CCCTC-binding factor (CTCF) is largely responsible for the 3D architecture of the genome, in concert with the action of cohesin, through the creation of long-range chromatin loops. Cohesin is hypothesized to be the main driver of these long-range chromatin interactions by the process of loop extrusion. Here, we performed ChIP-seq for CTCF and cohesin in two stages each of T and B cell differentiation and examined the binding pattern in all six antigen receptor (AgR) loci in these lymphocyte progenitors and in mature T and B cells, ES cells, and fibroblasts. The four large AgR loci have many bound CTCF sites, most of which are only occupied in lymphocytes, while only the CTCF sites at the end of each locus near the enhancers or J genes tend to be bound in non-lymphoid cells also. However, despite the generalized lymphocyte restriction of CTCF binding in AgR loci, the Igκ locus is the only locus that also shows significant lineage-specificity (T vs. B cells) and developmental stage-specificity (pre-B vs. pro-B) in CTCF binding. We show that cohesin binding shows greater lineage- and stage-specificity than CTCF at most AgR loci, providing more specificity to the loops. We also show that the culture of pro-B cells in IL7, a common practice to expand the number of cells before ChIP-seq, results in a CTCF-binding pattern resembling pre-B cells, as well as other epigenetic and transcriptional characteristics of pre-B cells. Analysis of the orientation of the CTCF sites show that all sites within the large V portions of the Igh and TCRβ loci have the same orientation. This suggests either a lack of requirement for convergent CTCF sites creating loops, or indicates an absence of any loops between CTCF sites within the V region portion of those loci but only loops to the convergent sites at the D-J-enhancer end of each locus. The V region portions of the Igκ and TCRα/δ loci, by contrast, have CTCF sites in both orientations, providing many options for creating CTCF-mediated convergent loops throughout the loci. CTCF/cohesin loops, along with transcription factors, drives contraction of AgR loci to facilitate the creation of a diverse repertoire of antibodies and T cell receptors.

Summary The genome is organized into topologically associated domains (TAD) that enclose smaller subTADs. Here we identify and characterize an enhancer that is located in the middle of the V gene region of the immunoglobulin kappa light chain (Igκ) locus that becomes active preceding the stage at which this locus undergoes V(D)J recombination. This enhancer is a hub of long-range interactions connecting subTADs in the V gene region with the recombination center at the J genes. Deletion of this element results in a highly altered long-range interaction pattern across the locus and, importantly, affects individual V gene utilization locus-wide. These results indicate the existence of an enhancer-dependent framework in the Igκ locus, and further suggest that the composition of the diverse antibody repertoire is regulated in a subTAD-specific manner. This enhancer thus plays a structural role orchestrating the proper folding of the Igκ locus in preparation for V(D)J recombination.

Antigen receptor loci are organized into variable (V), diversity (D) and joining ( J) gene segments that rearrange to generate antigen receptor repertoires. Here, we identified an enhancer (E34) in the murine immunoglobulin kappa (Igk) locus that instructed rearrangement of V κ genes located in a sub-topologically associating domain, including a V κ gene encoding for antibodies targeting bacterial phosphorylcholine. We show that E34 instructs the nuclear repositioning of the E34 sub-topologically associating domain from a recombination-repressive compartment to a recombination-permissive compartment that is marked by equivalent activating histone modifications. Finally, we found that E34-instructed V κ -J κ rearrangement was essential to combat Streptococcus pneumoniae but not methicillin-resistant Staphylococcus aureus or influenza infections. We propose that the merging of V κ genes with J κ elements is instructed by one-dimensional epigenetic information imposed by enhancers across V κ and J κ genomic regions. The data also reveal how enhancers generate distinct antibody repertoires that provide protection against lethal bacterial infection.B cells generate diverse antibody repertoires through somatic recombination of distinct gene segments. Antibodies are encoded by the immunoglobulin heavy chain (Igh) locus, and by the immunoglobulin kappa (Igk) or the immunoglobulin lambda (Igl) loci. The Igh locus is segregated into variable (V), diversity (D), joining ( J) and constant (C) regions. The Igk and Igl loci are composed of V, J and C regions. Igh and Igl locus recombination occurs by deletion, while Igk rearrangement is mediated by deletion or inversion 1 . Immunoglobulin loci rearrangement is mediated by the Recombination-Activating gene 1 (Rag1) and Rag2 enzymes which cleave recombination signal sequences (RSSs) flanking the V, D and J gene segments 1 . During B cell development, rearrangement is mostly sequential. At the pro-B cell stage, D H -J H rearrangement precedes that of V H -D H J H joining. Pro-B cells that undergo V κ gene rearrangement largely give rise to B1a cells, but the majority of V κ -J κ rearrangement is initiated at the pre-B cell stage 2,3 . If V κ -J κ rearrangement is nonproductive or auto-reactive, V κ regions continue to recombine until a functional non-self-reactive V κ -J κ gene product is generated or proceeds to generate V λ -J λ joints 4 .

Enhancers are defined as regulatory elements that control transcription in a cell-type and developmental stage-specific manner. They achieve this by physically interacting with their cognate gene promoters. Significantly, these interactions can occur through long genomic distances since enhancers may not be near their cognate promoters. The optimal coordination of enhancerregulated transcription is essential for the function and identity of the cell. Although great efforts to fully understand the principles of this type of regulation are ongoing, other potential functions of the long-range chromatin interactions (LRCIs) involving enhancers are largely unexplored. We recently uncovered a new role for enhancer elements in determining the three-dimensional (3D) structure of the immunoglobulin kappa (Igκ) light chain receptor locus suggesting a structural function for these DNA elements. This enhancer-mediated locus configuration shapes the resulting Igκ repertoire. We also propose a role for enhancers as critical components of subtopologically associating domain (subTAD) formation and nuclear spatial localization.