the insulator on various aspects of enhancer-mediated chromatin organization. First, enhancers at the AgR loci may arguably be more complex, as they regulate transcription as well as recombination.…”

“…The TCR␤ locus, in the wild-type and mutant TCR-ins and TCR-mut alleles (Fig. 1B) (14), provided a very useful system to dissect enhancerpromoter-insulator interaction in the wake of an enhancer activity of E␤ that differs from those of the enhancers at the Igf2/H19 locus.…”