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The ubiquitin-proteasome system plays important roles in various biological processes as it degrades the majority of cellular proteins. Adequate proteasomal degradation of crucial transcription regulators ensures the proper development of neutrophils. The ubiquitin E3 ligase of Growth factor independent 1 (GFI1), a key transcription repressor governing terminal granulopoiesis, remains obscure. Here we report that the deficiency of the ring finger protein Interferon regulatory factor 2 binding protein 2a (Irf2bp2a) leads to an impairment of neutrophils differentiation in zebrafish. Mechanistically, Irf2bp2a functions as a ubiquitin E3 ligase targeting Gfi1aa for proteasomal degradation. Moreover, irf2bp2a gene is repressed by Gfi1aa, thus forming a negative feedback loop between Irf2bp2a and Gfi1aa during neutrophils maturation. Different levels of GFI1 may turn it into a tumor suppressor or an oncogene in malignant myelopoiesis. Therefore, discovery of certain drug targets GFI1 for proteasomal degradation by IRF2BP2 might be an effective anti-cancer strategy., Author summary GFI1 is a key transcriptional repressor in hematopoiesis. The E3 ubiquitin ligase targeting GFI1 for proteasomal degradation remains obscure for many years. Here we show that the deficiency of the ring finger protein Irf2bp2a significantly impairs neutrophil maturation in zebrafish. Mechanistic studies reveal for the first time that instead of being a canonical transcription corepressor, Irf2bp2a functions as an E3 mediating the proteasomal degradation of Gfi1aa in neutrophil development.

Mutations in RanBP2 (also known as Nup358), one of the main components of the cytoplasmic filaments of the nuclear pore complex, contribute to the overproduction of acute necrotizing encephalopathy (ANE1)-associated cytokines. Here we report that RanBP2 represses the translation of the interleukin 6 (IL6) mRNA, which encodes a cytokine that is aberrantly up-regulated in ANE1. Our data indicates that soon after its production, the IL6 messenger ribonucleoprotein (mRNP) recruits Argonautes bound to let-7 microRNA. After this mRNP is exported to the cytosol, RanBP2 sumoylates mRNP-associated Argonautes, thereby stabilizing them and enforcing mRNA silencing. Collectively, these results support a model whereby RanBP2 promotes an mRNP remodelling event that is critical for the miRNA-mediated suppression of clinically relevant mRNAs, such as IL6., Author summary RanBP2 (also known as Nup358) is one of the main components of the cytoplasmic filaments of the nuclear pore complex. It has been speculated that RanBP2, which has a SUMO E3-ligase domain, may alter the composition of messenger ribonucleoprotein (mRNP) complexes as they emerge from the nuclear pore and thus regulate the ultimate fate of the mRNAs in the cytoplasm. Five separate missense mutations in RanBP2 cause acute necrotizing encephalopathy 1 (ANE1), which manifests as a sharp rise in cytokine production after common viral infections such as influenza and parainfluenza. However, how RanBP2 and its ANE1-associated mutations affect cytokine production is not well understood. Here we find that Argonaute proteins can be loaded onto mRNAs in the nucleus and upon their nuclear export become sumoylated by RanBP2. This in turn leads to the stabilization of Argonaute-mRNA complexes in the cytosol and enforces silencing of clinically relevant mRNAs, such as interleukin-6 (IL6). These finding presents one of the few biochemical characterizations of an mRNP remodeling event that occurs on the nuclear pore, and opens the possibility that cells control ANE1-associated cytokines via the interplay between RanBP2, microRNA-mediated silencing pathways, and post-translational regulation of proteins.

Much of our knowledge of the drivers of immune variation, and how these responses vary over time, comes from humans, domesticated livestock or laboratory organisms. While the genetic basis of variation in immune responses have been investigated in these systems, there is a poor understanding of how genetic variation influences immunity in natural, untreated populations living in complex environments. Here, we examine the genetic architecture of variation in immune traits in the Soay sheep of St Kilda, an unmanaged population of sheep infected with strongyle gastrointestinal nematodes. We assayed IgA, IgE and IgG antibodies against the prevalent nematode Teladorsagia circumcincta in the blood plasma of > 3,000 sheep collected over 26 years. Antibody levels were significantly heritable (h2 = 0.21 to 0.57) and highly stable over an individual’s lifespan. IgA levels were strongly associated with a region on chromosome 24 explaining 21.1% and 24.5% of heritable variation in lambs and adults, respectively. This region was adjacent to two candidate loci, Class II Major Histocompatibility Complex Transactivator (CIITA) and C-Type Lectin Domain Containing 16A (CLEC16A). Lamb IgA levels were also associated with the immunoglobulin heavy constant loci (IGH) complex, and adult IgE levels and lamb IgA and IgG levels were associated with the major histocompatibility complex (MHC). This study provides evidence of high heritability of a complex immunological trait under natural conditions and provides the first evidence from a genome-wide study that large effect genes located outside the MHC region exist for immune traits in the wild., Author summary Understanding how immune responses vary in natural populations can give an insight into how infection affects the ability of hosts and parasites to survive and reproduce, and how this drives evolutionary and ecological dynamics. Yet, very little is known about how immune responses vary over an individual’s lifetime and how genes contribute to this variation under natural conditions. Our study investigates the genetic architecture of variation in three antibody types, IgA, IgE and IgG in a wild population of Soay sheep on the St Kilda archipelago in North-West Scotland. Using data collected over 26 years, we show that antibody levels have a heritable basis in lambs and adults and are stable over an individual’s lifetime. We also identify several genomic regions with large effects on immune responses. Our study offers the first insights into the genetic control of immunity in a wild population, which is essential to understand how immune profiles vary in challenging natural conditions and how natural selection maintains genetic variation in complex immune traits.

Somatic mutations in protein-coding regions can generate ‘neoantigens’ causing developing cancers to be eliminated by the immune system. Quantitative estimates of the strength of this counterselection phenomenon have been lacking. We quantified the extent to which somatic mutations are depleted in peptides that are predicted to be displayed by major histocompatibility complex (MHC) class I proteins. The extent of this depletion depended on expression level of the neoantigenic gene, and on whether the patient had one or two MHC-encoding alleles that can display the peptide, suggesting MHC-encoding alleles are incompletely dominant. This study provides an initial quantitative understanding of counter-selection of identifiable subclasses of neoantigenic somatic variation., Author summary Cancer immunotherapy and personalized cancer vaccines depend on clearance of cancer and pre-cancer cells by the immune system. However, little is known about the strength of this phenomenon as it acts on the cell populations which give rise to tumors. Here we provide an initial quantitative estimate of the fraction of neo-antigen-containing cells in this population that are cleared by the MHC class I-dependent immune system. The impacts of both neo-antigenic gene expression and the number of neo-antigen-displaying MHC alleles on this clearance phenomenon were examined. A more complete understanding of immune clearance of neoantigenic cells and how this phenomenon varies between patients and cancers, has the potential to guide immunotherapy and cancer vaccines.

Human leukocyte antigen (HLA) is a key genetic factor conferring risk of systemic lupus erythematosus (SLE), but precise independent localization of HLA effects is extremely challenging. As a result, the contribution of specific HLA alleles and amino-acid residues to the overall risk of SLE and to risk of specific autoantibodies are far from completely understood. Here, we dissected (a) overall SLE association signals across HLA, (b) HLA-peptide interaction, and (c) residue-autoantibody association. Classical alleles, SNPs, and amino-acid residues of eight HLA genes were imputed across 4,915 SLE cases and 13,513 controls from Eastern Asia. We performed association followed by conditional analysis across HLA, assessing both overall SLE risk and risk of autoantibody production. DR15 alleles HLA-DRB1*15:01 (P = 1.4x10-27, odds ratio (OR) = 1.57) and HLA-DQB1*06:02 (P = 7.4x10-23, OR = 1.55) formed the most significant haplotype (OR = 2.33). Conditioned protein-residue signals were stronger than allele signals and mapped predominantly to HLA-DRB1 residue 13 (P = 2.2x10-75) and its proxy position 11 (P = 1.1x10-67), followed by HLA-DRB1-37 (P = 4.5x10-24). After conditioning on HLA-DRB1, novel associations at HLA-A-70 (P = 1.4x10-8), HLA-DPB1-35 (P = 9.0x10-16), HLA-DQB1-37 (P = 2.7x10-14), and HLA-B-9 (P = 6.5x10-15) emerged. Together, these seven residues increased the proportion of explained heritability due to HLA to 2.6%. Risk residues for both overall disease and hallmark autoantibodies (i.e., nRNP: DRB1-11, P = 2.0x10-14; DRB1-13, P = 2.9x10-13; DRB1-30, P = 3.9x10-14) localized to the peptide-binding groove of HLA-DRB1. Enrichment for specific amino-acid characteristics in the peptide-binding groove correlated with overall SLE risk and with autoantibody presence. Risk residues were in primarily negatively charged side-chains, in contrast with rheumatoid arthritis. We identified novel SLE signals in HLA Class I loci (HLA-A, HLA-B), and localized primary Class II signals to five residues in HLA-DRB1, HLA-DPB1, and HLA-DQB1. These findings provide insights about the mechanisms by which the risk residues interact with each other to produce autoantibodies and are involved in SLE pathophysiology., Author summary The Human leukocyte antigen (HLA) region is a key genetic factor conferring risk of systemic lupus erythematosus (SLE). In spite of multiple SLE association signals identified in the HLA region, only amino-acid residues within HLA-DRB1 have been specifically described previously. In this study, we performed an imputation-based analysis on individuals with East Asian ancestry, and characterized SLE risk within the HLA region for all involved independent genes (HLA-DRB1, HLA-DPB1, HLA-DQB1, HLA-A, and HLA-B). Furthermore, we identified a characteristic SLE risk residue signature as well as a pattern of specific nRNP and Ro/La autoantibody residues located in the peptide-binding grooves, suggesting their key involvement in autoantibody production.

Disrupting either the DNA annealing factor RAD52 or the A-family DNA polymerase POLQ can cause synthetic lethality with defects in BRCA1 and BRCA2, which are tumor suppressors important for homology-directed repair of DNA double-strand breaks (DSBs), and protection of stalled replication forks. A likely mechanism of this synthetic lethality is that RAD52 and/or POLQ are important for backup pathways for DSB repair and/or replication stress responses. The features of DSB repair events that require RAD52 vs. POLQ, and whether combined disruption of these factors causes distinct effects on genome maintenance, have been unclear. Using human U2OS cells, we generated a cell line with POLQ mutations upstream of the polymerase domain, a RAD52 knockout cell line, and a line with combined disruption of both genes. We also examined RAD52 and POLQ using RNA-interference. We find that combined disruption of RAD52 and POLQ causes at least additive hypersensitivity to cisplatin, and a synthetic reduction in replication fork restart velocity. We also examined the influence of RAD52 and POLQ on several DSB repair events. We find that RAD52 is particularly important for repair using ≥ 50 nt repeat sequences that flank the DSB, and that also involve removal of non-homologous sequences flanking the repeats. In contrast, POLQ is important for repair events using 6 nt (but not ≥ 18 nt) of flanking repeats that are at the edge of the break, as well as oligonucleotide microhomology-templated (i.e., 12–20 nt) repair events requiring nascent DNA synthesis. Finally, these factors show key distinctions with BRCA2, regarding effects on DSB repair events and response to stalled replication forks. These findings indicate that RAD52 and POLQ have distinct roles in genome maintenance, including for specific features of DSB repair events, such that combined disruption of these factors may be effective for genotoxin sensitization and/or synthetic lethal strategies., Author summary We have examined the role of two factors, RAD52 and POLQ, in genome maintenance pathways. While these factors are biochemically distinct, they are both synthetic lethal with loss of the BRCA1 and BRCA2 tumor suppressor genes, and hence are emerging therapeutic targets. Furthermore, RAD52 and POLQ have been implicated in chromosomal break repair events that use flanking repeats to restore the chromosome. We identified distinct features of chromosomal break repair events that are mediated by RAD52 vs. POLQ. Additionally, we have found that combined disruption of RAD52 and POLQ causes at least additive hypersensitivity to cisplatin and a synthetic reduction in replication fork restart velocity. These findings indicate that POLQ and RAD52 have distinct roles in genome maintenance, such that combined disruption of these factors could be a potential therapeutic strategy.

Drosophila Myc (dMyc), as a broad-spectrum transcription factor, can regulate the expression of a large number of genes to control diverse cellular processes, such as cell cycle progression, cell growth, proliferation and apoptosis. However, it remains largely unknown about whether dMyc can be involved in Drosophila innate immune response. Here, we have identified dMyc to be a negative regulator of Drosophila Imd pathway via the loss- and gain-of-function screening. We demonstrate that dMyc inhibits Drosophila Imd immune response via directly activating miR-277 transcription, which further inhibit the expression of imd and Tab2-Ra/b. Importantly, dMyc can improve the survival of flies upon infection, suggesting inhibiting Drosophila Imd pathway by dMyc is vital to restore immune homeostasis that is essential for survival. Taken together, our study not only reports a new dMyc-miR-277-imd/Tab2 axis involved in the negative regulation of Drosophila Imd pathway, and provides a new insight into the complex regulatory mechanism of Drosophila innate immune homeostasis maintenance., Author summary Innate immunity is the first line of defense against pathogenic microorganisms. Both overactivation and depression of immune response are detrimental to the organism. It is indispensable to regulate the duration and intensity of immune response. In this work, we find that Drosophila Myc (dMyc) as a transcription factor activates the transcription of miR-277 to negatively regulate the Imd pathway via inhibiting the expression of imd/Tab2 gene in the middle and later stage of Drosophila innate immune. dMyc is required to restore Drosophila immune homeostasis for the survival of flies upon infection. Since dMyc is well conserved in animals, our findings will be important in understanding the complex regulatory mechanisms of innate immune responses in animals.

Nucleophosmin (NPM1) is frequently mutated or subjected to chromosomal translocation in acute myeloid leukemia (AML). NPM protein is primarily located in the nucleus, but the recurrent NPMc+ mutation, which creates a nuclear export signal, is characterized by cytoplasmic localization and leukemogenic properties. Similarly, the NPM-MLF1 translocation product favors the partial cytoplasmic retention of NPM. Regardless of their common cellular distribution, NPM-MLF1 malignancies engender different effects on hematopoiesis compared to NPMc+ counterparts, highlighting possible aberrant nuclear function(s) of NPM in NPMc+ and NPM-MLF1 AML. We performed a proteomic analysis and found that NPM and NPM-MLF1 interact with various nuclear proteins including subunits of the chromatin remodeling complexes ISWI, NuRD and P/BAF. Accordingly, NPM and NPM-MLF1 are recruited to transcriptionally active or repressed genes along with NuRD subunits. Although the overall gene expression program in NPM knockdown cells is similar to that resulting from NPMc+, NPM-MLF1 expression differentially altered gene transcription regulated by NPM. The abnormal gene regulation imposed by NPM-MLF1 can be characterized by the enhanced recruitment of NuRD to gene regulatory regions. Thus, different mechanisms would orchestrate the dysregulation of NPM function in NPMc+- versus NPM1-MLF1-associated leukemia., Author summary NPMc+ mutation is the most common mutation in acute myeloid leukemia (AML) with prevalence in one third of all AML cases. NPM can also be involved in leukemogenic translocation including the t(3;5)(q25;q34) NPM-MLF1 translocation, which is associated to bad clinical course but remains poorly defined. We are reporting that NPM and the leukemogenic NPM-MLF1 play central role in chromatin organization and gene regulation in hematopoietic cells. A proteomic analysis provided the evidence that NPM and NPM-MLF1 are interacting with the chromatin remodeling complexes NuRD, P/BAF and ISWI in hematopoietic cells. The NPM nuclear depletion, such as imposed by the leukemogenic NPMc+ mutation, or the expression of NPM-MLF1 favors the uncontrolled recruitment of the CHD4/NuRD to chromatin and the abnormal regulation of NPM-target genes. Our results suggest that the abnormal gene regulation forced by NPM-MLF1 is different than the loss of nuclear function imposed by NPMc+, and it can be characterized by the enhanced recruitment of CHD4/NuRD to genes. Thus, NPM-MLF1 is likely to promote hematopoietic malignancies by disruption of gene regulation imposed by the NuRD activity.

Mechanistic differences in the development and function of adaptive, high-affinity antibody-producing B-2 cells and innate-like, “natural” antibody-producing B-1a cells remain poorly understood. Here we show that the multi-functional dynein light chain (DYNLL1/LC8) plays important roles in the establishment of B-1a cells in the peritoneal cavity and in the ongoing development of B-2 lymphoid cells in the bone marrow of mice. Epistasis analyses indicate that Dynll1 regulates B-1a and early B-2 cell development in a single, linear pathway with its direct transcriptional activator ASCIZ (ATMIN/ZNF822), and that the two genes also have complementary functions during late B-2 cell development. The B-2 cell defects caused by loss of DYNLL1 were associated with lower levels of the anti-apoptotic protein BCL-2, and could be supressed by deletion of pro-apoptotic BIM which is negatively regulated by both DYNLL1 and BCL-2. Defects in B cell development caused by loss of DYNLL1 could also be partially suppressed by a pre-arranged SWHEL Igm-B cell receptor transgene. In contrast to the rescue of B-2 cell numbers, the B-1a cell deficiency in Dynll1-deleted mice could not be suppressed by the loss of Bim, and was further compounded by the SWHEL transgene. Conversely, oncogenic MYC expression, which is synthetic lethal with Dynll1 deletion in B-2 cells, did not further reduce B-1a cell numbers in Dynll1-defcient mice. Finally, we found that the ASCIZ-DYNLL1 axis was also required for the early-juvenile development of aggressive MYC-driven and p53-deficient B cell lymphomas. These results identify ASCIZ and DYNLL1 as the core of a transcriptional circuit that differentially regulates the development of the B-1a and B-2 B lymphoid cell lineages and plays a critical role in lymphomagenesis., Author summary Antibody-producing B cells can be segregated into two major populations: The better known conventional B-2 cells typically produce high-affinity and mono-specific antibodies, but only after they encounter a particular pathogen or in response to vaccines. In contrast, the B-1a cells constitutively produce lower-affinity broad-specificity “natural” antibodies that serve as a preemptive defense against a wide range of microbes. Here we reveal that the transcription factor ASCIZ and its target DYNLL1 are essential for mice to have a normally sized pool of B-1a cells in place shortly after birth. We show that these two factors function in a single linear pathway during the development of B-1a cells. This interaction represents a rare example where the activity of a transcription factor, in this case ASCIZ, can be explained by the effects of a single target gene, in this case Dynll1. While ASCIZ and DYNLL1 are also required for producing normal numbers of B-2 cells, we discovered that they regulate B-1a cells and B-2 cells by distinct genetic mechanisms. Finally, we found that ASCIZ also contributes to the early onset of B-1a B cell-derived lymphoid cancers in juvenile mice. The results provide insight into the development of an important cell population of the immune system.

In C. elegans, removal of the germline triggers molecular events in the neighboring intestine, which sends an anti-aging signal to the rest of the animal. In this study, we identified an innate immunity related gene, named irg-7, as a novel mediator of longevity in germlineless animals. We consider irg-7 to be an integral downstream component of the germline longevity pathway because its expression increases upon germ cell removal and its depletion interferes with the activation of the longevity-promoting transcription factors DAF-16 and DAF-12 in germlineless animals. Furthermore, irg-7 activation by itself sensitizes the animals' innate immune response and extends the lifespan of animals exposed to live bacteria. This lifespan-extending pathogen resistance relies on the somatic gonad as well as on many genes previously associated with the reproductive longevity pathway. This suggests that these genes are also relevant in animals with an intact gonad, and can affect their resistance to pathogens. Altogether, this study demonstrates the tight association between germline homeostasis and the immune response of animals, and raises the possibility that the reproductive system can act as a signaling center to divert resources towards defending against putative pathogen attacks., Author summary Increased sensitivity to pathogens is one of the hallmarks of aging. Thus, pathways that slow down the aging process should provide a remedy to this challenge. In this study, we have used the model organism C. elegans to identify a new longevity gene whose activation improves the resistance of the animals to pathogenic food and extends the lifespan of the animals. This improved resistance is the result of activation of the innate immune response and requires many genes known to promote longevity in animals whose germline has been removed. Nevertheless, this improved survival in the presence of pathogenic food is achieved without depleting the germline of the animals. This suggests that these longevity-promoting genes, many of which are conserved between worms and humans, can also protect animals with an intact gonad by diverting resources towards defending against putative pathogen attacks.

During cytokinesis, a contractile ring generates the constricting force to divide a cell into two daughters. This ring is composed of filamentous actin and the motor protein myosin, along with additional structural and regulatory proteins, including anillin. Anillin is a required scaffold protein that links the actomyosin ring to membrane and its organizer, RhoA. However, the molecular basis for timely action of anillin at cytokinesis remains obscure. Here, we find that phosphorylation regulates efficient recruitment of human anillin to the equatorial membrane. Anillin is highly phosphorylated in mitosis, and is a substrate for mitotic kinases. We surveyed function of 46 residues on anillin previously found to be phosphorylated in human cells to identify those required for cytokinesis. Among these sites, we identified S635 as a key site mediating cytokinesis. Preventing S635 phosphorylation adjacent to the AH domain disrupts anillin concentration at the equatorial cortex at anaphase, whereas a phosphomimetic mutant, S635D, partially restores this localization. Time-lapse videomicroscopy reveals impaired recruitment of S635A anillin to equatorial membrane and a transient unstable furrow followed by ultimate failure in cytokinesis. A phosphospecific antibody confirms phosphorylation at S635 in late cytokinesis, although it does not detect phosphorylation in early cytokinesis, possibly due to adjacent Y634 phosphorylation. Together, these findings reveal that anillin recruitment to the equatorial cortex at anaphase onset is enhanced by phosphorylation and promotes successful cytokinesis., Author Summary Human diseases such as cancer and congenital trisomies arise from loss of genetic material during cell division. Yet in most divisions, cells preserve their genetic integrity by strict coordination of cell membrane cleavage (cytokinesis), with accurate separation of genetic material (mitosis). Thus, understanding how mitosis and cytokinesis are coordinated can provide insight into human disease. Anillin is one central integrator of cytokinesis that is recruited in a ring-like structure on the membrane of dividing cells. Protein phosphorylation is a common mechanism regulating timing of events in mitosis; although 46 phosphorylation sites have been mapped on anillin, their functional significance is unknown. Here, we evaluated the effect of blocking anillin phosphorylation on human cell division. Surprisingly, most phosphorylation events appeared dispensable for cytokinesis in the assay used. By contrast, phosphorylation of S635 is important for early recruitment of anillin to the midzone membrane, furrow stabilization and efficient cytokinesis. Our findings highlight a central mechanism regulating the timing of human cytokinesis.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of horses and other equid species. Following natural infection, 10–70% of the infected stallions can become persistently infected and continue to shed EAV in their semen for periods ranging from several months to life. Recently, we reported that some stallions possess a subpopulation(s) of CD3+ T lymphocytes that are susceptible to in vitro EAV infection and that this phenotypic trait is associated with long-term carrier status following exposure to the virus. In contrast, stallions not possessing the CD3+ T lymphocyte susceptible phenotype are at less risk of becoming long-term virus carriers. A genome wide association study (GWAS) using the Illumina Equine SNP50 chip revealed that the ability of EAV to infect CD3+ T lymphocytes and establish long-term carrier status in stallions correlated with a region within equine chromosome 11. Here we identified the gene and mutations responsible for these phenotypes. Specifically, the work implicated three allelic variants of the equine orthologue of CXCL16 (EqCXCL16) that differ by four non-synonymous nucleotide substitutions (XM_00154756; c.715 A → T, c.801 G → C, c.804 T → A/G, c.810 G → A) within exon 1. This resulted in four amino acid changes with EqCXCL16S (XP_001504806.1) having Phe, His, Ile and Lys as compared to EqCXL16R having Tyr, Asp, Phe, and Glu at 40, 49, 50, and 52, respectively. Two alleles (EqCXCL16Sa, EqCXCL16Sb) encoded identical protein products that correlated strongly with long-term EAV persistence in stallions (P, Author Summary A variable proportion of EAV infected stallions (10–70%) may become persistently infected and continuously shed the virus exclusively in their semen after recovery from acute infection. Previous studies in our laboratory have shown that stallions with the CD3+ T lymphocyte susceptibility phenotype to in vitro EAV infection are at higher risk of becoming persistently infected carriers compared to those that lack this phenotype. Here genetic and experimental studies were used to demonstrate that CXCL16 in the horse codes for two proteins, one associated with resistance and the other associated with susceptibility of CD3+ T lymphocytes to EAV infection. The two proteins are the result of four nucleotide substitutions in exon 1 of the equine CXCL16 gene. These alleles determine the outcome of in vitro infection of CD3+ T lymphocytes with EAV and are strongly associated with the establishment and maintenance of long-term carrier state in stallions. In vitro studies demonstrated that one form of CXCL16 protein (CXCL16S) is one of the cellular receptors for EAV and has higher scavenger activity and adhesion ability as compared to the form of the protein associated with resistance (CXCL16R).

The vertebrate adaptive immune system provides a flexible and diverse set of molecules to neutralize pathogens. Yet, viruses such as HIV can cause chronic infections by evolving as quickly as the adaptive immune system, forming an evolutionary arms race. Here we introduce a mathematical framework to study the coevolutionary dynamics between antibodies and antigens within a host. We focus on changes in the binding interactions between the antibody and antigen populations, which result from the underlying stochastic evolution of genotype frequencies driven by mutation, selection, and drift. We identify the critical viral and immune parameters that determine the distribution of antibody-antigen binding affinities. We also identify definitive signatures of coevolution that measure the reciprocal response between antibodies and viruses, and we introduce experimentally measurable quantities that quantify the extent of adaptation during continual coevolution of the two opposing populations. Using this analytical framework, we infer rates of viral and immune adaptation based on time-shifted neutralization assays in two HIV-infected patients. Finally, we analyze competition between clonal lineages of antibodies and characterize the fate of a given lineage in terms of the state of the antibody and viral populations. In particular, we derive the conditions that favor the emergence of broadly neutralizing antibodies, which may have relevance to vaccine design against HIV., Author Summary We normally think of evolution occurring in a population of organisms, in response to their external environment. Rapid evolution of cellular populations also occurs within our bodies when the adaptive immune system works to eliminate infections. Some viruses, such as HIV, are able to evolve as quickly as our immune response, resulting in a chronic infection with both viral and immune populations perpetually adapting. Here we develop a mathematical description of this coevolutionary process, discover key parameters that govern the distribution of interactions between the two populations, introduce principled measures of adaptation, and analyze the conditions under which highly potent broadly neutralizing antibodies will emerge and dominate the immune response.

During postnatal life the cerebral cortex passes through critical periods of plasticity allowing its physiological adaptation to the environment. In the visual cortex, critical period onset and closure are influenced by the non-cell autonomous activity of the Otx2 homeoprotein transcription factor, which regulates the maturation of parvalbumin-expressing inhibitory interneurons (PV cells). In adult mice, the maintenance of a non-plastic adult state requires continuous Otx2 import by PV cells. An important source of extra-cortical Otx2 is the choroid plexus, which secretes Otx2 into the cerebrospinal fluid. Otx2 secretion and internalization requires two small peptidic domains that are part of the DNA-binding domain. Thus, mutating these “transfer” sequences also modifies cell autonomous transcription, precluding this approach to obtain a cell autonomous-only mouse. Here, we develop a mouse model with inducible secretion of an anti-Otx2 single-chain antibody to trap Otx2 in the extracellular milieu. Postnatal secretion of this single-chain antibody by PV cells delays PV maturation and reduces plasticity gene expression. Induced adult expression of this single-chain antibody in cerebrospinal fluid decreases Otx2 internalization by PV cells, strongly induces plasticity gene expression and reopens physiological plasticity. We provide the first mammalian genetic evidence for a signaling mechanism involving intercellular transfer of a homeoprotein transcription factor. Our single-chain antibody mouse model is a valid strategy for extracellular neutralization that could be applied to other homeoproteins and signaling molecules within and beyond the nervous system., Author Summary Classically, cell signaling is based on the secretion of molecules that bind cell surface receptors. Lipophilic agents can do without cell-surface receptors due to their ability to diffuse through the plasma membrane, but this is normally not the case for proteins, which cannot pass the membrane barrier. However, homeoprotein transcription factors represent an exception as they are secreted and internalized by live cells owing to two peptidic domains. An important illustration of this novel signaling mechanism is provided by Otx2, a homeoprotein that travels from the choroid plexus to specific inhibitory neurons in the cerebral cortex, where it regulates physiological plasticity throughout life. Because the two transfer peptides are in the DNA-binding domain of Otx2, it is impossible to mutate them without altering both cell signaling and cell-autonomous functions. We have therefore developed a mouse in which a secreted anti-Otx2 single-chain antibody can be induced to trap extracellular Otx2 while leaving its cell autonomous function untouched. We show that neutralizing extracellular Otx2 modifies the expression of plasticity genes in the visual cortex, thus providing the first genetic demonstration for homeoprotein signaling in a mammal.

Elimination of the proliferating germline extends lifespan in C. elegans. This phenomenon provides a unique platform to understand how complex metazoans retain metabolic homeostasis when challenged with major physiological perturbations. Here, we demonstrate that two conserved transcription regulators essential for the longevity of germline-less adults, DAF-16/FOXO3A and TCER-1/TCERG1, concurrently enhance the expression of multiple genes involved in lipid synthesis and breakdown, and that both gene classes promote longevity. Lipidomic analyses revealed that key lipogenic processes, including de novo fatty acid synthesis, triglyceride production, desaturation and elongation, are augmented upon germline removal. Our data suggest that lipid anabolic and catabolic pathways are coordinately augmented in response to germline loss, and this metabolic shift helps preserve lipid homeostasis. DAF-16 and TCER-1 also perform essential inhibitory functions in germline-ablated animals. TCER-1 inhibits the somatic gene-expression program that facilitates reproduction and represses anti-longevity genes, whereas DAF-16 impedes ribosome biogenesis. Additionally, we discovered that TCER-1 is critical for optimal fertility in normal adults, suggesting that the protein acts as a switch supporting reproductive fitness or longevity depending on the presence or absence of the germline. Collectively, our data offer insights into how organisms adapt to changes in reproductive status, by utilizing the activating and repressive functions of transcription factors and coordinating fat production and degradation., Author Summary The balance between production and breakdown of fats is critical for health, especially during reproduction-related changes such as onset of puberty or menopause. However, little is known about how animals retain a balanced metabolism when undergoing major life events. Here, we have used a C. elegans mutant that successfully adapts to loss of reproductive cells to address this question. Our data suggest that the conserved proteins DAF-16/FOXO3A and TCER-1/TCERG1 mediate a coordinated increase in fat synthesis and degradation when the reproductive cells are lost. This coupling likely helps the animal to manage the lipids that would have been deposited in eggs as yolk, thus preventing metabolic disarray. These proteins also inhibit processes that would have normally supported reproduction. Together the activities of these transcription regulators allow the mutant to convert a debilitating loss of fertility into improved health and longevity. We also report that TCER-1 promotes reproductive health in normal adults, whereas when procreation is impeded, it switches roles to repress fertility and enhance lipid equilibrium. These observations offer insights into how complex organisms coordinate their metabolism to suit their reproductive needs.

Heme-iron recycling from senescent red blood cells (erythrophagocytosis) accounts for the majority of total body iron in humans. Studies in cultured cells have ascribed a role for HRG1/SLC48A1 in heme-iron transport but the in vivo function of this heme transporter is unclear. Here we present genetic evidence in a zebrafish model that Hrg1 is essential for macrophage-mediated heme-iron recycling during erythrophagocytosis in the kidney. Furthermore, we show that zebrafish Hrg1a and its paralog Hrg1b are functional heme transporters, and genetic ablation of both transporters in double knockout (DKO) animals shows lower iron accumulation concomitant with higher amounts of heme sequestered in kidney macrophages. RNA-seq analyses of DKO kidney revealed large-scale perturbation in genes related to heme, iron metabolism and immune functions. Taken together, our results establish the kidney as the major organ for erythrophagocytosis and identify Hrg1 as an important regulator of heme-iron recycling by macrophages in the adult zebrafish., Author summary Total body iron stores in mammals is a composite of iron absorption from diet and iron recycled by macrophages from dying red blood cells (RBCs). Upon erythrophagocytosis of RBCs, the hemoglobin is degraded and heme is imported from the phagosomal compartment into the cytoplasm so that the iron can be released from heme. Defects in these pathways can lead to aberrant iron homeostasis. The Heme Responsive Gene-1 (HRG1, SLC48A1) was identified previously as a heme importer in the intestine of the roundworm, Caenorhabditis elegans. In cell culture studies, HRG1 was demonstrated to mobilize heme from the erythrophagosome of mouse macrophages into the cytosol. However, the in vivo function of HRG1 remains to be elucidated. The zebrafish is a powerful genetic animal model for studying vertebrate development and ontogeny of hematopoiesis. In zebrafish, the kidney marrow is the adult hematopoietic organ that is functionally analogous to the mammalian bone marrow. In this study, we show that Hrg1 plays an essential in vivo role in recycling of damaged RBCs, and that the kidney macrophages are primarily responsible for recycling heme-iron in the adult zebrafish.

The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3’ third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants and suggest there are alternate forms of the Tfp assembly apparatus that mediate various functions including transformation., Author Summary Neisseria gonorrhoeae, the bacterium responsible for the disease gonorrhea, is capable of undergoing natural DNA transformation, a critical mechanism by which bacteria can take up DNA from the environment. Uptake of foreign DNA can lead to adaptation to a changing environment and allow the spread of antibiotic resistance, a particularly relevant issue in N. gonorrhoeae. It has been proposed that the Type IV pilus directly mediates transformation, however, our data show that the extended pilus is not required for transformation. Only a portion (the Ntd) of the pilin protein is required to maintain transformation ability and this domain is released by a unique proteolysis event termed S-pilin cleavage. Release of the Ntd allows cells to maintain competence during pilin antigenic variation, a process vital for immune escape that frequently produces cells that cannot form Type IV pili. While these cells were previously thought to be incapable of DNA transformation, our data suggest that S-pilin cleavage evolved as a mechanism that allows DNA transformation and horizontal gene transfer to occur in these cells lacking pili. We propose that a structurally similar but distinct pseudopilus mediates transformation.