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The establishment of a diverse B cell antigen receptor (BCR) repertoire by V(D)J recombination also generates autoreactive B cells. Anergy is one tolerance mechanism; it renders autoreactive B cells insensitive to stimulation by self-antigen, whereas Toll-like receptor (TLR) signaling can reactivate anergic B cells. Here, we describe a critical role of the transcription factor Ikaros in controlling BCR anergy and TLR signaling. Mice with specific deletion of Ikaros in mature B cells developed systemic autoimmunity. Ikaros regulated many anergy-associated genes, including Zfp318 , which is implicated in the attenuation of BCR responsiveness by promoting immunoglobulin D expression in anergic B cells. TLR signaling was hyperactive in Ikaros-deficient B cells, which failed to upregulate feedback inhibitors of the MyD88–nuclear factor κB signaling pathway. Systemic inflammation was lost on expression of a non-self-reactive BCR or loss of MyD88 in Ikaros-deficient B cells. Thus, Ikaros acts as a guardian preventing autoimmunity by promoting BCR anergy and restraining TLR signaling. Immature B cells expressing self-reactive BCRs induce anergy programs to promote tolerance. Busslinger and colleagues show that the transcription factor Ikaros enforces anergy by inducing transcription of negative-feedback regulators of the BCR and TLR–MyD88 pathways.

Nuclear processes, such as V(D)J recombination, are orchestrated by the three-dimensional organization of chromosomes at multiple levels, including compartments 1 and topologically associated domains (TADs) 2 , 3 consisting of chromatin loops 4 . TADs are formed by chromatin-loop extrusion 5 – 7 , which depends on the loop-extrusion function of the ring-shaped cohesin complex 8 – 12 . Conversely, the cohesin-release factor Wapl 13 , 14 restricts loop extension 10 , 15 . The generation of a diverse antibody repertoire, providing humoral immunity to pathogens, requires the participation of all V genes in V(D)J recombination 16 , which depends on contraction of the 2.8-Mb-long immunoglobulin heavy chain ( Igh ) locus by Pax5 17 , 18 . However, how Pax5 controls Igh contraction in pro-B cells remains unknown. Here we demonstrate that locus contraction is caused by loop extrusion across the entire Igh locus. Notably, the expression of Wapl is repressed by Pax5 specifically in pro-B and pre-B cells, facilitating extended loop extrusion by increasing the residence time of cohesin on chromatin. Pax5 mediates the transcriptional repression of Wapl through a single Pax5-binding site by recruiting the polycomb repressive complex 2 to induce bivalent chromatin at the Wapl promoter. Reduced Wapl expression causes global alterations in the chromosome architecture, indicating that the potential to recombine all V genes entails structural changes of the entire genome in pro-B cells. Pax5 regulates contraction of the immunoglobulin heavy chain ( Igh ) locus—an essential step in V(D)J recombination—by promoting chromatin loop extrusion via repression of Wapl expression.

Blimp-1 is known to act as a transcriptional repressor by suppressing genes associated with mature and activated germinal center B cells. Busslinger and colleagues show that Blimp-1 can also directly activate gene expression in plasma cells, including those encoding proteins that regulate the production of membrane-bound immunoglobulin versus secreted immunoglobulin. The transcription factor Blimp-1 is necessary for the generation of plasma cells. Here we studied its functions in plasmablast differentiation by identifying regulated Blimp-1 target genes. Blimp-1 promoted the migration and adhesion of plasmablasts. It directly repressed genes encoding several transcription factors and Aicda (which encodes the cytidine deaminase AID) and thus silenced B cell–specific gene expression, antigen presentation and class-switch recombination in plasmablasts. It directly activated genes, which led to increased expression of the plasma cell regulator IRF4 and proteins involved in immunoglobulin secretion. Blimp-1 induced the transcription of immunoglobulin genes by controlling the 3′ enhancers of the loci encoding the immunoglobulin heavy chain ( Igh ) and κ-light chain ( Igk ) and, furthermore, regulated the post-transcriptional expression switch from the membrane-bound form of the immunoglobulin heavy chain to its secreted form by activating Ell2 (which encodes the transcription-elongation factor ELL2). Notably, Blimp-1 recruited chromatin-remodeling and histone-modifying complexes to regulate its target genes. Hence, many essential functions of plasma cells are under the control of Blimp-1.

Extended loop extrusion across the immunoglobulin heavy-chain ( Igh ) locus facilitates V H -DJ H recombination following downregulation of the cohesin-release factor Wapl by Pax5, resulting in global changes in the chromosomal architecture of pro-B cells. Here, we demonstrate that chromatin looping and V K -J K recombination at the Igk locus were insensitive to Wapl upregulation in pre-B cells. Notably, the Wapl protein was expressed at a 2.2-fold higher level in pre-B cells compared with pro-B cells, which resulted in a distinct chromosomal architecture with normal loop sizes in pre-B cells. High-resolution chromosomal contact analysis of the Igk locus identified multiple internal loops, which likely juxtapose V K and J K elements to facilitate V K -J K recombination. The higher Wapl expression in Igμ-transgenic pre-B cells prevented extended loop extrusion at the Igh locus, leading to recombination of only the 6 most 3’ proximal V H genes and likely to allelic exclusion of all other V H genes in pre-B cells. These results suggest that pro-B and pre-B cells with their distinct chromosomal architectures use different chromatin folding principles for V gene recombination, thereby enabling allelic exclusion at the Igh locus, when the Igk locus is recombined. V gene recombination at the immunoglobulin heavy chain locus ( Igh ) is facilitated by extended loop extrusion. In this study, the authors find that, unlike Igh , the κ light chain locus does not involve extended loop extrusion but instead involves multiple, short-range loops for V gene combination.

Depletion of the cohesin-associated protein Wapl in mice is shown to increase the residence time of cohesin on DNA, which leads to clustering of cohesin in axial structures, and causes chromatin condensation in interphase chromosomes; the findings suggest that cohesin could have an architectural role in interphase chromosome organization. Wapl protein's role in chromosome organization Cohesin has important functions during chromosome segregation but is also thought to help organize chromatin fibres into loops during interphase. Here, Jan-Michael Peters and colleagues show that depletion of the cohesin-associated protein Wapl increases the residence time of cohesin on DNA, which in turn leads to clustering of cohesin in axial structures and causes chromatin condensation in interphase chromosomes. Depletion of Wapl also affects gene expression and leads to defects in chromosome segregation. These findings indicate that the dynamic interaction of cohesin with DNA, as mediated by Wapl, is an important determinant of chromatin structure, and that cohesin could have an architectural role in interphase chromosome organization. Mammalian genomes contain several billion base pairs of DNA that are packaged in chromatin fibres. At selected gene loci, cohesin complexes have been proposed to arrange these fibres into higher-order structures 1 , 2 , 3 , 4 , 5 , 6 , 7 , but how important this function is for determining overall chromosome architecture and how the process is regulated are not well understood. Using conditional mutagenesis in the mouse, here we show that depletion of the cohesin-associated protein Wapl 8 , 9 stably locks cohesin on DNA, leads to clustering of cohesin in axial structures, and causes chromatin condensation in interphase chromosomes. These findings reveal that the stability of cohesin–DNA interactions is an important determinant of chromatin structure, and indicate that cohesin has an architectural role in interphase chromosome territories. Furthermore, we show that regulation of cohesin–DNA interactions by Wapl is important for embryonic development, expression of genes such as c-myc (also known as Myc ), and cell cycle progression. In mitosis, Wapl-mediated release of cohesin from DNA is essential for proper chromosome segregation and protects cohesin from cleavage by the protease separase, thus enabling mitotic exit in the presence of functional cohesin complexes.

s Early B cell lymphopoiesis depends on E2A, Ebf1, Pax5 and Ikaros family members. In the present study, we used acute protein degradation in mice to identify direct target genes of these transcription factors in pro-B, small pre-B and immature B cells. E2A, Ebf1 and Pax5 predominantly function as transcriptional activators by inducing open chromatin at their target genes, have largely unique functions and are essential for early B cell maintenance. Ikaros and Aiolos act as dedicated repressors to cooperatively control early B cell development. The surrogate light-chain genes Igll1 and Vpreb1 are directly activated by Ebf1 and Pax5 in pro-B cells and directly repressed by Ikaros and Aiolos in small pre-B cells. Pax5 and E2A contribute to V(D)J recombination by activating Rag1 , Rag2 , Dntt , Irf4 and Irf8 . Similar to Pax5, Ebf1 also represses the cohesin-release factor gene Wapl to mediate prolonged loop extrusion across the Igh locus. In summary, in vivo protein degradation has provided unprecedented insight into the control of early B cell lymphopoiesis by five transcription factors. By use of a degron-mediated acute protein degradation model, Schwickert and colleagues are able to distinguish between direct and indirect gene targets of multiple transcription factors involved in early B cell development.

B-1a B cells are a distinct subset of mature B cells that provide innate-like protection against pathogens. Busslinger and colleagues identify the transcription factor Bhlhe41 as being essential for B-1a development and self-renewal. Innate-like B-1a cells provide a first line of defense against pathogens, yet little is known about their transcriptional control. Here we identified an essential role for the transcription factor Bhlhe41, with a lesser contribution by Bhlhe40, in controlling B-1a cell differentiation. Bhlhe41 −/− Bhlhe40 −/− B-1a cells were present at much lower abundance than were their wild-type counterparts. Mutant B-1a cells exhibited an abnormal cell-surface phenotype and altered B cell receptor (BCR) repertoire exemplified by loss of the phosphatidylcholine-specific V H 12V κ 4 BCR. Expression of a pre-rearranged V H 12V κ 4 BCR failed to 'rescue' the mutant phenotype and revealed enhanced proliferation accompanied by increased cell death. Bhlhe41 directly repressed the expression of cell-cycle regulators and inhibitors of BCR signaling while enabling pro-survival cytokine signaling. Thus, Bhlhe41 controls the development, BCR repertoire and self-renewal of B-1a cells.

The transcription factor Ikaros is required for lymphopoiesis. Busslinger and colleagues show Ikaros positively regulates genes encoding pre-BCR signal transducers and thereby promotes pro-B to pre-B cell progression and proliferation. The transcription factor Ikaros is an essential regulator of lymphopoiesis. Here we studied its B cell–specific function by conditional inactivation of the gene encoding Ikaros ( Ikzf1 ) in pro-B cells. B cell development was arrested at an aberrant 'pro-B cell' stage characterized by increased cell adhesion and loss of signaling via the pre-B cell signaling complex (pre-BCR). Ikaros activated genes encoding signal transducers of the pre-BCR and repressed genes involved in the downregulation of pre-BCR signaling and upregulation of the integrin signaling pathway. Unexpectedly, derepression of expression of the transcription factor Aiolos did not compensate for the loss of Ikaros in pro-B cells. Ikaros induced or suppressed active chromatin at regulatory elements of activated or repressed target genes. Notably, binding of Ikaros and expression of its target genes were dynamically regulated at distinct stages of early B lymphopoiesis.

Pax5 is a critical regulator of B‐cell commitment. Here, we identified direct Pax5 target genes by streptavidin‐mediated ChIP‐chip analysis of pro‐B cells expressing in vivo biotinylated Pax5. By binding to promoters and enhancers, Pax5 directly regulates the expression of multiple transcription factor, cell surface receptor and signal transducer genes. One of the newly identified enhancers was shown by transgenic analysis to confer Pax5‐dependent B‐cell‐specific activity to the Nedd9 gene controlling B‐cell trafficking. Profiling of histone modifications in Pax5‐deficient and wild‐type pro‐B cells demonstrated that Pax5 induces active chromatin at activated target genes, while eliminating active chromatin at repressed genes in committed pro‐B cells. Pax5 rapidly induces these chromatin and transcription changes by recruiting chromatin‐remodelling, histone‐modifying and basal transcription factor complexes to its target genes. These data provide novel insight into the regulatory network and epigenetic regulation, by which Pax5 controls B‐cell commitment. The mechanism of Pax5‐mediated gene activation and repression during early B‐cell development is unclear. This study identifies Pax5‐binding sites in pro‐B cells and the changes in chromatin modifications induced by the recruitment of chromatin‐modifying and transcription factors.

Antibody secretion by plasma cells provides acute and long-term protection against pathogens. The high secretion potential of plasma cells depends on the unfolded protein response, which is controlled by the transcription factor Xbp1. Here, we analyzed the Xbp1-dependent gene expression program of plasma cells and identified Bhlha15 (Mist1) as the most strongly activated Xbp1 target gene. As Mist1 plays an important role in other secretory cell types, we analyzed in detail the phenotype of Mist1-deficient plasma cells in Cd23 -Cre Bhlha15 fl/fl mice under steady-state condition or upon NP-KLH immunization. Under both conditions, Mist1-deficient plasma cells were 1.4-fold reduced in number and exhibited increased IgM production and antibody secretion compared to control plasma cells. At the molecular level, Mist1 regulated a largely different set of target genes compared with Xbp1. Notably, expression of the Blimp1 protein, which is known to activate immunoglobulin gene expression and to contribute to antibody secretion, was 1.3-fold upregulated in Mist1-deficient plasma cells, which led to a moderate downregulation of most Blimp1-repressed target genes in the absence of Mist1. Importantly, a 2-fold reduction of Blimp1 ( Prdm1 ) expression was sufficient to restore the cell number and antibody expression of plasma cells in Prdm1 Gfp/+ Cd23 -Cre Bhlha15 fl/fl mice to the same level seen in control mice. Together, these data indicate that Mist1 restricts antibody secretion by restraining Blimp1 expression, which likely contributes to the viability of plasma cells.