Site Specific and Orientation Dependent CTCF Binding Determines VDJ Recombination at Murine Tcrb Locus

Abstract

CTCF is a multifunctional protein that mediates long-range cis-DNA interactions in mammalian genomes. Chromatin architecture governs spatial and functional interactions of gene regulatory elements at various loci and is impacted by the ability of CTCF to restrict cohesin complex dependent chromatin extrusion. In addition, at antigen receptor loci, long-range interactions facilitate spatial proximity of gene segments for VDJ recombination that generates functional genes encoding immunoglobulins and T-cell receptors in developing lymphocytes. To investigate the role of CTCF in VDJ recombination, we mutated CTCF binding sites (CBS) of murine Tcrb locus. Our analysis revealed that CBS interspersed in the domain encompassing variable gene segments (Vb) are not redundant. They exhibit independent but additive effects on dynamic chromatin organization leading to distinct VDJ recombination profiles in CBS mutants depending on positions of mutated CBS relative to Vb segments. Further, inversion of a single CBS drastically altered the chromatin loop organization and VDJ recombination profile. Our results demonstrate the critical importance of chromatin extrusion for generation of chromatin loops for VDJ recombination and underscore its dynamic impediment by CTCF binding at specific points within Vb segment domain to be essential to diversify the usage of Vb segments for VDJ recombination at Tcrb locus.

Keywords: CTCF; Chromatin organization; VDJ recombination; antigen receptor loci; chromatin loop; chromosome conformation.