Targeting BMI-1 in B cells restores effective humoral immune responses and controls chronic viral infection.

Ineffective antibody-mediated responses are a key characteristic of chronic viral infection. However, our understanding of the intrinsic mechanisms that drive this dysregulation are unclear. Here, we identify that targeting the epigenetic modifier BMI-1 in mice improves humoral responses to chronic lymphocytic choriomeningitis virus. BMI-1 was upregulated by germinal center B cells in chronic viral infection, correlating with changes to the accessible chromatin landscape, compared to acute infection. B cell-intrinsic deletion of Bmi1 accelerated viral clearance, reduced splenomegaly and restored splenic architecture. Deletion of Bmi1 restored c-Myc expression in B cells, concomitant with improved quality of antibody and coupled with reduced antibody-secreting cell numbers. Specifically, BMI-1-deficiency induced antibody with increased neutralizing capacity and enhanced antibody-dependent effector function. Using a small molecule inhibitor to murine BMI-1, we could deplete antibody-secreting cells and prohibit detrimental immune complex formation in vivo. This study defines BMI-1 as a crucial immune modifier that controls antibody-mediated responses in chronic infection.

Type I interferons induce an epigenetically distinct memory B cell subset in chronic viral infection.

Memory B cells (MBCs) are key providers of long-lived immunity against infectious disease, yet in chronic viral infection, they do not produce effective protection. How chronic viral infection disrupts MBC development and whether such changes are reversible remain unknown. Through single-cell (sc)ATAC-seq and scRNA-seq during acute versus chronic lymphocytic choriomeningitis viral infection, we identified a memory subset enriched for interferon (IFN)-stimulated genes (ISGs) during chronic infection that was distinct from the T-bet+ subset normally associated with chronic infection. Blockade of IFNAR-1 early in infection transformed the chromatin landscape of chronic MBCs, decreasing accessibility at ISG-inducing transcription factor binding motifs and inducing phenotypic changes in the dominating MBC subset, with a decrease in the ISG subset and an increase in CD11c+CD80+ cells. However, timing was critical, with MBCs resistant to intervention at 4 weeks post-infection. Together, our research identifies a key mechanism to instruct MBC identity during viral infection.

Single-cell profiling of transcriptome and histone modifications with EpiDamID.

Recent advances in single-cell sequencing technologies have enabled simultaneous measurement of multiple cellular modalities, but the combined detection of histone post-translational modifications and transcription at single-cell resolution has remained limited. Here, we introduce EpiDamID, an experimental approach to target a diverse set of chromatin types by leveraging the binding specificities of single-chain variable fragment antibodies, engineered chromatin reader domains, and endogenous chromatin-binding proteins. Using these, we render the DamID technology compatible with the genome-wide identification of histone post-translational modifications. Importantly, this includes the possibility to jointly measure chromatin marks and transcription at the single-cell level. We use EpiDamID to profile single-cell Polycomb occupancy in mouse embryoid bodies and provide evidence for hierarchical gene regulatory networks. In addition, we map H3K9me3 in early zebrafish embryogenesis, and detect striking heterochromatic regions specific to notochord. Overall, EpiDamID is a new addition to a vast toolbox to study chromatin states during dynamic cellular processes.

B Cells Hit a Class Ceiling in the Germinal Center.

Class-switch recombination (CSR) provides diverse effector functions to B cells. In this issue of Immunity, Roco et al. (2019) refute long-standing dogma that germinal centers are the principal location for CSR; instead, CSR predominantly occurs early post immunization and declines during germinal center formation.

Nuclear chromosome locations dictate segregation error frequencies.

Chromosome segregation errors during cell divisions generate aneuploidies and micronuclei, which can undergo extensive chromosomal rearrangements such as chromothripsis1-5. Selective pressures then shape distinct aneuploidy and rearrangement patterns-for example, in cancer6,7-but it is unknown whether initial biases in segregation errors and micronucleation exist for particular chromosomes. Using single-cell DNA sequencing8 after an error-prone mitosis in untransformed, diploid cell lines and organoids, we show that chromosomes have different segregation error frequencies that result in non-random aneuploidy landscapes. Isolation and sequencing of single micronuclei from these cells showed that mis-segregating chromosomes frequently also preferentially become entrapped in micronuclei. A similar bias was found in naturally occurring micronuclei of two cancer cell lines. We find that segregation error frequencies of individual chromosomes correlate with their location in the interphase nucleus, and show that this is highest for peripheral chromosomes behind spindle poles. Randomization of chromosome positions, Cas9-mediated live tracking and forced repositioning of individual chromosomes showed that a greater distance from the nuclear centre directly increases the propensity to mis-segregate. Accordingly, chromothripsis in cancer genomes9 and aneuploidies in early development10 occur more frequently for larger chromosomes, which are preferentially located near the nuclear periphery. Our findings reveal a direct link between nuclear chromosome positions, segregation error frequencies and micronucleus content, with implications for our understanding of tumour genome evolution and the origins of specific aneuploidies during development.

Combinatorial single-cell profiling of major chromatin types with MAbID.

Gene expression programs result from the collective activity of numerous regulatory factors. Studying their cooperative mode of action is imperative to understand gene regulation, but simultaneously measuring these factors within one sample has been challenging. Here we introduce Multiplexing Antibodies by barcode Identification (MAbID), a method for combinatorial genomic profiling of histone modifications and chromatin-binding proteins. MAbID employs antibody-DNA conjugates to integrate barcodes at the genomic location of the epitope, enabling combined incubation of multiple antibodies to reveal the distributions of many epigenetic markers simultaneously. We used MAbID to profile major chromatin types and multiplexed measurements without loss of individual data quality. Moreover, we obtained joint measurements of six epitopes in single cells of mouse bone marrow and during mouse in vitro differentiation, capturing associated changes in multifactorial chromatin states. Thus, MAbID holds the potential to gain unique insights into the interplay between gene regulatory mechanisms, especially for low-input samples and in single cells.

CD80 and PD-L2 define functionally distinct memory B cell subsets that are independent of antibody isotype.

Memory B cells (MBCs) are long-lived sources of rapid, isotype-switched secondary antibody-forming cell (AFC) responses. Whether MBCs homogeneously retain the ability to self-renew and terminally differentiate or if these functions are compartmentalized into MBC subsets has remained unclear. It has been suggested that antibody isotype controls MBC differentiation upon restimulation. Here we demonstrate that subcategorizing MBCs on the basis of their expression of CD80 and PD-L2, independently of isotype, identified MBC subsets with distinct functions upon rechallenge. CD80(+)PD-L2(+) MBCs differentiated rapidly into AFCs but did not generate germinal centers (GCs); conversely, CD80(-)PD-L2(-) MBCs generated few early AFCs but robustly seeded GCs. The gene-expression patterns of the subsets supported both the identity and function of these distinct MBC types. Hence, the differentiation and regeneration of MBCs are compartmentalized.

Telerehabilitation consultations with a physiotherapist for chronic knee pain versus in-person consultations in Australia: the PEAK non-inferiority randomised controlled trial.

BACKGROUND: Telerehabilitation whether perceived as less effective than in-person care for musculoskeletal problems. We aimed to determine if physiotherapy video conferencing consultations were non-inferior to in-person consultations for chronic knee pain. METHODS: In this non-inferiority randomised controlled trial, we recruited primary care physiotherapists from 27 Australian clinics. Using computer-generated blocks, participants with chronic knee pain consistent with osteoarthritis were randomly assigned (1:1, stratified by physiotherapist and clinic) in-person or telerehabilitation (ie, video conferencing) physiotherapist consultations. Participants and physiotherapists were unmasked to group assignment. Both groups had five consultations over 3 months for strengthening, physical activity, and education. Primary outcomes were knee pain (on a numerical rating scale of 0-10) and physical function (using the Western Ontario and McMaster Universities osteoarthritis index of 0-68) at 3 months after randomisation. Primary analysis was by modified intention-to-treat using all available data. This trial is registered with the Australian and New Zealand Clinical Trials Registry, ACTRN12619001240134. FINDINGS: Between Dec 10, 2019, and June 17, 2022, 394 adults were enrolled, with 204 allocated to in-person care and 190 to telerehabilitation. 15 primary care physiotherapists were recruited. At 3 months, 383 (97%) participants provided information for primary outcomes and both groups reported improved pain (mean change 2·98, SD 2·23 for in-person care and 3·14, 1·87 for telerehabilitation) and function (10·20, 11·63 and 10·75, 9·62, respectively). Telerehabilitation was non-inferior for pain (mean difference 0·16, 95% CI -0·26 to 0·57) and function (1·65, -0·23 to 3·53). The number of participants reporting adverse events was similar between groups (40 [21%] for in-person care and 35 [19%] for telerehabilitation) and none were serious. INTERPRETATION: Telerehabilitation with a physiotherapist is non-inferior to in-person care for chronic knee pain. FUNDING: National Health and Medical Research Council.

snpAIMeR: R package for evaluating ancestry informative marker contributions in non-model population diagnostics.

MOTIVATION: Single nucleotide polymorphism (SNP) markers are increasingly popular for population genomics and inferring ancestry for individuals of unknown origin. Because large SNP datasets are impractical for rapid and routine analysis, diagnostics rely on panels of highly informative markers. Strategies exist for selecting these markers, however, resources for efficiently evaluating their performance are limited for non-model systems. RESULTS: snpAIMeR is a user-friendly R package that evaluates the efficacy of genomic markers for the cluster assignment of unknown individuals. It is intended to help minimize panel size and genotyping effort by determining the informativeness of candidate diagnostic markers. Provided genotype data from individuals of known origin, it uses leave-one-out cross-validation to determine population assignment rates for individual markers and marker combinations. AVAILABILITY AND IMPLEMENTATION: snpAIMeR is available on CRAN (https://CRAN.R-project.org/package=snpAIMeR).

Whole Genomes Reveal Evolutionary Relationships and Mechanisms Underlying Gene-Tree Discordance in Neodiprion Sawflies.

Rapidly evolving taxa are excellent models for understanding the mechanisms that give rise to biodiversity. However, developing an accurate historical framework for comparative analysis of such lineages remains a challenge due to ubiquitous incomplete lineage sorting and introgression. Here, we use a whole-genome alignment, multiple locus-sampling strategies, and summary-tree and SNP-based species-tree methods to infer a species tree for eastern North American Neodiprion species, a clade of pine-feeding sawflies (Order: Hymenopteran; Family: Diprionidae). We recovered a well-supported species tree that-except for three uncertain relationships-was robust to different strategies for analyzing whole-genome data. Nevertheless, underlying gene-tree discordance was high. To understand this genealogical variation, we used multiple linear regression to model site concordance factors estimated in 50-kb windows as a function of several genomic predictor variables. We found that site concordance factors tended to be higher in regions of the genome with more parsimony-informative sites, fewer singletons, less missing data, lower GC content, more genes, lower recombination rates, and lower D-statistics (less introgression). Together, these results suggest that incomplete lineage sorting, introgression, and genotyping error all shape the genomic landscape of gene-tree discordance in Neodiprion. More generally, our findings demonstrate how combining phylogenomic analysis with knowledge of local genomic features can reveal mechanisms that produce topological heterogeneity across genomes.

Requirements of IL-4 during the Generation of B Cell Memory.

IL-4 has long been established as a key regulator of Th cells and for promoting effective B cell survival and isotype class switching. Yet, despite having been extensively studied, the specific role of IL-4 in generating humoral memory in vivo is unclear. In this review, we explore the recent studies that unravel the cellular sources and spatiotemporal production of IL-4, the relationship between IL-4 and IL-21 during germinal center responses and the formation of Ab-secreting cells, and the current understanding of whether IL-4 promotes or suppresses memory B cell generation in vitro and in vivo.

Genome-lamina interactions are established de novo in the early mouse embryo.

In mammals, the emergence of totipotency after fertilization involves extensive rearrangements of the spatial positioning of the genome1,2. However, the contribution of spatial genome organization to the regulation of developmental programs is unclear3. Here we generate high-resolution maps of genomic interactions with the nuclear lamina (a filamentous meshwork that lines the inner nuclear membrane) in mouse pre-implantation embryos. We reveal that nuclear organization is not inherited from the maternal germline but is instead established de novo shortly after fertilization. The two parental genomes establish lamina-associated domains (LADs)4 with different features that converge after the 8-cell stage. We find that the mechanism of LAD establishment is unrelated to DNA replication. Instead, we show that paternal LAD formation in zygotes is prevented by ectopic expression of Kdm5b, which suggests that LAD establishment may be dependent on remodelling of H3K4 methylation. Our data suggest a step-wise assembly model whereby early LAD formation precedes consolidation of topologically associating domains.

CRISPR-Cas9 editing of the arginine-vasopressin V1a receptor produces paradoxical changes in social behavior in Syrian hamsters.

Studies from a variety of species indicate that arginine­vasopressin (AVP) and its V1a receptor (Avpr1a) play a critical role in the regulation of a range of social behaviors by their actions in the social behavior neural network. To further investigate the role of AVPRs in social behavior, we performed CRISPR-Cas9­mediated editing at the Avpr1a gene via pronuclear microinjections in Syrian hamsters (Mesocricetus auratus), a species used extensively in behavioral neuroendocrinology because they produce a rich suite of social behaviors. Using this germ-line gene-editing approach, we generated a stable line of hamsters with a frame-shift mutation in the Avpr1a gene resulting in the null expression of functional Avpr1as. Avpr1a knockout (KO) hamsters exhibited a complete lack of Avpr1a-specific autoradiographic binding throughout the brain, behavioral insensitivity to centrally administered AVP, and no pressor response to a peripherally injected Avpr1a-specific agonist, thus confirming the absence of functional Avpr1as in the brain and periphery. Contradictory to expectations, Avpr1a KO hamsters exhibited substantially higher levels of conspecific social communication (i.e., odor-stimulated flank marking) than their wild-type (WT) littermates. Furthermore, sex differences in aggression were absent, as both male and female KOs exhibited more aggression toward same-sex conspecifics than did their WT littermates. Taken together, these data emphasize the importance of comparative studies employing gene-editing approaches and suggest the startling possibility that Avpr1a-specific modulation of the social behavior neural network may be more inhibitory than permissive.

Exome sequencing in multiplex families with left-sided cardiac defects has high yield for disease gene discovery.

Congenital heart disease (CHD) is a common group of birth defects with a strong genetic contribution to their etiology, but historically the diagnostic yield from exome studies of isolated CHD has been low. Pleiotropy, variable expressivity, and the difficulty of accurately phenotyping newborns contribute to this problem. We hypothesized that performing exome sequencing on selected individuals in families with multiple members affected by left-sided CHD, then filtering variants by population frequency, in silico predictive algorithms, and phenotypic annotations from publicly available databases would increase this yield and generate a list of candidate disease-causing variants that would show a high validation rate. In eight of the nineteen families in our study (42%), we established a well-known gene/phenotype link for a candidate variant or performed confirmation of a candidate variant's effect on protein function, including variants in genes not previously described or firmly established as disease genes in the body of CHD literature: BMP10, CASZ1, ROCK1 and SMYD1. Two plausible variants in different genes were found to segregate in the same family in two instances suggesting oligogenic inheritance. These results highlight the need for functional validation and demonstrate that in the era of next-generation sequencing, multiplex families with isolated CHD can still bring high yield to the discovery of novel disease genes.

Myocardial glycophagy flux dysregulation and glycogen accumulation characterize diabetic cardiomyopathy.

Diabetic heart disease morbidity and mortality is escalating. No specific therapeutics exist and mechanistic understanding of diabetic cardiomyopathy etiology is lacking. While lipid accumulation is a recognized cardiomyocyte phenotype of diabetes, less is known about glycolytic fuel handling and storage. Based on in vitro studies, we postulated the operation of an autophagy pathway in the myocardium specific for glycogen homeostasis - glycophagy. Here we visualize occurrence of cardiac glycophagy and show that the diabetic myocardium is characterized by marked glycogen elevation and altered cardiomyocyte glycogen localization. We establish that cardiac glycophagy flux is disturbed in diabetes. Glycophagy may represent a potential therapeutic target for alleviating the myocardial impacts of metabolic disruption in diabetic heart disease.

Elongated galactan side chains mediate cellulose-pectin interactions in engineered Arabidopsis secondary cell walls.

The plant secondary cell wall is a thickened matrix of polysaccharides and lignin deposited at the cessation of growth in some cells. It forms the majority of carbon in lignocellulosic biomass, and it is an abundant and renewable source for forage, fiber, materials, fuels, and bioproducts. The complex structure and arrangement of the cell wall polymers mean that the carbon is difficult to access in an economical and sustainable way. One solution is to alter the cell wall polymer structure so that it is more suited to downstream processing. However, it remains difficult to predict what the effects of this engineering will be on the assembly, architecture, and properties of the cell wall. Here, we make use of Arabidopsis plants expressing a suite of genes to increase pectic galactan chain length in the secondary cell wall. Using multi-dimensional solid-state nuclear magnetic resonance, we show that increasing galactan chain length enhances pectin-cellulose spatial contacts and increases cellulose crystallinity. We also found that the increased galactan content leads to fewer spatial contacts of cellulose with xyloglucan and the backbone of pectin. Hence, we propose that the elongated galactan side chains compete with xyloglucan and the pectic backbone for cellulose interactions. Due to the galactan topology, this may result in comparatively weak interactions and disrupt the cell wall architecture. Therefore, introduction of this strategy into trees or other bioenergy crops would benefit from cell-specific expression strategies to avoid negative effects on plant growth.

Fern cell walls and the evolution of arabinogalactan proteins in streptophytes.

Significant changes have occurred in plant cell wall composition during evolution and diversification of tracheophytes. As the sister lineage to seed plants, knowledge on the cell wall of ferns is key to track evolutionary changes across tracheophytes and to understand seed plant-specific evolutionary innovations. Fern cell wall composition is not fully understood, including limited knowledge of glycoproteins such as the fern arabinogalactan proteins (AGPs). Here, we characterize the AGPs from the leptosporangiate fern genera Azolla, Salvinia, and Ceratopteris. The carbohydrate moiety of seed plant AGPs consists of a galactan backbone including mainly 1,3- and 1,3,6-linked pyranosidic galactose, which is conserved across the investigated fern AGPs. Yet, unlike AGPs of angiosperms, those of ferns contained the unusual sugar 3-O-methylrhamnose. Besides terminal furanosidic arabinose, Ara (Araf), the main linkage type of Araf in the ferns was 1,2-linked Araf, whereas in seed plants 1,5-linked Araf is often dominating. Antibodies directed against carbohydrate epitopes of AGPs supported the structural differences between AGPs of ferns and seed plants. Comparison of AGP linkage types across the streptophyte lineage showed that angiosperms have rather conserved monosaccharide linkage types; by contrast bryophytes, ferns, and gymnosperms showed more variability. Phylogenetic analyses of glycosyltransferases involved in AGP biosynthesis and bioinformatic search for AGP protein backbones revealed a versatile genetic toolkit for AGP complexity in ferns. Our data reveal important differences across AGP diversity of which the functional significance is unknown. This diversity sheds light on the evolution of the hallmark feature of tracheophytes: their elaborate cell walls.