Systemic Human ILC Precursors Provide a Substrate for Tissue ILC Differentiation.

Innate lymphoid cells (ILCs) represent innate versions of T helper and cytotoxic T cells that differentiate from committed ILC precursors (ILCPs). How ILCPs give rise to mature tissue-resident ILCs remains unclear. Here, we identify circulating and tissue ILCPs in humans that fail to express the transcription factors and cytokine outputs of mature ILCs but have these signature loci in an epigenetically poised configuration. Human ILCPs robustly generate all ILC subsets in vitro and in vivo. While human ILCPs express low levels of retinoic acid receptor (RAR)-related orphan receptor C (RORC) transcripts, these cells are found in RORC-deficient patients and retain potential for EOMES+ natural killer (NK) cells, interferon gamma-positive (IFN-γ+) ILC1s, interleukin (IL)-13+ ILC2s, and for IL-22+, but not for IL-17A+ ILC3s. Our results support a model of tissue ILC differentiation ("ILC-poiesis"), whereby diverse ILC subsets are generated in situ from systemically distributed ILCPs in response to local environmental signals.

Transcription factors and 3D genome conformation in cell-fate decisions.

How cells adopt different identities has long fascinated biologists. Signal transduction in response to environmental cues results in the activation of transcription factors that determine the gene-expression program characteristic of each cell type. Technological advances in the study of 3D chromatin folding are bringing the role of genome conformation in transcriptional regulation to the fore. Characterizing this role of genome architecture has profound implications, not only for differentiation and development but also for diseases including developmental malformations and cancer. Here we review recent studies indicating that the interplay between transcription and genome conformation is a driving force for cell-fate decisions.

Transcriptional Regulation by (Super)Enhancers: From Discovery to Mechanisms.

Accurate control of gene expression in the right cell at the right moment is of fundamental importance to animal development and homeostasis. At the heart of gene regulation lie the enhancers, a class of gene regulatory elements that ensures precise spatiotemporal activation of gene transcription. Mammalian genomes are littered with enhancers, which are frequently organized in cooperative clusters such as locus control regions and superenhancers. Here, we discuss our current knowledge of enhancer biology, including an overview of the discovery of the various enhancer subsets and the mechanistic models used to explain their gene regulatory function.

Epigenomic partitioning of a polygenic risk score for asthma reveals distinct genetically driven disease pathways.

BACKGROUND: Individual differences in susceptibility to develop asthma, a heterogeneous chronic inflammatory lung disease, are poorly understood. It remains debated whether genetics can predict asthma risk and how genetic variants modulate the complex pathophysiology of asthma. AIM: To build polygenic risk scores (PRSs) for asthma risk prediction and epigenomically link predictive genetic variants to pathophysiological mechanisms. METHODS: Restricted PRSs were constructed using single nucleotide variants derived from genome-wide association studies and validated using data generated in the Rotterdam Study, a Dutch prospective cohort of 14 926 individuals. Outcomes used were asthma, childhood-onset asthma (COA), adulthood-onset asthma (AOA), eosinophilic asthma, and asthma exacerbations. Genome-wide chromatin analysis data from 19 disease-relevant cell types were used for epigenomic PRS partitioning. RESULTS: PRSs obtained predicted asthma and related outcomes, with the strongest associations observed for COA (2.55 odds ratios per PRS standard deviation, area under the curve of 0.760). PRSs allowed for the classification of individuals into high and low-risk groups. PRS partitioning using epigenomic profiles identified 5 clusters of variants within putative gene regulatory regions linked to specific asthma-relevant cells, genes, and biological pathways. CONCLUSIONS: PRSs were associated with asthma(-related traits) in a Dutch prospective cohort, with substantially higher predictive power observed for COA than for AOA. Importantly, PRS variants could be epigenomically partitioned into clusters of regulatory variants with different pathophysiological association patterns and effect estimates, which likely represent distinct genetically driven disease pathways. Our findings have potential implications for personalized risk mitigation and treatment strategies.

Decoding the genetic and epigenetic basis of asthma.

Asthma is a complex and heterogeneous chronic inflammatory disease of the airways. Alongside environmental factors, asthma susceptibility is strongly influenced by genetics. Given its high prevalence and our incomplete understanding of the mechanisms underlying disease susceptibility, asthma is frequently studied in genome-wide association studies (GWAS), which have identified thousands of genetic variants associated with asthma development. Virtually all these genetic variants reside in non-coding genomic regions, which has obscured the functional impact of asthma-associated variants and their translation into disease-relevant mechanisms. Recent advances in genomics technology and epigenetics now offer methods to link genetic variants to gene regulatory elements embedded within non-coding regions, which have started to unravel the molecular mechanisms underlying the complex (epi)genetics of asthma. Here, we provide an integrated overview of (epi)genetic variants associated with asthma, focusing on efforts to link these disease associations to biological insight into asthma pathophysiology using state-of-the-art genomics methodology. Finally, we provide a perspective as to how decoding the genetic and epigenetic basis of asthma has the potential to transform clinical management of asthma and to predict the risk of asthma development.

Multi-source data approach for personalized outcome prediction in lung cancer screening: update from the NELSON trial.

Trials show that low-dose computed tomography (CT) lung cancer screening in long-term (ex-)smokers reduces lung cancer mortality. However, many individuals were exposed to unnecessary diagnostic procedures. This project aims to improve the efficiency of lung cancer screening by identifying high-risk participants, and improving risk discrimination for nodules. This study is an extension of the Dutch-Belgian Randomized Lung Cancer Screening Trial, with a focus on personalized outcome prediction (NELSON-POP). New data will be added on genetics, air pollution, malignancy risk for lung nodules, and CT biomarkers beyond lung nodules (emphysema, coronary calcification, bone density, vertebral height and body composition). The roles of polygenic risk scores and air pollution in screen-detected lung cancer diagnosis and survival will be established. The association between the AI-based nodule malignancy score and lung cancer will be evaluated at baseline and incident screening rounds. The association of chest CT imaging biomarkers with outcomes will be established. Based on these results, multisource prediction models for pre-screening and post-baseline-screening participant selection and nodule management will be developed. The new models will be externally validated. We hypothesize that we can identify 15-20% participants with low-risk of lung cancer or short life expectancy and thus prevent ~140,000 Dutch individuals from being screened unnecessarily. We hypothesize that our models will improve the specificity of nodule management by 10% without loss of sensitivity as compared to assessment of nodule size/growth alone, and reduce unnecessary work-up by 40-50%.

Overexpression of SH2-Containing Inositol Phosphatase Contributes to Chronic Lymphocytic Leukemia Survival.

Balanced activity of kinases and phosphatases downstream of the BCR is essential for B cell differentiation and function and is disturbed in chronic lymphocytic leukemia (CLL). In this study, we employed IgH.TEµ mice, which spontaneously develop CLL, and stable EMC CLL cell lines derived from these mice to explore the role of phosphatases in CLL. Genome-wide expression profiling comparing IgH.TEµ CLL cells with wild-type splenic B cells identified 96 differentially expressed phosphatase genes, including SH2-containing inositol phosphatase (Ship2). We found that B cell-specific deletion of Ship2, but not of its close homolog Ship1, significantly reduced CLL formation in IgH.TEµ mice. Treatment of EMC cell lines with Ship1/2 small molecule inhibitors resulted in the induction of caspase-dependent apoptosis. Using flow cytometry and Western blot analysis, we observed that blocking Ship1/2 abrogated EMC cell survival by exerting dual effects on the BCR signaling cascade. On one hand, specific Ship1 inhibition enhanced calcium signaling and thereby abrogated an anergic response to BCR stimulation in CLL cells. On the other hand, concomitant Ship1/Ship2 inhibition or specific Ship2 inhibition reduced constitutive activation of the mTORC1/ribosomal protein S6 pathway and downregulated constitutive expression of the antiapoptotic protein Mcl-1, in both EMC cell lines and primary IgH.TEµ CLL cells. Importantly, also in human CLL, we found overexpression of many phosphatases including SHIP2. Inhibition of SHIP1/SHIP2 reduced cellular survival and S6 phosphorylation and enhanced basal calcium levels in human CLL cells. Taken together, we provide evidence that SHIP2 contributes to CLL pathogenesis in mouse and human CLL.

Increased group 2 innate lymphoid cells in peripheral blood of adults with mastocytosis.

BACKGROUND: Systemic mastocytosis is a hematological disease in which aberrant mast cells accumulate because of gain-of-function mutations in the KIT receptor. Group 2 innate lymphoid cells (ILC2s) are effector cells of type 2 immune responses that also express KIT and colocalize with mast cells at barrier tissue sites. In mouse models, mast cell-ILC2 crosstalk can drive local inflammation. However, a possible role for ILC2s in the pathophysiology of mastocytosis remains unexplored. OBJECTIVE: We sought to characterize circulating ILC2s in a clinically diverse cohort of patients with mastocytosis. METHODS: We included 21 adults with systemic mastocytosis and 18 healthy controls. Peripheral blood ILC2 abundance and phenotype were analyzed by flow cytometry and correlated to clinical characteristics, including the presence of the D816V KIT mutation. RESULTS: ILC2 levels were significantly higher in D816V+ patients with mastocytosis compared with D816V- patients or healthy controls. We observed increased proportions of KIT+ ILC2s among patients with mastocytosis, regardless of D816V status. Patients with skin involvement and itch showed the highest levels of ILC2s, which was independent from atopy or serum tryptase levels. Allele-specific quantitative PCR showed that the vast majority of ILC2s did not carry the D816V mutation. CONCLUSIONS: Our findings suggest a role for ILC2s and pathogenic ILC2-mast cell crosstalk in mastocytosis. We hypothesize that a high cutaneous D816V+ mast cell burden alters the skin microenvironment to induce chronic local ILC2 activation and their dissemination into the circulation. Activated ILC2s could contribute to skin symptoms by producing inflammatory mediators and by further augmenting mast cell mediator release.

Bacterial lysate add-on therapy to reduce exacerbations in severe asthma: A double-blind placebo-controlled trial.

BACKGROUND: Asthma exacerbations are frequently induced by respiratory tract infections (RTIs). Bacterial lysates have been described to possess immune-modulatory effects and reduce RTIs as well as asthma symptoms in children. However, whether bacterial lysates have similar effects in adult asthma patients is unknown. AIMS: To reduce asthma exacerbations by add-on bacterial lysate therapy in adults with severe asthma and to characterize the clinical and immune-modulatory effects of this treatment. METHODS: Asthma patients (GINA 4) with ≥2 annual exacerbations in the previous year were included. The intervention regimen consisted of OM-85/placebo for 10 consecutive days per month for 6 months during two winter seasons. Primary end-point was the number of severe asthma exacerbations within 18 months. The study was approved by the national and local ethical review board and registered in the Dutch Trial Registry (NL5752). All participants provided written informed consent. RESULTS: Seventy-five participants were included (38 OM-85; 37 placebo). Exacerbation frequencies were not different between the groups after 18 months (incidence rate ratio 1.07, 95%CI [0.68-1.69], p = 0.77). With the use of OM-85, FEV1% increased by 3.81% (p = 0.04) compared with placebo. Nasopharyngeal swabs taken during RTIs detected a virus less frequently in patients using OM-85 compared to placebo (30.5% vs. 48.0%, p = 0.02). In subjects with type 2 inflammation adherent to the protocol (22 OM-85; 20 placebo), a non-statistically significant decrease in exacerbations in the OM-85 group was observed (IRR = 0.71, 95%CI [0.39-1.26], p = 0.25). Immune-modulatory effects included an increase in several plasma cytokines in the OM-85 group, especially IL-10 and interferons. Peripheral blood T- and B cell subtyping, including regulatory T cells, did not show differences between the groups. CONCLUSION: Although OM-85 may have immune-modulatory effects, it did not reduce asthma exacerbations in this heterogeneous severe adult asthma group. Post hoc analysis showed a potential clinical benefit in patients with type 2 inflammation.

The dynamic emergence of GATA1 complexes identified in in vitro embryonic stem cell differentiation and in vivo mouse fetal liver.

GATA1 is an essential transcriptional regulator of myeloid hematopoietic differentiation towards red blood cells. During erythroid differentiation, GATA1 forms different complexes with other transcription factors such as LDB1, TAL1, E2A and LMO2 ("the LDB1 complex") or with FOG1. The functions of GATA1 complexes have been studied extensively in definitive erythroid differentiation; however, the temporal and spatial formation of these complexes during erythroid development is unknown. We applied proximity ligation assay (PLA) to detect, localize and quantify individual interactions during embryonic stem cell differentiation and in mouse fetal liver (FL) tissue. We show that GATA1/LDB1 interactions appear before the proerythroblast stage and increase in a subset of the CD71+/TER119- cells to activate the terminal erythroid differentiation program in 12.5 day FL. Using Ldb1 and Gata1 knockdown FL cells, we studied the functional contribution of the GATA1/LDB1 complex during differentiation. This shows that the active LDB1 complex appears quite late at the proerythroblast stage of differentiation and confirms the power of PLA in studying the dynamic interaction of proteins in cell differentiation at the single cell level. We provide dynamic insight into the temporal and spatial formation of the GATA1 and LDB1 transcription factor complexes during hematopoietic development and differentiation.

Butyrate inhibits human mast cell activation via epigenetic regulation of FcεRI-mediated signaling.

BACKGROUND: Short-chain fatty acids (SCFAs) are fermented dietary components that regulate immune responses, promote colonic health, and suppress mast cell-mediated diseases. However, the effects of SCFAs on human mast cell function, including the underlying mechanisms, remain unclear. Here, we investigated the effects of the SCFAs (acetate, propionate, and butyrate) on mast cell-mediated pathology and human mast cell activation, including the molecular mechanisms involved. METHOD: Precision-cut lung slices (PCLS) of allergen-exposed guinea pigs were used to assess the effects of butyrate on allergic airway contraction. Human and mouse mast cells were co-cultured with SCFAs and assessed for degranulation after IgE- or non-IgE-mediated stimulation. The underlying mechanisms involved were investigated using knockout mice, small molecule inhibitors/agonists, and genomics assays. RESULTS: Butyrate treatment inhibited allergen-induced histamine release and airway contraction in guinea pig PCLS. Propionate and butyrate, but not acetate, inhibited IgE- and non-IgE-mediated human or mouse mast cell degranulation in a concentration-dependent manner. Notably, these effects were independent of the stimulation of SCFA receptors GPR41, GPR43, or PPAR, but instead were associated with inhibition of histone deacetylases. Transcriptome analyses revealed butyrate-induced downregulation of the tyrosine kinases BTK, SYK, and LAT, critical transducers of FcεRI-mediated signals that are essential for mast cell activation. Epigenome analyses indicated that butyrate redistributed global histone acetylation in human mast cells, including significantly decreased acetylation at the BTK, SYK, and LAT promoter regions. CONCLUSION: Known health benefits of SCFAs in allergic disease can, at least in part, be explained by epigenetic suppression of human mast cell activation.

The DNA-binding protein CTCF limits proximal Vκ recombination and restricts κ enhancer interactions to the immunoglobulin κ light chain locus.

Regulation of immunoglobulin (Ig) V(D)J gene rearrangement is dependent on higher-order chromatin organization. Here, we studied the in vivo function of the DNA-binding zinc-finger protein CTCF, which regulates interactions between enhancers and promoters. By conditional deletion of the Ctcf gene in the B cell lineage, we demonstrate that loss of CTCF allowed Ig heavy chain recombination, but pre-B cell proliferation and differentiation was severely impaired. In the absence of CTCF, the Igκ light chain locus showed increased proximal and reduced distal Vκ usage. This was associated with enhanced proximal Vκ and reduced Jκ germline transcription. Chromosome conformation capture experiments demonstrated that CTCF limits interactions of the Igκ enhancers with the proximal V(κ) gene region and prevents inappropriate interactions between these strong enhancers and elements outside the Igκ locus. Thus, although Ig gene recombination can occur in the absence of CTCF, it is a critical factor determining Vκ segment choice for recombination.

OneD: increasing reproducibility of Hi-C samples with abnormal karyotypes.

The three-dimensional conformation of genomes is an essential component of their biological activity. The advent of the Hi-C technology enabled an unprecedented progress in our understanding of genome structures. However, Hi-C is subject to systematic biases that can compromise downstream analyses. Several strategies have been proposed to remove those biases, but the issue of abnormal karyotypes received little attention. Many experiments are performed in cancer cell lines, which typically harbor large-scale copy number variations that create visible defects on the raw Hi-C maps. The consequences of these widespread artifacts on the normalized maps are mostly unexplored. We observed that current normalization methods are not robust to the presence of large-scale copy number variations, potentially obscuring biological differences and enhancing batch effects. To address this issue, we developed an alternative approach designed to take into account chromosomal abnormalities. The method, called OneD, increases reproducibility among replicates of Hi-C samples with abnormal karyotype, outperforming previous methods significantly. On normal karyotypes, OneD fared equally well as state-of-the-art methods, making it a safe choice for Hi-C normalization. OneD is fast and scales well in terms of computing resources for resolutions up to 5 kb.

The presence of CLL-associated stereotypic B cell receptors in the normal BCR repertoire from healthy individuals increases with age.

BACKGROUND: Aging is known to induce immunosenescence, resulting in alterations in both the innate and adaptive immune system. Here we evaluated the effects of aging on B cell subsets in peripheral blood of 155 immunologically healthy individuals in four age categories (range 20-95y) via multi-parameter flow cytometry. Furthermore, we studied the naive and antigen-experienced B cell receptor (BCR) repertoire of different age groups and compared it to the clonal BCR repertoire of chronic lymphocytic leukemia (CLL), a disease typically presenting in elderly individuals. RESULTS: Total numbers and relative frequencies of B cells were found to decline upon aging, with reductions in transitional B cells, memory cell types, and plasma blasts in the 70 + y group. The BCR repertoire of naive mature B cells and antigen-experienced B cells did not clearly alter until age 70y. Clear changes in IGHV gene usage were observed in naive mature B cells of 70 + y individuals, with a transitional pattern in the 50-70y group. IGHV gene usage of naive mature B cells of the 50-70y, but not the 70 + y, age group resembled that of both younger (50-70y) and older (70 + y) CLL patients. Additionally, CLL-associated stereotypic BCR were found as part of the healthy control BCR repertoire, with an age-associated increase in frequency of several stereotypic BCR (particularly subsets #2 and #5). CONCLUSION: Composition of the peripheral B cell compartment changes with ageing, with clear reductions in non-switched and CD27 + IgG+ switched memory B cells and plasma blasts in especially the 70 + y group. The BCR repertoire is relatively stable until 70y, whereafter differences in IGHV gene usage are seen. Upon ageing, an increasing trend in the occurrence of particular CLL-associated stereotypic BCR is observed.

Epigenome analysis links gene regulatory elements in group 2 innate lymphocytes to asthma susceptibility.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are major producers of the cytokines driving allergic asthma, and increased ILC2 numbers have been detected in blood and sputum of asthmatic patients. Asthma susceptibility has a strong genetic component, but the underlying mechanisms and whether asthma genetics affect ILC2 biology remain unclear. OBJECTIVE: We sought to study the ILC2 transcriptome and epigenome during airway inflammation (AI) to couple these to genes and genetic variants associated with asthma pathogenesis. METHODS: Mice harboring a reporter for the key ILC2 transcription factor GATA-3 were subjected to IL-33-driven AI, and ILC2s were isolated from bronchoalveolar lavage fluid and mediastinal lymph nodes. Human ILC2s were purified from peripheral blood and activated in vitro. We used RNA sequencing, genome-wide identification of histone-3 lysine-4 dimethylation-marked chromatin, and computational approaches to study the ILC2 transcriptome and epigenome. RESULTS: Activated ILC2s in mice displayed a tissue-specific gene expression signature that emerged from remarkably similar epigenomes. We identified superenhancers implicated in controlling ILC2 identity and asthma-associated genes. More than 300 asthma-associated genetic polymorphisms identified in genome-wide association studies localized to H3K4Me2+ gene regulatory elements in ILC2s. A refined set of candidate causal asthma-associated variants was uniquely enriched in ILC2, but not TH2 cell, regulatory regions. CONCLUSIONS: ILC2s in AI use a flexible epigenome that couples adaptation to new microenvironments with functional plasticity. Importantly, we reveal strong correlations between gene regulatory mechanisms in ILC2s and the genetic basis of asthma, supporting a pathogenic role for ILC2s in patients with allergic asthma.

A cellular and molecular view of T helper 17 cell plasticity in autoimmunity.

Since the original identification of the T helper 17 (Th17) subset in 2005, it has become evident that these cells do not only contribute to host defence against pathogens, such as bacteria and fungi, but that they are also critically involved in the pathogenesis of many autoimmune diseases. In contrast to the classic Th1 and Th2 cells, which represent rather stably polarized subsets, Th17 cells display remarkable heterogeneity and plasticity. This has been attributed to the characteristics of the key transcription factor that guides Th17 differentiation, retinoic acid receptor-related orphan nuclear receptor gamma (RORγ). Unlike the 'master regulators' T-bet and GATA3 that orchestrate Th1 and Th2 differentiation, respectively, RORγ controls transcription at relatively few loci in Th17 cells. Moreover, its expression is not stabilized by positive feedback loops but rather influenced by environmental cues, allowing for substantial functional plasticity. Importantly, a subset of IL-17/IFNγ double-producing Th17 cells was identified in both human and mouse models. Evidence is accumulating that these IL-17/IFNγ double-producing cells are pathogenic drivers in autoimmune diseases, including rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. In addition, IL-17/IFNγ double-producing cells have been identified in disorders in which the role of autoimmunity remains unclear, such as sarcoidosis. The observed plasticity of Th17 cells towards the Th1 phenotype can be explained by extensive epigenetic priming of the IFNG locus in Th17 cells. In fact, Th17 cells display an IFNG chromatin landscape that is remarkably similar to that of Th1 cells. On the other hand, pathogenic capabilities of Th17 cells can be restrained by stimulating IL-10 production and transdifferentiation into IL-10 producing T regulatory type 1 (Tr1) cells. In this review, we discuss recent advances in our knowledge on the cellular and molecular mechanisms involved in Th17 differentiation, heterogeneity and plasticity. We focus on transcriptional regulation of the Th17 expression program, the epigenetic dynamics involved, and how genetic variants associated with autoimmunity may affect immune responses through distal gene regulatory elements. Finally, the implications of Th17 cell plasticity for the pathogenesis and treatment of human autoimmune diseases will be discussed.

The genome-wide dynamics of the binding of Ldb1 complexes during erythroid differentiation.

One of the complexes formed by the hematopoietic transcription factor Gata1 is a complex with the Ldb1 (LIM domain-binding protein 1) and Tal1 proteins. It is known to be important for the development and differentiation of the erythroid cell lineage and is thought to be implicated in long-range interactions. Here, the dynamics of the composition of the complex-in particular, the binding of the negative regulators Eto2 and Mtgr1-are studied, in the context of their genome-wide targets. This shows that the complex acts almost exclusively as an activator, binding a very specific combination of sequences, with a positioning relative to transcription start site, depending on the type of the core promoter. The activation is accompanied by a net decrease in the relative binding of Eto2 and Mtgr1. A Chromosome Conformation Capture sequencing (3C-seq) assay also shows that the binding of the Ldb1 complex marks genomic interaction sites in vivo. This establishes the Ldb1 complex as a positive regulator of the final steps of erythroid differentiation that acts through the shedding of negative regulators and the active interaction between regulatory sequences.

Long-range gene regulation and novel therapeutic applications.

An intimate relationship exists between nuclear architecture and gene activity. Unraveling the fine-scale three-dimensional structure of the genome and its impact on gene regulation is a major goal of current epigenetic research, one with direct implications for understanding the molecular mechanisms underlying human phenotypic variation and disease susceptibility. In this context, the novel revolutionary genome editing technologies and emerging new ways to manipulate genome folding offer new promises for the treatment of human disorders.

Pre-B cell receptor signaling induces immunoglobulin κ locus accessibility by functional redistribution of enhancer-mediated chromatin interactions.

During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline V(κ) transcription. To investigate whether pre-BCR signaling modulates V(κ) accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ∼3.2 Mb V(κ) region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3'κ enhancer with V(κ) genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and V(κ) genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used V(κ) genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the V(κ) region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the V(κ) region, whereby the two enhancers play distinct roles.