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Age-associated/autoimmune B cells (ABCs) are an emerging B cell subset with aberrant expansion in systemic lupus erythematosus. ABC generation and differentiation exhibit marked sexual dimorphism, and Toll-like receptor 7 (TLR-7) engagement is a key contributor to these sex differences. ABC generation is also controlled by interleukin-21 (IL-21) and its interplay with interferon-γ and IL-4. This study was undertaken to investigate whether IL-13 receptor α1 (IL-13Rα1), an X-linked receptor that transmits IL-4/IL-13 signals, regulates ABCs and lupus pathogenesis.Mice lacking DEF-6 and switch-associated protein 70 (double-knockout [DKO]), which preferentially develop lupus in females, were crossed with IL-13Rα1-knockout mice. IL-13Rα1-knockout male mice were also crossed with Y chromosome autoimmune accelerator (Yaa) DKO mice, which overexpress TLR-7 and develop severe disease. ABCs were assessed using flow cytometry and RNA-Seq. Lupus pathogenesis was evaluated using serologic and histologic analyses.ABCs expressed higher levels of IL-13Rα1 than follicular B cells. The absence of IL-13Rα1 in either DKO female mice or Yaa DKO male mice decreased the accumulation of ABCs, the differentiation of ABCs into plasmablasts, and autoantibody production. Lack of IL-13Rα1 also prolonged survival and delayed the development of tissue inflammation. IL-13Rα1 deficiency diminished in vitro generation of ABCs, an effect that, surprisingly, could be observed in response to IL-21 alone. RNA-Seq revealed that ABCs lacking IL-13Rα1 down-regulated some histologic characteristics of B cells but up-regulated myeloid markers and proinflammatory mediators.Our findings indicate a novel role for IL-13Rα1 in controlling ABC generation and differentiation, suggesting that IL-13Rα1 contributes to these effects by regulating a subset of IL-21-mediated signaling events. These results also suggest that X-linked genes besides TLR7 participate in the regulation of ABCs in lupus.

Proper chromosome segregation is essential to avoid aneuploidy, yet this process fails with increasing age in mammalian oocytes. Here we report a role for the scarcely described protein CENP-V in oocyte spindle formation and chromosome segregation. We show that depending on the oocyte maturation state, CENP-V localizes to centromeres, to microtubule organizing centers, and to spindle microtubules. We find that Cenp-V−/− oocytes feature severe deficiencies, including metaphase I arrest, strongly reduced polar body extrusion, increased numbers of mis-aligned chromosomes and aneuploidy, multipolar spindles, unfocused spindle poles and loss of kinetochore spindle fibres. We also show that CENP-V protein binds, diffuses along, and bundles microtubules in vitro. The spindle assembly checkpoint arrests about half of metaphase I Cenp-V−/− oocytes from young adults only. This finding suggests checkpoint weakening in ageing oocytes, which mature despite carrying mis-aligned chromosomes. Thus, CENP-V is a microtubule bundling protein crucial to faithful oocyte meiosis, and Cenp-V−/− oocytes reveal age-dependent weakening of the spindle assembly checkpoint., Chromosome segregation is essential to avoid aneuploidy, yet in mammalian oocytes it progressively fails in an age-dependent manner. Here the authors identify CENP-V as a microtubule binding and bundling protein crucial to faithful oocyte meiosis, and present Cenp-V−/− oocytes as revealing age-dependent weakening of the spindle assembly checkpoint.

Crosslinking of FceRI-bound IgE triggers the release of a large number of biologically active, potentially anaphylactic compounds by mast cells. FceRI activation ought to be well-controlled to restrict adverse activation. As mast cells are embedded in tissues, adhesion molecules may contribute to limiting premature activation. Here, we report that E-Cadherin serves that purpose. Having confirmed that cultured mast cells express E-Cadherin, a mast-cell-specific E-Cadherin deficiency, Mcpt5-Cre E-Cdhfl/fl mice, was used to analyze mast cell degranulation in vitro and in vivo. Cultured peritoneal mast cells from Mcpt5-Cre E-Cdhfl/fl mice were normal with respect to many parameters but showed much-enhanced degranulation in three independent assays. Soluble E-Cadherin reduced the degranulation of control cells. The release of some newly synthesized inflammatory cytokines was decreased by E-Cadherin deficiency. Compared to controls, Mcpt5-Cre E-Cdhfl/fl mice reacted much stronger to IgE-dependent stimuli, developing anaphylactic shock. We suggest E-Cadherin-mediated tissue interactions restrict mast cell degranulation to prevent their precocious activation.

Previous data showed that meiotic cohesin SMC1β protects spermatocyte telomeres from damage. The underlying reason, however, remained unknown as the expressions of telomerase and shelterin components were normal inSmc1β−/−spermatocytes. Here. we report that SMC1β restricts expression of the long noncoding RNA TERRA (telomeric repeat containing RNA) in spermatocytes. In somatic cell lines increased TERRA was reported to cause telomere damage through altering telomere chromatin structure. InSmc1β−/−spermatocytes, we observed strongly increased levels of TERRA which accumulate on damaged chromosomal ends, where enhanced R-loop formation was found. This suggested a more open chromatin configuration near telomeres inSmc1β−/−spermatocytes, which was confirmed by ATAC-seq. Telomere-distal regions were not affected by the absence of SMC1β but RNA-seq revealed increased transcriptional activity in telomere-proximal regions. Thus, SMC1β promotes closed chromatin specifically near telomeres and limits TERRA expression in spermatocytes.

Although GM-CSF has been widely used in dendritic cell (DC) research, the mechanisms, factors, and signals regulating steady-state differentiation and maturation of GM-CSF–dependent DCs are insufficiently known. We found that the absence, individually or combined, of the related proteins DEF6 and SWAP-70 strongly enhances differentiation of murine GM-CSF–derived DCs. Contrasting SWAP-70, control through DEF6 does not depend on RHOA activation. DEF6 deficiency leads to expression of the DC-specific transcription factor ZBTB46 and prolonged STAT5 activation in GM-CSF cultures. SWAP-70 and DEF6-mediated restriction of DC differentiation converges mechanistically at the NF-κB pathway. DEF6 acts at early stages of DC differentiation in CD115−cKIT+ myeloid DC progenitors, whereas SWAP-70 acts subsequently. SWAP-70 and DEF6 regulate steady-state DC cytokine expression as well as in vivo accumulation in lymphatic tissue of migratory DCs. Our studies thus elucidate previously unknown roles of two closely related factors with distinct and complementary activities in DC differentiation and steady-state DC function.

In T cells, processes such as migration and immunological synapse formation are accompanied by the dynamic reorganization of the actin cytoskeleton, which has been suggested to be mediated by regulators of RhoGTPases and by F-actin bundlers. SWAP-70 controls F-actin dynamics in various immune cells, but its role in T cell development and function has remained incompletely understood. CD4

Differences in immune responses to viruses and autoimmune diseases such as systemic lupus erythematosus (SLE) can show sexual dimorphism. Age-associated B cells (ABC) are a population of CD11c+T-bet+ B cells critical for antiviral responses and autoimmune disorders. Absence of DEF6 and SWAP-70, two homologous guanine exchange factors, in double-knock-out (DKO) mice leads to a lupus-like syndrome in females marked by accumulation of ABCs. Here we demonstrate that DKO ABCs show sex-specific differences in cell number, upregulation of an ISG signature, and further differentiation. DKO ABCs undergo oligoclonal expansion and differentiate into both CD11c+ and CD11c− effector B cell populations with pathogenic and pro-inflammatory function as demonstrated by BCR sequencing and fate-mapping experiments. Tlr7 duplication in DKO males overrides the sex-bias and further augments the dissemination and pathogenicity of ABCs, resulting in severe pulmonary inflammation and early mortality. Thus, sexual dimorphism shapes the expansion, function and differentiation of ABCs that accompanies TLR7-driven immunopathogenesis., Autoimmunity mediated by age-associated B cells (ABC) can affect males and females differently. Here, using a lupus-like mouse model that affects females more severely, the authors observe an ABC mediated and guanine nucleotide exchange factor (GEF) restrained pathogenic process involving TLR7.

Sex differences characterize immune responses to viruses and autoimmune diseases like SLE. ABCs are an emerging population of CD11c+ T-bet+ B cells critical for antiviral responses and autoimmune disorders. DEF6 and SWAP70, are two homologous molecules whose combined absence in double-knock-out mice (DKOs) leads to a lupus syndrome in females marked by an accumulation of ABCs. Here we demonstrate that DKO ABCs exhibit sex-specific differences in their expansion, upregulation of an ISG signature, and further differentiation. BCR sequencing and fate mapping experiments reveal that DKO ABCs undergo oligoclonal expansion and differentiate into both CD11c+ and CD11c- effector populations with pathogenic and proinflammatory potential. Tlr7 duplication in DKO males overrides the sex-bias and further augments the dissemination and pathogenicity of ABCs resulting in severe pulmonary inflammation and early mortality. Thus, sexual dimorphism shapes the expansion, function, and differentiation of ABCs contributing to the sex-bias that accompanies TLR7-driven immunopathogenesis.

Defects of filaggrin (FLG) compromise epidermal barrier function and represent an important known genetic risk factor for atopic dermatitis (AD), but also for systemic atopy, including allergic sensitization and asthma. The flaky tail mouse model, widely used to address mechanisms of atopy induction by barrier-defective skin, harbors two mutations that affect the skin barrier, the mutation Flgft, resulting in near-complete loss of FLG expression, and the matted mutation inactivating transmembrane protein 79 (Tmem79). Upon separation of the two mutant loci, which are closely linked on chromosome 3, mice defective only for Tmem79 featured pronounced dermatitis and systemic atopy. Upon extensive backcross to BALB/c, also Flgft/ft mice (assumed to be wild type for Tmem79), developed AD-like dermatitis and reproduced the human ‘atopic march’, with high IgE levels and spontaneous asthma, suggesting a key role for functional Flg in protection from atopy also in mice. In contrast, BALB/c mice congenic for a targeted Flg knock out mutation did not develop skin inflammation or atopy. To resolve this discrepancy, we generated Flg-deficient mice on a pure BALB/c background by inactivating the Flg gene in BALB/c embryos. These animals feature an ichthyosis phenotype, but do not develop spontaneous dermatitis or systemic atopy. We sequenced the genome of the atopic Flgft BALB/c congenics and discovered that they were unexpectedly homozygous for the atopy-causing Tmem79matted mutation. In summary, we show that Flg-deficiency does not cause atopy in mice. This finding is in line with lack of atopic disease in a fraction of Ichthyosis vulgaris patients carrying two FLG null alleles. However, absence of FLG may promote and modulate dermatitis caused by other genetic barrier defects, as skin inflammation in Tmem79ma/maFlgft/ft BALB/c congenics is qualitatively different compared to Tmem79ma/ma mice.

In mitotic cells, establishment of sister chromatid cohesion requires acetylation of the cohesin subunit SMC3 (acSMC3) by ESCO1 and/or ESCO2. Meiotic cohesin plays additional but poorly understood roles in the formation of chromosome axial elements (AEs) and synaptonemal complexes. Here, we show that levels of ESCO2, acSMC3, and the pro-cohesion factor sororin increase on meiotic chromosomes as homologs synapse. These proteins are less abundant on the largely unsynapsed sex chromosomes, whose sister chromatid cohesion appears weaker throughout the meiotic prophase. Using three distinct conditionalEsco2knockout mouse strains, we demonstrate that ESCO2 is essential for male gametogenesis. Partial depletion of ESCO2 in prophase I spermatocytes delays chromosome synapsis and further weakens cohesion along sex chromosomes, which show extensive separation of AEs into single chromatids. Unsynapsed regions of autosomes are associated with the sex chromatin and also display split AEs. This study provides the first evidence for a specific role of ESCO2 in mammalian meiosis, identifies a particular ESCO2 dependence of sex chromosome cohesion and suggests support of autosomal synapsis by acSMC3-stabilized cohesion.

F-actin binding and bundling are crucial to a plethora of cell processes, including morphogenesis, migration, adhesion and many others. SWAP-70 was recently described as an in vitro F-actin-binding and -bundling protein. Fluorescence cross-correlation spectroscopy measurements with purified recombinant SWAP-70 confirmed that it forms stable oligomers that facilitate F-actin bundling. However, it remained unclear how SWAP-70 oligomerization and F-actin binding are controlled in living cells. We addressed this by biophysical approaches, including seFRET, FACS-FRET and FLIM-FRET. PIP3-mediated association with the cytoplasmic membrane and non-phosphorylated Y426 are required for SWAP-70 to dimerize and to bind F-actin. The dimerization region was identified near the C terminus where R546 is required for dimerization and, thus, F-actin bundling. The in vitro and in vivo data presented here reveal the functional relationship between the cytoplasm-to-membrane translocation and dimerization of SWAP-70, and F-actin binding and bundling, and demonstrate that SWAP-70 is a finely controlled modulator of membrane-proximal F-actin dynamics.This article has an associated First Person interview with the first author of the paper.

Introduction: Cohesin complex genes are commonly mutated in cancer particularly in myeloid malignancies. Yet patients with germline mutations in cohesin genes, leading to cohesinopathies like Cornelia-de-Lange syndrome (CdLS) are generally not known to be tumor-prone. The complex plays a major role in chromosome alignment and segregation (Uhlmann, Nature Reviews Molecular Cell Biology, 2016), homologous recombination-driven DNA repair (Ström et al., Molecular Cell, 2004) and regulation of gene expression (Busslinger et al., Nature, 2017). To deepen the understanding of cohesin variants in cancer predisposition, we performed TRIO Sequencing in two independent pediatric cancer cohorts. Thereby, we identified a novel recurrent heterozygous germline variant in the cohesin gene RAD21 not described in CdLS patients , located in the binding domain of the cofactors WAPL and PDS5B . Methods: Whole exome sequencing (WES) in a TRIO (child-parent datasets) setting was carried out in two independent, unselected cancer cohorts (TRIO-D, n=158 (Wagener et al., European Journal of Human Genetics, 2021) and TRIO-DD, n=60). To investigate the oncogenic potential of the novel RAD21 variant molecular and functional assessment was performed focusing on potential implications on the complex. Results: The newly identified RAD21 variant at amino acid position 298 resulting in a Proline to Serine (p.P298S) and a Proline to Alanine exchange, respectively, (p.P298A) is only rarely mutated in the general population (gnomAD database n=118,479; RAD21 p.P298S MAF To assess the influence of RAD21 p.P298S/A on the binding capacity of the complex, RAD21 variants and the wildtype (WT) were cloned and transfected into HEK293T cells, respectively. Immunoprecipitation analysis of RAD21 with the cofactors WAPL and PDS5B showed no differential binding between the WT and the variants, suggesting that RAD21 p.P298S/A does not impact the formation of the complex. Nevertheless, on a transcriptional level 83 genes were significantly differentially expressed in RAD21 p.P298S and p.P298A compared to the wildtype (fc>1.5, adj. p-value For cross-validation of the germline variant RAD21 p.P298S/A and its potential role in pediatric lymphoblastic malignancies, we analysed a third cohort of 150 children with relapsed ALL (IntReALL) for RAD21 p.P298S/A. We again identified RAD21 p.P298A in a boy (12y) with B-cell precursor acute lymphoblastic leukemia. To compare our data to a non-pediatric cancer setting, a cohort of 2300 young adults ( Conclusion: Taken together, we present for the first time the potential role of RAD21 germline variants in pediatric lymphoblastic malignancies. This may shed new light on the many roles of the cohesin complex and its implication outside the typical syndromal presentation. Disclosures No relevant conflicts of interest to declare.

Loss of FLG causes ichthyosis vulgaris. Reduced FLG expression compromises epidermal barrier function and is associated with atopic dermatitis, allergy, and asthma. The flaky tail mouse harbors two mutations that affect the skin barrier, Flgft, resulting in hypomorphic FLG expression, and Tmem79ma, inactivating TMEM79. Mice defective only for TMEM79 featured dermatitis and systemic atopy, but also Flgft/ft BALB/c congenic mice developed eczema, high IgE, and spontaneous asthma, suggesting that FLG protects from atopy. In contrast, a targeted Flg-knockout mutation backcrossed to BALB/c did not result in dermatitis or atopy. To resolve this discrepancy, we generated FLG-deficient mice on pure BALB/c background by inactivating Flg in BALB/c embryos. These mice feature an ichthyosis phenotype, barrier defect, and facilitated percutaneous sensitization. However, they do not develop dermatitis or atopy. Whole-genome sequencing of the atopic Flgft BALB/c congenics revealed that they were homozygous for the atopy-causing Tmem79matted mutation. In summary, we show that FLG deficiency does not cause atopy in mice, in line with lack of atopic disease in a fraction of patients with ichthyosis vulgaris carrying two Flg null alleles. However, the absence of FLG likely promotes and modulates dermatitis caused by other genetic barrier defects.

Lipids affect the membrane properties determining essential biological processes. Earlier studies have suggested a role of switch-activated protein 70 (SWAP-70) in lipid raft formation of dendritic cells. We used lipidomics combined with genetic and biochemical assays to analyze the role of SWAP-70 in lipid dynamics. TLR activation using LPS as a ligand represented a pathogenic immunogenic stimulus, physical disruption of cell–cell contacts a tolerogenic stimulus. Physical disruption, but not LPS, caused an increase of phosphatidylcholine ether and cholesteryl esters in CD11c+ immune cells. An increase of ceramide (Cer) was a hallmark for LPS activation. SWAP-70 was required for regulating the increase and localization of Cers in the cell membrane. SWAP-70 controls Cer accumulation through the regulation of pH-dependent acid-sphingomyelinase activity and of RhoA-dependent transport of endosomal contents to the plasma membrane. Poor accumulation of Cers in Swap70−/− cells caused decreased apoptosis. This shows that two different pathways of activation, immunogenic and tolerogenic, induce different changes in the lipid composition of cultured CD11c+ cells, and highlights the important role of SWAP-70 in Cer dynamics in dendritic cells.

The nucleoporin ALADIN is required for multiple stages of oocyte maturation and for supporting embryonic divisions postfertilization., Asymmetric cell divisions depend on the precise placement of the spindle apparatus. In mammalian oocytes, spindles assemble close to the cell’s center, but chromosome segregation takes place at the cell periphery where half of the chromosomes are expelled into small, nondeveloping polar bodies at anaphase. By dividing so asymmetrically, most of the cytoplasmic content within the oocyte is preserved, which is critical for successful fertilization and early development. Recently we determined that the nucleoporin ALADIN participates in spindle assembly in somatic cells, and we have also shown that female mice homozygously null for ALADIN are sterile. In this study we show that this protein is involved in specific meiotic stages, including meiotic resumption, spindle assembly, and spindle positioning. In the absence of ALADIN, polar body extrusion is compromised due to problems in spindle orientation and anchoring at the first meiotic anaphase. ALADIN null oocytes that mature far enough to be fertilized in vitro are unable to support embryonic development beyond the two-cell stage. Overall, we find that ALADIN is critical for oocyte maturation and appears to be far more essential for this process than for somatic cell divisions.

This study reveals a role for the cohesin modifier ESCO2 for maintenance of sister chromatid cohesion in mammalian meiosis and a specific weakness of cohesion within male sex chromosome chromatin, In mitotic cells, establishment of sister chromatid cohesion requires acetylation of the cohesin subunit SMC3 (acSMC3) by ESCO1 and/or ESCO2. Meiotic cohesin plays additional but poorly understood roles in the formation of chromosome axial elements (AEs) and synaptonemal complexes. Here, we show that levels of ESCO2, acSMC3, and the pro-cohesion factor sororin increase on meiotic chromosomes as homologs synapse. These proteins are less abundant on the largely unsynapsed sex chromosomes, whose sister chromatid cohesion appears weaker throughout the meiotic prophase. Using three distinct conditional Esco2 knockout mouse strains, we demonstrate that ESCO2 is essential for male gametogenesis. Partial depletion of ESCO2 in prophase I spermatocytes delays chromosome synapsis and further weakens cohesion along sex chromosomes, which show extensive separation of AEs into single chromatids. Unsynapsed regions of autosomes are associated with the sex chromatin and also display split AEs. This study provides the first evidence for a specific role of ESCO2 in mammalian meiosis, identifies a particular ESCO2 dependence of sex chromosome cohesion and suggests support of autosomal synapsis by acSMC3-stabilized cohesion.

The journal of allergy and clinical immunology 144(4, Supplement) S31-S45 (2019). doi:10.1016/j.jaci.2019.02.004 special issue: "Mast cells in health and disease: summary of the German Research Foundation (DFG) priority programme : SPP 1394", Published by Elsevier, Amsterdam [u.a.]

Signal peptide peptidase (SPP) and the four homologous SPP‐like (SPPL) proteases constitute a family of intramembrane aspartyl proteases with selectivity for type II‐oriented transmembrane segments. Here, we analyse the physiological function of the orphan protease SPPL2c, previously considered to represent a non‐expressed pseudogene. We demonstrate proteolytic activity of SPPL2c towards selected tail‐anchored proteins. Despite shared ER localisation, SPPL2c and SPP exhibit distinct, though partially overlapping substrate spectra and inhibitory profiles, and are organised in different high molecular weight complexes. Interestingly, SPPL2c is specifically expressed in murine and human testis where it is primarily localised in spermatids. In mice, SPPL2c deficiency leads to a partial loss of elongated spermatids and reduced motility of mature spermatozoa, but preserved fertility. However, matings of male and female SPPL2c −/− mice exhibit reduced litter sizes. Using proteomics we identify the sarco/endoplasmic reticulum Ca 2+ ‐ATPase (SERCA2)‐regulating protein phospholamban (PLN) as a physiological SPPL2c substrate. Accumulation of PLN correlates with a decrease in intracellular Ca 2+ levels in elongated spermatids that likely contribute to the compromised male germ cell differentiation and function of SPPL2c −/− mice.

Mouse embryo fibroblasts (MEFs) grow slowly after cultivation from animals, however, after an extended period of cultivation, their growth accelerates. We found that SWAP-70 deficient MEFs failed to increase growth rates. They maintain normal growth rates and proliferation cycles for at least 5 years. Complementing SWAP-70 deficiency in one of these MEF clones, MEF1F2, by expressing human SWAP-70 resulted in fast growth of the cells after further cultivation for a long period. The resulting cells show a transformation phenotype, since they grow on top of each other and do not show contact inhibition. This phenotype was reverted when sanguinarine, a putative SWAP-70 inhibitor, was added. Two SWAP-70 expressing clones were examined in detail. Even after cell density became very high their cdc2 and NFκB were still activated suggesting that they do not stop growing. One of the clones formed colonies in soft agar and formed tumors in nude mice. Lately, one more clone became transformed being able to make colonies in soft agar. We maintain 4 human SWAP-70 expressing MEF1F2 cell lines. Three out of 4 clones exhibited transforming phenotypes. The mouse SWAP-70 gene also promoted transformation of MEFs. Taken together our data suggest that SWAP-70 is not a typical oncogene, but is required for spontaneous transformation of MEFs.

Objective Treg cells need to acquire an effector phenotype to function in settings of inflammation. Whether effector Treg cells can limit disease severity in lupus is unknown. Interferon regulatory factor 4 (IRF-4) is an essential controller of effector Treg cells and regulates their ability to express interleukin-10 (IL-10). In non-Treg cells, IRF-4 activity is modulated by interactions with DEF-6 and its homolog switch-associated protein 70 (SWAP-70). Although mice lacking both DEF-6 and SWAP-70 (double-knockout [DKO] mice) develop lupus, they display normal survival, suggesting that in DKO mice, Treg cells can moderate disease development. The purpose of this study was to investigate whether Treg cells from DKO mice have an increased capacity to become effector Treg cells due to the ability of DEF-6 and SWAP-70 to restrain IRF-4 activity. Methods Treg cells were evaluated by fluorescence-activated cell sorting. The B lymphocyte-induced maturation protein 1 (BLIMP-1)/IL-10 axis was assessed by crossing DKO mice with BLIMP-1-YFP-10BiT dual-reporter mice. Deletion of IRF-4 in Treg cells from DKO mice was achieved by generating FoxP3(Cre) IRF-4(fl/fl) DKO mice. Results The concomitant absence of DEF-6 and SWAP-70 led to increased numbers of Treg cells, which acquired an effector phenotype in a cell-intrinsic manner. In addition, Treg cells from DKO mice exhibited enhanced expression of the BLIMP-1/IL-10 axis. Notably, DKO effector Treg cells survived and expanded as disease progressed. The accumulation of Treg cells from DKO mice was associated with the up-regulation of genes controlling autophagy. IRF-4 was required for the expansion and function of effector Treg cells from DKO mice. Conclusion This study revealed the existence of mechanisms that, by acting on IRF-4, can fine-tune the function and survival of effector Treg cells in lupus. These findings suggest that the existence of a powerful effector Treg cell compartment that successfully survives in an unfavorable inflammatory environment could limit disease development.

Post-transcriptional modifications can control protein abundance, but the extent to which these alterations contribute to the expression of T helper (TH) lineage-defining factors is unknown. Tight regulation of Bcl6 expression, an essential transcription factor for T follicular helper (TFH) cells, is critical as aberrant TFH cell expansion is associated with autoimmune diseases, such as systemic lupus erythematosus (SLE). Here we show that lack of the SLE risk variant Def6 results in deregulation of Bcl6 protein synthesis in T cells as a result of enhanced activation of the mTORC1–4E-BP–eIF4E axis, secondary to aberrant assembly of a raptor–p62–TRAF6 complex. Proteomic analysis reveals that this pathway selectively controls the abundance of a subset of proteins. Rapamycin or raptor deletion ameliorates the aberrant TFH cell expansion in mice lacking Def6. Thus deregulation of mTORC1-dependent pathways controlling protein synthesis can result in T-cell dysfunction, indicating a mechanism by which mTORC1 can promote autoimmunity., Excessive expansion of the T follicular helper (TFH) cell pool is associated with autoimmune disease and Def6 has been identified as an SLE risk variant. Here the authors show that Def6 limits proliferation of TFH cells in mice via alteration of mTORC1 signaling and inhibition of Bcl6 expression.

Lipids affect the membrane properties determining essential biological processes. Earlier studies have suggested a role of switch-activated protein 70 (SWAP-70) in lipid raft formation of dendritic cells. We used lipidomics combined with genetic and biochemical assays to analyze the role of SWAP-70 in lipid dynamics. TLR activation using LPS as a ligand represented a pathogenic immunogenic stimulus, physical disruption of cell-cell contacts a tolerogenic stimulus. Physical disruption, but not LPS, caused an increase of phosphatidylcholine ether and cholesteryl esters in CD11c

Meiosis is a highly specialized cell division that facilitates the production of haploid gametes from diploid mother cells. It is characterized by unique chromatin structures and chromatin associated protein complexes. The analysis of these structures and complexes has greatly benefited from and relied on the visualization of meiotic proteins in diverse preparations of meiocytes. In this chapter we summarize methods that can be used for the characterization of the behavior and localization of meiotic proteins in mammalian meiocytes.

Background: Precise spatiotemporal rearrangement of the actin cytoskeleton is essential for cell migration and adhesion. Results: SWAP-70 bundles actin filaments and interacts with cofilin in vitro and in murine mast cells. Conclusion: SWAP-70 is a parallel and anti-parallel bundling protein that participates in the organization of F-actin networks. Significance: Identification and characterization of a regulator of F-actin dynamics improve our understanding on the spatiotemporal control of cytoskeletal processes. Coordinated assembly and disassembly of actin into filaments and higher order structures such as stress fibers and lamellipodia are fundamental for cell migration and adhesion. However, the precise spatiotemporal regulation of F-actin structures is not completely understood. SWAP-70, a phosphatidylinositol 3,4,5-trisphosphate-interacting, F-actin-binding protein, participates in actin rearrangements through yet unknown mechanisms. Here, we show that SWAP-70 is an F-actin-bundling protein that oligomerizes through a Gln/Glu-rich stretch within a coiled-coil region. SWAP-70 bundles filaments in parallel and anti-parallel fashion through its C-terminal F-actin binding domain and delays dilution-induced F-actin depolymerization. We further demonstrate that SWAP-70 co-localizes and directly interacts with cofilin, an F-actin severing and depolymerization factor, and contributes to the regulation of cofilin activity in vivo. In line with these activities, upon stem cell factor stimulation, murine bone marrow-derived mast cells lacking SWAP-70 display aberrant regulation of F-actin and actin free barbed ends dynamics. Moreover, proper stem cell factor-dependent cofilin activation via dephosphorylation and subcellular redistribution into a detergent-resistant cytoskeletal compartment also require SWAP-70. Together, these findings reveal an important role of SWAP-70 in the dynamic spatiotemporal regulation of F-actin networks.

Asthma and allergies are major health concerns in which Ig isotype E plays a pivotal role. Ag-bound IgE drives mast cells and basophils into exocytosis, thereby promoting allergic and potentially anaphylactic reactions. The importance of tightly regulated IgE production is underscored by severe immunological conditions in humans with elevated IgE levels. Cytokines direct IgH class-switching to a particular isotype by initiation of germline transcription (GLT) from isotype-specific intronic (I) promoters. The switch to IgE depends on IL-4, which stimulates GLT of the Iε promoter, but is specifically and strongly impaired in Swap-70−/− mice. Although early events in IL-4 signal transduction (i.e., activation of the JAK/STAT6 pathway) do not require SWAP-70, SWAP-70 deficiency results in impaired Iε GLT. The affinity of STAT6 to chromatin is reduced in absence of SWAP-70. Chromatin immunoprecipitation revealed that SWAP-70 binds to Iε and is required for association of STAT6 with Iε. BCL6, known to antagonize STAT6 particularly at Iε, is increased on Iε in absence of SWAP-70. Other promoters bound by BCL6 and STAT6 were found unaffected. We conclude that SWAP-70 controls IgE production through regulation of the antagonistic STAT6 and BCL6 occupancy of Iε. The identification of this mechanism opens new avenues to inhibit allergic reactions triggered by IgE.

Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the telomerase complex to telomeres through a not yet fully understood mechanism. Factors promoting telomerase–telomere interaction are expected to directly bind telomeres and physically interact with the telomerase complex. In search for such a factor we carried out a SILAC-based DNA–protein interaction screen and identified HMBOX1, hereafter referred to as homeobox telomere-binding protein 1 (HOT1). HOT1 directly and specifically binds double-stranded telomere repeats, with the in vivo association correlating with binding to actively processed telomeres. Depletion and overexpression experiments classify HOT1 as a positive regulator of telomere length. Furthermore, immunoprecipitation and cell fractionation analyses show that HOT1 associates with the active telomerase complex and promotes chromatin association of telomerase. Collectively, these findings suggest that HOT1 supports telomerase-dependent telomere elongation., A homeobox protein binds double-stranded telomeric DNA sequences independent of the shelterin complex, and interacts with active telomerase to positively regulate telomere length.

Meiosis is a highly specialized cell division that facilitates the production of haploid gametes from diploid mother cells. It is characterized by unique chromatin structures and chromatin associated protein complexes. The analysis of these structures and complexes has greatly benefited from and relied on the visualization of meiotic proteins in diverse preparations of meiocytes. In this chapter we summarize methods that can be used for the characterization of the behavior and localization of meiotic proteins in mammalian meiocytes.

Centromeres control genetic inheritance by directing chromosome segregation but are not genetically encoded themselves. Rather, centromeres are defined by nucleosomes containing CENP-A, a histone H3 variant [1]. In cycling somatic cells, centromere identity is maintained by an established cell cycle-coupled CENP-A chromatin assembly pathway, but how centromeres are inherited through the mammalian female germline is unclear because of the long (months to decades) prophase I arrest. We show that mouse oocytes retain the pool of CENP-A nucleosomes assembled before birth, and this pool is sufficient for centromere function, fertility, and genome transmission to embryos. Indeed, oocytes lack any measurable CENP-A nucleosome assembly through the entire fertile lifespan of the female (>1 year). Thus, the remarkable stability of CENP-A nucleosomes confers transgenerational centromere identity in mammals.

Mammalian meiocytes feature four meiosis-specific cohesin proteins in addition to ubiquitous ones, but the roles of the individual cohesin complexes are incompletely understood. To decipher the functions of the two meiosis-specific kleisins, REC8 or RAD21L, together with the only meiosis-specific SMC protein SMC1β, we generated Smc1βRec8 and Smc1βRad21L mouse mutants. Analysis of spermatocyte chromosomes revealed that besides SMC1β complexes, SMC1α/RAD21 and to a small extent SMC1α/REC8 contribute to chromosome axis length. Removal of SMC1β and RAD21L almost completely abolishes all chromosome axes. The sex chromosomes do not pair in single or double mutants, and autosomal synapsis is impaired in all mutants. Super resolution microscopy revealed synapsis-associated SYCP1 aberrantly deposited between sister chromatids and on single chromatids in Smc1βRad21L cells. All mutants show telomere length reduction and structural disruptions, while wild-type telomeres feature a circular TRF2 structure reminiscent of t-loops. There is no loss of centromeric cohesion in both double mutants at leptonema/early zygonema, indicating that, at least in the mutant backgrounds, an SMC1α/RAD21 complex provides centromeric cohesion at this early stage. Thus, in early prophase I the most prominent roles of the meiosis-specific cohesins are in axis-related features such as axis length, synapsis and telomere integrity rather than centromeric cohesion., This work was supported by grants fropm the Deutsche Forschungsgemeinschaft SPP1384 (RJ) and by the BFU (2014-59307-R) and JCyLe (AP).

Cells use ring-like structured protein complexes for various tasks in DNA dynamics. The tripartite cohesin ring is particularly suited to determine chromosome architecture, for it is large and dynamic, may acquire different forms, and is involved in several distinct nuclear processes. This review focuses on cohesin's role in structuring chromosomes during mitotic and meiotic cell divisions and during interphase.

The trend of women to become pregnant when older than in previous generations poses a paramount medical problem, for oocytes are particularly prone to chromosome missegregation, and aneuploidy increases with age. Recent data strongly suggest that as oocyte age increases sister chromatid cohesion is weakened or lost. Cohesin deterioration seems to contribute significantly to age-dependent aneuploidy, as discussed in this review.