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

Abstract

CTCF-mediated chromatin interactions influence organization and function of mammalian genome in diverse ways. We analyzed the interactions among CTCF binding sites (CBS) at the murine TCRb locus to discern the role of CTCFmediated interactions in the regulation of transcription and VDJ recombination. Chromosome conformation capture analysis revealed thymocyte-specific long-range intrachromosomal interactions among various CBS across the locus that were relevant for defining the limit of the enhancer Eb-regulated recombination center (RC) and for facilitating the spatial proximity of TCRb variable (V) gene segments to the RC. Ectopic CTCF binding in the RC region, effected via genetic manipulation, altered CBS-directed chromatin loops, interfered with RC establishment, and reduced the spatial proximity of the RC with Trbv segments. Changes in chromatin loop organization by ectopic CTCF binding were relatively modest but influenced transcription and VDJ recombination dramatically. Besides revealing the importance of CTCF-mediated chromatin organization for TCRb regulation, the observed chromatin loops were consistent with the emerging idea that CBS orientations influence chromatin loop organization and underscored the importance of CBS orientations for defining chromatin architecture that supports VDJ recombination. Further, our study suggests that in addition to mediating long-range chromatin interactions, CTCF influences intricate configuration of chromatin loops that govern functional interactions between elements.KEYWORDS antigen receptor loci, CTCF, VDJ recombination, chromatin loop, chromatin organization S patial and temporal regulation of nuclear processes is intricately related to chromatin structure and organization since it can influence the interactions among regulatory elements. The mechanisms underlying these complex interactions are not completely understood. CTCF was identified to be the trans-acting factor that can organize an insulator and block enhancer-promoter interaction in a position dependent manner (1-3). Subsequently, CTCF has emerged as an important architectural protein that can influence interchromosomal and intrachromosomal interactions and impact nuclear functions largely in collaboration with cohesin (4, 5). Genome-wide investigations, including chromatin immunoprecipitation (ChIP), 4C, Hi-C, and chromatin interaction analysis by paired-end tag sequencing (ChIA-PET), etc., have established an important role of CTCF in organization of topologically associated domains (TADs) (6, 7). In addition, CTCF binding sites (CBS) located within TADs can contribute to celltype-specific chromatin loop organization by facilitating, as well as inhibiting, the interactions between regulatory elements. Locus-specific genetic analysis relying on deletion and inversion of CBS has revealed CBS orientation-dependent chromatin loop organization and its influence on transcriptional regulation (8-11). However, several aspects regarding the ability of CBS to organize chromatin and regulate function

DamC reveals principles of chromatin folding in vivo without crosslinking and ligation

“…However, it is unclear whether new loops can be established between endogenous and ectopically inserted CTCF sites. Early 3C observations suggested that ectopic sequences containing CTCF sites can change the surrounding chromosomal interactions39,40; however, experimental resolution in 3C did not allow to resolve single CTCF loops, and inserted sequences contained additional regulatory regions. Since piggyBac-TetO constructs alone do not perturb chromosome structure, we further engineered them to insert ectopic CTCF sites in the genome and detect resulting structural modifications without confounding effects.…”

Variable Extent of Lineage-Specificity and Developmental Stage-Specificity of Cohesin and CCCTC-Binding Factor Binding Within the Immunoglobulin and T Cell Receptor Loci

“…Also, in the TCRβ locus, the CTCF site near Eβ has been reported to interact very strongly with the poorly occupied CTCF site just upstream of the promoter of Dβ1, PDβ1, even though those CTCF sites are in tandem orientation. However, the CTCF site near PDβ1 is in the same HindIII fragment as PDβ1, so it is not clear if those 3C experiments are reveal CTCF–CTCF interactions or CTCF interactions with the PDβ1 promoter itself ( 81 ). We have previously documented that Eμ is recruited to the interacting CTCF sites at IGCR1 and 3′RR, and the same could be happening here ( 36 ).…”

Regulation of T-cell Receptor Gene Expression by Three-Dimensional Locus Conformation and Enhancer Function

“…Similar to the architectural changes observed at the TCRα/TCRδ locus during thymocyte development [ 50 ], distinct genomic conformations have been observed by confocal microscopy at the TCRβ locus during DN3a to DP differentiation [ 51 ] ( Figure 6 b). In DN3a thymocytes, the entire TCRβ locus presents a completely compacted configuration, bringing all Vβ gene segments into spatial proximity to the 25-kb TCRβ D-J region to assure a diverse TCRβ repertoire [ 51 , 59 , 60 , 61 , 62 , 63 ]. This locus architecture is thought to be mediated by CTCF/cohesin binding to CBSs associated with half of the Vβ gene segments (such as the A, B, and C sites), two intergenic sites located upstream of the Dβ gene segments (5′PC and D sites), and another one (E site) located downstream of the TCRβ enhancer (Eβ).…”

“…This locus architecture is thought to be mediated by CTCF/cohesin binding to CBSs associated with half of the Vβ gene segments (such as the A, B, and C sites), two intergenic sites located upstream of the Dβ gene segments (5′PC and D sites), and another one (E site) located downstream of the TCRβ enhancer (Eβ). CBSs associated to the Vβ gene segments are all oriented convergently toward the 5′PC, D, and E sites [ 47 , 63 ], allowing CTCF-CTCF looping formation. These loops bring the Vβ gene segments into the vicinity of the DβJβ region via locus contraction.…”

“…The TCRβ RC is defined by Eβ-Dβ1 promoter and Eβ-Dβ2 promoter loops formed in a 25-kb region [ 61 , 72 ], emphasizing Eβ as the crucial element in RC establishment ( Figure 6 b). In contrast with the TCRδ and TCRα RCs, where CTCF and cohesin bind to convergent CBSs and play a functional role in RC formation [ 52 , 53 , 71 ], no convergently oriented CBSs are found within the TCRβ RC (5′PC, D, and E sites have the same orientation) [ 47 , 63 ]. However, tandem CBSs flanking the TCRβ RC can create coiled loops that could contribute to the formation and stabilization of this RC as well as to the configuration of specific loops that facilitate RSS synapsis and recombination.…”