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DNase I-hypersensitive site analysis of the murine IgH gene locus. ( A ) Schematic representation of the IgH locus with DNase I-hypersensitive sites. The complete locus including V H gene segments and all heavy chain isotypes is shown on the top line. The second line is a detailed schematic of part of the locus starting from the 5 ¢ D FL16.1 gene segment to 7 kb downstream of the C d membrane exon. The restriction endonuclease sites Eco RI (R), Ase I (A), Sac I (S), Kpn I (K), Sal I (Sa), Xho I (X) and Bgl II (B) were used for Southern blot analysis of DNase I-treated genomic DNA. Probes used in the study are labeled 1±6, and are shown below their corresponding hybridizing fragments. The position of the intronic m enhancer is marked with an oval m E. The nuclease-hypersenstive sites identi®ed are indicated by the bold arrowheads. ( B and C ) DNase I sensitivity assays. Nuclei from
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The immunoglobulin heavy chain (IgH) gene locus spans several megabases. We show that IgH activation during B-cell differentiation, as measured by histone acetylation, occurs in discrete, independently regulated domains. Initially, a 120 kb domain of germline DNA is hyperacetylated, that extends from DFL16.1, the 5′-most DH gene segment, to the int...
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... B-and T-cell developments are strictly controlled processes that ensure the selection of mature lymphocytes expressing a correctly rearranged and not autoreactive B-or T-cell receptor (BCR or TCR). B-cell precursors in the bone marrow (BM) are firstly selected on the basis of the expression of a functional pre-BCR (1)(2)(3)(4)(5), passing from large pre-B to small pre-B stages (Hardy fractions C′ and D (6)). Finally, those precursors that correctly express a functional IgM receptor on their surface (positive selection) and that do not recognize self-antigens (negative selection) are selected and develop into mature B cells. ...
The functional relevance of K⁺ and Ca²⁺ ion channels in the “Store Operated Calcium Entry” (SOCE) during B and T lymphocyte activation is well proven. However, their role in the process of T- and B- cell development and selection is still poorly defined. In this scenario, our aim was to characterize the expression of the ether à-go-go-related gene 1 (ERG1) and KV1.3 K⁺ channels during the early stages of mouse lymphopoiesis and analyze how they affect Ca²⁺signaling, or other signaling pathways, known to mediate selection and differentiation processes of lymphoid clones. We provide here evidence that the mouse (m)ERG1 is expressed in primary lymphoid organs, bone marrow (BM), and thymus of C57BL/6 and SV129 mice. This expression is particularly evident in the BM during the developmental stages of B cells, before the positive selection (large and small PreB). mERG1 is also expressed in all thymic subsets of both strains, when lymphocyte positive and negative selection occurs. Partially overlapping results were obtained for KV1.3 expression. mERG1 and KV1.3 were expressed at significantly higher levels in B-cell precursors of mice developing an experimental autoimmune encephalomyelitis (EAE). The pharmacological blockage of ERG1 channels with E4031 produced a significant reduction in intracellular Ca²⁺ after lymphocyte stimulation in the CD4⁺ and double-positive T-cell precursors’ subsets. This suggests that ERG1 might contribute to maintaining the electrochemical gradient responsible for driving Ca²⁺ entry, during T-cell receptor signaling which sustains lymphocyte selection checkpoints. Such role mirrors that performed by the shaker-type KV1.3 potassium channel during the activation process of mature lymphocytes. No effects on Ca²⁺ signaling were observed either in B-cell precursors after blocking KV1.3 with PSORA-4. In the BM, the pharmacological blockage of ERG1 channels produced an increase in ERK phosphorylation, suggesting an effect of ERG1 in regulating B-lymphocyte precursor clones’ proliferation and checkpoint escape. Overall, our results suggest a novel physiological function of ERG1 in the processes of differentiation and selection of lymphoid precursors, paving the way to further studies aimed at defining the expression and role of ERG1 channels in immune-based pathologies in addition to that during lymphocyte neoplastic transformation.
... IL-7Ra À/À pro-B cells in vivo displayed decreased non-coding transcription and recombination of 5 0 V H genes (Corcoran et al., 1998), prompting the hypothesis that the IL-7R influences Igh recombination through increasing accessibility of 5 0 V H genes, supported by studies linking IL-7R signaling and active histone modifications in the Igh locus (Chowdhury and Sen, 2001;Johnson et al., 2003;Xu et al., 2008), and suggesting that IL-7R activation of the Igh locus is mediated by STAT5 (Bertolino et al., 2005). However, conditional deletion of STAT5 was rescued by the pro-survival factor B-Cell Lymphoma-2 (BCL-2) with no deficiency in 5 0 V H recombination, suggesting that the dominant role of STAT5 was pro-B cell survival (Malin et al., 2010). ...
Generation of the primary antibody repertoire requires V(D)J recombination of hundreds of gene segments in the immunoglobulin heavy chain (Igh) locus. The role of interleukin-7 receptor (IL-7R) signaling in Igh recombination has been difficult to partition from its role in B cell survival and proliferation. With a detailed description of the Igh repertoire in murine IL-7Rα−/− bone marrow B cells, we demonstrate that IL-7R signaling profoundly influences VH gene selection during VH-to-DJH recombination. We find skewing toward 3′ VH genes during de novo VH-to-DJH recombination more severe than the fetal liver (FL) repertoire and uncover a role for IL-7R signaling in DH-to-JH recombination. Transcriptome and accessibility analyses suggest reduced expression of B lineage transcription factors (TFs) and targets and loss of DH and VH antisense transcription in IL-7Rα−/− B cells. Thus, in addition to its roles in survival and proliferation, IL-7R signaling shapes the Igh repertoire by activating underpinning mechanisms.
... To determine the impact of loss of Setd2/H3K36me3 on the Igh locus at the proB stage, we conducted chromatin immunoprecipitation sequencing (ChIP-Seq) and found both a global loss of H3K36me3 across the genome, as well as a focal loss on the Igh locus where a wellstudied critical regulatory region near the Eµ enhancer resides (Chowdhury et al., 2001) ( Figure 4A and Figure S5A). As accessibility of this region is critical for B cell development (Chowdhury et al., 2001;Chakraborty et al., 2009), we wanted to ascertain if the loss of H3K36me3 impacted local chromatin architecture or accessibility. ...
... To determine the impact of loss of Setd2/H3K36me3 on the Igh locus at the proB stage, we conducted chromatin immunoprecipitation sequencing (ChIP-Seq) and found both a global loss of H3K36me3 across the genome, as well as a focal loss on the Igh locus where a wellstudied critical regulatory region near the Eµ enhancer resides (Chowdhury et al., 2001) ( Figure 4A and Figure S5A). As accessibility of this region is critical for B cell development (Chowdhury et al., 2001;Chakraborty et al., 2009), we wanted to ascertain if the loss of H3K36me3 impacted local chromatin architecture or accessibility. In proB cells, ablation of H3K36me3 did not affect chromatin accessibility ( Figure 4B and Figure S5B) nor did it disrupt the local levels of H3K4me3 and H3K9ac ( Figure 4C), two histone modifications essential for maintaining an open and actively transcribed chromatin structure at this regulatory region (Chowdhury et al., 2001;Chakraborty et al., 2009) and, for H3K4me3, the recruitment and activation of the Rag2 protein itself (Shimazaki et al., 2014;Johnson et al., 2010;Ji et al., 2010;Matheson et al., 2012;Bettridge et al., 2017). ...
... As accessibility of this region is critical for B cell development (Chowdhury et al., 2001;Chakraborty et al., 2009), we wanted to ascertain if the loss of H3K36me3 impacted local chromatin architecture or accessibility. In proB cells, ablation of H3K36me3 did not affect chromatin accessibility ( Figure 4B and Figure S5B) nor did it disrupt the local levels of H3K4me3 and H3K9ac ( Figure 4C), two histone modifications essential for maintaining an open and actively transcribed chromatin structure at this regulatory region (Chowdhury et al., 2001;Chakraborty et al., 2009) and, for H3K4me3, the recruitment and activation of the Rag2 protein itself (Shimazaki et al., 2014;Johnson et al., 2010;Ji et al., 2010;Matheson et al., 2012;Bettridge et al., 2017). Loss of H3K36me3 did not significantly affect the methylation states of mono-, di-or tri-methyl lysine-27 or mono-and di-methyl lysine-36 residues in this region ( Figure S5C). ...
Repair of DNA double-stranded breaks (DSBs) during lymphocyte development is essential for V(D)J recombination and forms the basis of immunoglobulin variable region diversity. Understanding of this process in lymphogenesis has historically centered on the study of RAG1/2 recombinases and a set of classical non-homologous end-joining factors. Much less has been reported regarding the role of chromatin modifications on this process. Here, we show a role for the non-redundant histone H3 lysine methyltransferase, Setd2, and its modification of lysine-36 tri-methylation (H3K36me3), in the processing and joining of DNA ends during V(D)J recombination. Loss leads to mis-repair of Rag-induced DNA DSBs, especially when combined with loss of Atm kinase activity. Furthermore, loss reduces immune repertoire and a severe block in lymphogenesis as well as causing post-mitotic neuronal apoptosis. Together, these studies are suggestive of an important role of Setd2/H3K36me3 in these two mammalian developmental processes that are influenced by double-stranded break repair.
... joining (J) gene segments of the IgH locus with DH-JH rearrangement occurring prior to VH to DJH rearrangement (Chowdhury & Sen 2001). This process is reliant on multiple factors including IL-7, the transcription factors PAX5 and Yin Yang 1 as well as RAG1 and RAG2 expression (Liu et al. 2007;Oettinger et al. 1990;Xu et al. 2008). ...
Fc receptor-like 6 (FCRL6) is an immunoreceptor tyrosine-based inhibitory motif-bearing transmembrane receptor upregulated on human cytotoxic T and NK cells during chronic immune activation. It has been suggested to act as an inhibitory receptor by possibly interacting with human leukocyte antigen-DR (HLA-DR), but its function remains largely unknown. We initially investigated the role of FCRL6 in T cells using its murine counterpart. However, Fcrl6 expression was absent in developing, mature or activated mouse T cells. Therefore, we generated a human FCRL6 (hFCRL6) expressing transgenic mouse to investigate the function of this receptor. The expression of HLA-DR on antigen presenting cells resulted in reduced in vitro proliferation of hFCRL6+ CD4+ T cells. However, we were unable to recapitulate this inhibitory effect of the hFCRL6:HLA-DR axis in an in vivo environment. Further characterisation of mouse immune populations revealed Fcrl6 expression in natural killer (NK) and developing B cells. Analysis of Fcrl6-/- mice showed that the development of these cells as well as T cells and the splenic proportion of most major immune populations remained unaffected by FCRL6 deletion. We observed a reduction in the proportion of splenic macrophages and NK cells in Fcrl6-/- and NK conditional Fcrl6-/- mice, respectively. However, NK cell responses in a chronic retroviral infection model as well as a tumour model remained unaffected by FCRL6 deletion. Similarly, B cell antibody production in response to retroviral infection was also unaffected by FCRL6 deletion. Overall, data obtained here suggested that the mouse ortholog of FCRL6 might not possess the potential immunoregulatory capacity of its human counterpart and thus may have evolved to mediate non-immunoregulatory functions. Further studies will be required to define the role of mouse FCRL6 in NK cells and macrophages in addition to hFCRL6 in NK cells and T cells.
... In contrast, B-cell development within the fetal liver can occur independent of IL-7 38,39 . In terms of directing recombination, IL-7 and its downstream signaling component STAT5 have been shown to promote Igh accessibility and recombination [40][41][42] while actively inhibiting recombination of the Igk locus 34,43 . Activated STAT5 enters the nucleus and forms a complex with PRC2/EZH2, which binds to the intronic enhancer of Igk (iEκ) and induces H3K27me3-mediated repression to inhibit recombination of this locus 44,45 . ...
B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This ‘alternate pathway’ of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties. B-1a B cells are innate-like cells with biased reactivity to bacteria and self-antigens. Here the authors show that reduced interleukin-7 in developing fetal liver-derived pro-B cells induces premature immunoglobulin κ rearrangement, alleviating the requirement for a pre-BCR selection stage and allowing the generation of autoreactive B1-a B cells.
... In contrast, B-cell development within the fetal liver can occur independent of IL-7 38,39 . In terms of directing recombination, IL-7 and its downstream signaling component STAT5 have been shown to promote Igh accessibility and recombination [40][41][42] while actively inhibiting recombination of the Igk locus 34,43 . Activated STAT5 enters the nucleus and forms a complex with PRC2/EZH2, which binds to the intronic enhancer of Igk (iEκ) and induces H3K27me3-mediated repression to inhibit recombination of this locus 44,45 . ...
... The explanation given suggested that activation of particular V gene families for rearrangement might require different signals. Proximal V gene segments should become accessible first because V genes more distal to Cµ would require the presence of IL-7 for accessibility (14). Similar suggestions were made for early progenitors during adulthood (15,16). ...
... As in normal WT mice B cell development is not synchronized, this could not be clearly defined in earlier experiments. Along this line, it was also shown before that in B cell precursors isolated from Rag2 −/− mice the histone assembly of distal VH genes allowing their active recombination was highly dependent on IL-7 (14). In the present mouse model, it would be possible to investigate this phenomenon in more molecular details in vivo. ...
We employed the B-Indu-Rag1 model in which the coding exon of recombination-activating gene 1 (Rag1) is inactivated by inversion. It is flanked by inverted loxP sites. Accordingly, B cell development is stopped at the pro/pre B-I cell precursor stage. A B cell-specific Cre recombinase fused to a mutated estrogen receptor allows the induction of RAG1 function and B cell development by application of Tamoxifen. Since Rag1 function is recovered in a non-self-renewing precursor cell, only single waves of development can be induced. Using this system, we could determine that B cells minimally require 5 days to undergo development from pro/preB-I cells to the large and 6 days to the small preB-II cell stage. First immature transitional (T) 1 and T2 B cells could be detected in the bone marrow at day 6 and day 7, respectively, while their appearance in the spleen took one additional day. We also tested a contribution of adult bone marrow to the pool of B-1 cells. Sublethally irradiated syngeneic WT mice were adoptively transferred with bone marrow of B-Indu-Rag1 mice and B cell development was induced after 6 weeks. A significant portion of donor derived B-1 cells could be detected in such adult mice. Finally, early VH gene usage was tested after induction of B cell development. During the earliest time points the VH genes proximal to D/J were found to be predominantly rearranged. At later time points, the large family of the most distal VH prevailed.
... This result further supports a role for NFATc1 downstream of IL-7 signaling in the regulation of B cell developmental events, as IL-7 signaling has been reported to regulate IgH locus accessibility. 52,53 A threshold level of NFATc1 activity is essential for B cell differentiation NFATc1 is expressed from two distinct promoters. NFATc1α isoforms are directed from the distal P1 and NFATc1β isoform expression is directed from the proximal P2 promoter. ...
... 16,55 IL-7 signaling and EBF1 have been shown to regulate Ig gene rearrangement. 16,52,53 Again, the similarity in this aspect suggests that IL-7, NFATc1, and EBF1 all act in a linear axis (Supplementary Figure S5a) and that a deficiency in any one will lead to similar defects in B cell development. ...
B cell development in bone marrow is a precisely regulated complex process. Through successive stages of differentiation, which are regulated by a multitude of signaling pathways and an array of lineage-specific transcription factors, the common lymphoid progenitors ultimately give rise to mature B cells. Similar to early thymocyte development in the thymus, early B cell development in bone marrow is critically dependent on IL-7 signaling. During this IL-7-dependent stage of differentiation, several transcription factors, such as E2A, EBF1, and Pax5, among others, play indispensable roles in B lineage specification and maintenance. Although recent studies have implicated several other transcription factors in B cell development, the role of NFATc1 in early B cell developmental stages is not known. Here, using multiple gene-manipulated mouse models and applying various experimental methods, we show that NFATc1 activity is vital for early B cell differentiation. Lack of NFATc1 activity in pro-B cells suppresses EBF1 expression, impairs immunoglobulin gene rearrangement, and thereby preBCR formation, resulting in defective B cell development. Overall, deficiency in NFATc1 activity arrested the pro-B cell transition to the pre-B cell stage, leading to severe B cell lymphopenia. Our findings suggest that, along with other transcription factors, NFATc1 is a critical component of the signaling mechanism that facilitates early B cell differentiation.
... Precisely orchestrated assemblies of V, D, and J gene segments generate vast diversities of antigen receptors in developing B cells, and epigenetic modifications play pivotal roles in these V(D) J recombinations. Permissive histone modifications allow access to target sites by recombination complexes, therefore antigen receptor loci that are poised for V(D)J rearrangement are typically marked by permissive histone modifications, including H3Ac, H4Ac, H3K4me2, H3K4me3, and H3K79me2 [146][147][148][149][150][151][152][153][154][155]. H3K4me3 is directly recognized by the plant homeodomain (PHD) domain of RAG2. ...
... In contrast, B cell development within the fetal liver can occur independent of IL-7 37,38 . In terms of directing recombination, IL-7 and its downstream signaling component STAT5, have been shown to promote Igh accessibility and recombination [39][40][41] while actively inhibiting recombination of the Igk locus 33, 42 . Activated STAT5 enters the nucleus and forms a complex with PRC2/EZH2 which binds to the intronic enhancer . ...
B-1a cells are long-lived, self-renewing innate like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells they have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver (FL) versus bone marrow (BM) environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa ( Igk ) recombination at the early pro-B cell stage. As a result, B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain (SLC). This ‘alternate pathway’ of development enables the production of B cells with self reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing models of B-1a cell development and explain how these cells acquire their unique properties.
