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