Methods for analyzing chromosome conformation in mammalian cells are either low resolution or low throughput and are technically challenging. In next-generation (NG) Capture-C, we have redesigned the ...Capture-C method to achieve unprecedented levels of sensitivity and reproducibility. NG Capture-C can be used to analyze many genetic loci and samples simultaneously. High-resolution data can be produced with as few as 100,000 cells, and single-nucleotide polymorphisms can be used to generate allele-specific tracks. The method is straightforward to perform and should greatly facilitate the investigation of many questions related to gene regulation as well as the functional dissection of traits examined in genome-wide association studies.
Conservation interventions require evaluation to understand what factors predict success or failure. To date, there has been little systematic investigation of the effect of social and cultural ...context on conservation success, although a large body of literature argues it is important. We investigated whether local cultural context, particularly local institutions and the efforts of interventions to engage with this culture significantly influence conservation outcomes. We also tested the effects of community participation, conservation education, benefit provision, and market integration. We systematically reviewed the literature on community-based conservation and identified 68 interventions suitable for inclusion. We used a protocol to extract and code information and evaluated a range of measures of outcome success (attitudinal, behavioral, ecological, and economic). We also examined the association of each predictor with each outcome measure and the structure of predictor covariance. Local institutional context influenced intervention outcomes, and interventions that engaged with local institutions were more likely to succeed. Nevertheless, there was limited support for the role of community participation, conservation education, benefit provision, and market integration on intervention success. We recommend that conservation interventions seek to understand the societies they work with and tailor their activities accordingly. Systematic reviews are a valuable approach for assessing conservation evidence, although sensitive to the continuing lack of high-quality reporting on conservation interventions.
Adaptive immunity involves the development of bespoke antibodies in germinal centers (GCs) through immunoglobulin somatic hypermutation (SHM) in GC dark zones (DZs) and clonal selection in light ...zones (LZs). Accurate selection requires that cells fully replace surface B cell receptors (BCRs) following SHM, but whether this happens before LZ entry is not clear. We found that most GC B cells degrade pre-SHM receptors before leaving the DZ, and that B cells acquiring crippling mutations during SHM rarely reached the LZ. Instead, apoptosis was triggered preferentially in late G1, a stage wherein cells with functional BCRs re-entered cell cycle or reduced surface expression of the chemokine receptor CXCR4 to enable LZ migration. Ectopic expression of the anti-apoptotic gene Bcl2 was not sufficient for cells with damaging mutations to reach the LZ, suggesting that BCR-dependent cues may actively facilitate the transition. Thus, BCR replacement and pre-screening in DZs prevents the accumulation of clones with non-functional receptors and facilitates selection in the LZ.
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•GC B cells exchange surface BCRs in dark zones (DZ) following somatic hypermutation•GC B cells acquiring damaging BCR mutations genes rarely transition to the light zone (LZ)•Apoptosis of GC B cells in DZ occurs preferentially in the late G1 stage of cell cycle•Ectopic expression of Bcl2 fails to rescue LZ entry by B cells with damaged BCRs
Somatic hypermutation is important for the generation of high-affinity antibodies, but this mutational process is also likely to negatively impact the functional integrity of B cell receptors (BCRs). Stewart et al. find that germinal center B cells replace surface BCRs in dark zones (DZ) and present evidence for a DZ checkpoint that prevents the accumulation of clones with non-functional BCRs, thus facilitating selection in the LZ.
Germinal centers (GCs) form in lymph nodes after immunization or infection to facilitate antibody affinity maturation and memory and plasma cell (PC) development. PC differentiation is thought to ...involve stringent selection for GC B cells expressing the highest-affinity antigen receptors, but how this plays out during complex polyclonal responses is unclear. We combine temporal lineage tracing with antibody characterization to gain a snapshot of PCs developing during influenza infection. GCs co-mature B cell clones with antibody affinities spanning multiple orders of magnitude; however, each generates PCs with similar efficiencies, including weak binders. Within lineages, PC selection is not restricted to variants with the highest-affinity antibodies. Differentiation is commonly associated with proliferative expansion to produce “nodes” of identical PCs. Immunization-induced GCs generate fewer PCs but still of low- and high-antibody affinities. We propose that generating low-affinity antibody PCs reflects an evolutionary compromise to facilitate diverse serum antibody responses.
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•Germinal center B cells are fate-mapped to study plasma cell selection during infection•Antibody affinities of PCs simultaneously exiting GCs span several orders of magnitude•Substantial GC clonal diversity is retained through PC selection•GC PC output after protein immunization is much reduced but remains affinity permissive
B cells are known to undergo sequential rounds of mutation and affinity-based selection, in sites known as germinal centers, to produce higher-affinity antibodies over time. This study finds that germinal centers formed in response to complex immune challenges support the differentiation of clonally diverse plasma cells, including some of low-antibody affinity, suggesting that high affinity is not the only factor driving the maturation of the antibody repertoire.
A substantial amount of organismal complexity is thought to be encoded by enhancers which specify the location, timing, and levels of gene expression. In mammals there are more enhancers than ...promoters which are distributed both between and within genes. Here we show that activated, intragenic enhancers frequently act as alternative tissue-specific promoters producing a class of abundant, spliced, multiexonic poly(A)+ RNAs (meRNAs) which reflect the host gene's structure. meRNAs make a substantial and unanticipated contribution to the complexity of the transcriptome, appearing as alternative isoforms of the host gene. The low protein-coding potential of meRNAs suggests that many meRNAs may be byproducts of enhancer activation or underlie as-yet-unidentified RNA-encoded functions. Distinguishing between meRNAs and mRNAs will transform our interpretation of dynamic changes in transcription both at the level of individual genes and of the genome as a whole.
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► Intragenic enhancers act as unidirectional, cell-specific, alternative promoters ► Activated intragenic enhancers produce a class of multiexonic poly(A)+ RNAs, meRNAs ► meRNAs appear as isoforms of the host gene ► meRNAs add significantly to transcriptome complexity
Multiple cytokines, including interleukin 6 (IL-6), IL-11, IL-27, oncostatin M (OSM), and leukemia inhibitory factor (LIF), signal via the common GP130 cytokine receptor subunit. In this study, we ...describe a patient with a homozygous mutation of
(encoding GP130 p.N404Y) who presented with recurrent infections, eczema, bronchiectasis, high IgE, eosinophilia, defective B cell memory, and an impaired acute-phase response, as well as skeletal abnormalities including craniosynostosis. The p.N404Y missense substitution is associated with loss of IL-6, IL-11, IL-27, and OSM signaling but a largely intact LIF response. This study identifies a novel immunodeficiency with phenotypic similarities to STAT3 hyper-IgE syndrome caused by loss of function of GP130.
We used the paradigmatic GATA-PU.1 axis to explore, at the systems level, dynamic relationships between transcription factor (TF) binding and global gene expression programs as multipotent cells ...differentiate. We combined global ChIP-seq of GATA1, GATA2, and PU.1 with expression profiling during differentiation to erythroid and neutrophil lineages. Our analysis reveals (1) differential complexity of sequence motifs bound by GATA1, GATA2, and PU.1; (2) the scope and interplay of GATA1 and GATA2 programs within, and during transitions between, different cell compartments, and the extent of their hard-wiring by DNA motifs; (3) the potential to predict gene expression trajectories based on global associations between TF-binding data and target gene expression; and (4) how dynamic modeling of DNA-binding and gene expression data can be used to infer regulatory logic of TF circuitry. This rubric exemplifies the utility of this cross-platform resource for deconvoluting the complexity of transcriptional programs controlling stem/progenitor cell fate in hematopoiesis.
•Cross-platform resource for TF-network regulation of multipotent blood cell fate•DNA motif dependence and changing specificity of GATA factors in lineage choice•Modeling-based inference identifies GATA2 repression of PU.1 in multipotent cells•Priming, recruitment, and switching modes of GATA interplay during differentiation
A systems-level resource of the GATA-PU.1 axis provides insight into the dynamics of transcriptional programs during hematopoietic lineage commitment and differentiation.
Antibody affinity maturation occurs in germinal centers (GCs) through iterative rounds of somatic hypermutation and selection. Selection involves B cells competing for T cell help based on the amount ...of antigen they capture and present on their MHC class II (MHCII) proteins. How GC B cells are able to rapidly and repeatedly transition between mutating their B cell receptor genes and then being selected shortly after is not known. We report that MHCII surface levels and degradation are dynamically regulated in GC B cells. Through ectopic expression of a photoconvertible MHCII-mKikGR chimeric gene, we found that individual GC B cells differed in the rates of MHCII protein turnover. Fluctuations in surface MHCII levels were dependent on ubiquitination and the E3 ligase March1. Increases in March1 expression in centroblasts correlated with decreases in surface MHCII levels, whereas CD83 expression in centrocytes helped to stabilize MHCII at that stage. Defects in MHCII ubiquitination caused GC B cells to accumulate greater amounts of a specific peptide-MHCII (pMHCII), suggesting that MHCII turnover facilitates the replacement of old complexes. We propose that pMHCII complexes are periodically targeted for degradation in centroblasts to favor the presentation of recently acquired antigens, thereby promoting the fidelity and efficiency of selection.
Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle ...weakness. We performed whole-exome sequencing to determine the underlying defect in a group of individuals with an inherited limb-girdle pattern of myasthenic weakness. We identify DPAGT1 as a gene in which mutations cause a congenital myasthenic syndrome. We describe seven different mutations found in five individuals with DPAGT1 mutations. The affected individuals share a number of common clinical features, including involvement of proximal limb muscles, response to treatment with cholinesterase inhibitors and 3,4-diaminopyridine, and the presence of tubular aggregates in muscle biopsies. Analyses of motor endplates from two of the individuals demonstrate a severe reduction of endplate acetylcholine receptors. DPAGT1 is an essential enzyme catalyzing the first committed step of N-linked protein glycosylation. Our findings underscore the importance of N-linked protein glycosylation for proper functioning of the neuromuscular junction. Using the DPAGT1-specific inhibitor tunicamycin, we show that DPAGT1 is required for efficient glycosylation of acetylcholine-receptor subunits and for efficient export of acetylcholine receptors to the cell surface. We suggest that the primary pathogenic mechanism of DPAGT1 mutations is reduced levels of acetylcholine receptors at the endplate region. These individuals share clinical features similar to those of congenital myasthenic syndrome due to GFPT1 mutations, and their disorder might be part of a larger subgroup comprising the congenital myasthenic syndromes that result from defects in the N-linked glycosylation pathway and that manifest through impaired neuromuscular transmission.
De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within ...the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39–90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.
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