The cytokine genes of Oncorhynchus masou formosanus include a defective interleukin-4/13A gene.

Oncorhynchus masou formosanus (Formosa landlocked salmon) is a critically endangered salmonid fish endemic to Taiwan. To begin to understand how its drastic change in lifestyle from anadromous to exclusively river-dwelling is reflected in its immune genes, we characterized the genes encoding six cytokines (IL-2A, IL-2B, IL-4/13A, IL-4/13B1, IL-4/13B2, and IL-17A/F2a) important for T cell responses as no genomic data is available for this fish. Interestingly, all genes appeared homozygous indicative of a genetic bottleneck. The IL2 and IL17A/F2a genes and their products are highly similar to their characterized homologs in Oncorhynchus mykiss (rainbow trout) and other salmonid fish. Two notable differences were observed in IL4/13 family important for type 2 immune responses. First, O. m. formosanus carries not only one but two genes encoding IL-4/13B1 proteins and expansions of these genes are present in other salmonid fish. Second, the OmfoIL4/13A gene carries a 228 bp deletion that results in a premature stop codon and hence a non-functional IL-4/13A cytokine. This suggests a reduced ability for T cell responses against parasitic infections in this species.

Polystyrene-colonizing bacteria are enriched for long-chain alkane degradation pathways.

One of the most promising strategies for the management of plastic waste is microbial biodegradation, but efficient degraders for many types of plastics are still lacking, including those for polystyrene (PS). Genomics has emerged as a powerful tool for mining environmental microbes that may have the ability to degrade different types of plastics. In this study, we use 16S rRNA sequencing to analyze the microbiomes for multiple PS samples collected from sites with different vegetation in Taiwan to reveal potential common properties between species that exhibit growth advantages on PS surfaces. Phylum enrichment analysis identified Cyanobacteria and Deinococcus-Thermus as being the most over-represented groups on PS, and both phyla include species known to reside in extreme environments and could encode unique enzymes that grant them properties suitable for colonization on PS surfaces. Investigation of functional enrichment using reference genomes of PS-enriched species highlighted carbon metabolic pathways, especially those related to hydrocarbon degradation. This is corroborated by the finding that genes encoding long-chain alkane hydroxylases such as AlmA are more prevalent in the genomes of PS-associated bacteria. Our analyses illustrate how plastic in the environment support the colonization by different microbes compared to surrounding soil. In addition, our results point to the possibility that alkane hydroxylases could confer growth advantages of microbes on PS.

Functional characterization of the MyD88 homologs in Strongylocentrotus purpuratus.

Toll-like receptor signaling is an evolutionarily conserved pathway to induce the expression of immune mediators in response to encounters with pathogens. MyD88 is a central adapter connecting the intracellular domain of the receptors to downstream kinases. Here, we conducted a comprehensive assessment of the MyD88 family in an echinoderm, Strongylocentrotus purpuratus. Of five SpMyD88s only two closely related proteins, SpMyD88A and SpMyD88B, are functional in mammalian cell lines as their overexpression facilitates the activation of the downstream transcription factor NF-κB. This requires the presence of the endogenous mammalian MyD88s, and domain swapping indicated that the death domains of S. purpuratus MyD88 are unable to efficiently connect to the respective domains of the vertebrate IRAK kinases. This suggests that the interaction surfaces between the signaling mediators in this conserved signaling pathway are not as conserved as previously thought but were likely shaped and evolved by pathogenic selection over evolutionary timescales.

Measuring Mutator Enzyme Activity Using an E. coli-Based Colony Formation Assay.

Mutator enzymes alter the nucleotide sequences of DNA or RNA molecules; immune systems utilize them to destroy the integrity of pathogen genomes and to optimize immune mediators of the host. Their dysregulation has been linked to tumorigenesis in various tissues. Defining and comparing the activities of such mutator enzymes requires a robust versatile assay that is independent of their biological context as in vivo mutation rates are typically low. Here we provide detailed protocols for two widely used E. coli-based approaches that detect the activities of ectopically expressed cytidine deaminases on two distinct reporter genes: an extrachromosomal kanamycin-resistance gene or an endogenous chromosomal substrate, the rpoB gene-encoding RNA polymerase. The generation of mutations is in both cases measured in a colony formation assay. With appropriate modifications, these assays can be extended to study other mutator enzymes.

Trajectory mapping of the early Drosophila germline reveals controls of zygotic activation and sex differentiation.

Germ cells in Drosophila melanogaster are specified maternally shortly after fertilization and are transcriptionally quiescent until their zygotic genome is activated to sustain further development. To understand the molecular basis of this process, we analyzed the progressing transcriptomes of early male and female germ cells at the single-cell level between germline specification and coalescence with somatic gonadal cells. Our data comprehensively cover zygotic activation in the germline genome, and analyses on genes that exhibit germline-restricted expression reveal that polymerase pausing and differential RNA stability are important mechanisms that establish gene expression differences between the germline and soma. In addition, we observe an immediate bifurcation between the male and female germ cells as zygotic transcription begins. The main difference between the two sexes is an elevation in X Chromosome expression in females relative to males, signifying incomplete dosage compensation, with a few select genes exhibiting even higher expression increases. These indicate that the male program is the default mode in the germline that is driven to female development with a second X Chromosome.

AID/APOBEC-like cytidine deaminases are ancient innate immune mediators in invertebrates.

In the course of both innate and adaptive immunity, cytidine deaminases within the activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) family modulate immune responses by mutating specific nucleic acid sequences of hosts and pathogens. The evolutionary emergence of these mediators, however, seems to coincide precisely with the emergence of adaptive immunity in vertebrates. Here, we show a family of genes in species within two divergent invertebrate phyla-the echinoderm Strongylocentrotus purpuratus and the brachiopod Lingula anatina-that encode proteins with similarities in amino acid sequence and enzymatic activities to the vertebrate AID/APOBECs. The expression of these invertebrate factors is enriched in tissues undergoing constant, direct interactions with microbes and can be induced upon pathogen challenge. Our findings suggest that AID/APOBEC proteins, and their function in immunity, emerged far earlier than previously thought. Thus, cytidine deamination is probably an ancient innate immune mechanism that predates the protostome/deuterostome divergence.

Lectins identify distinct populations of coelomocytes in Strongylocentrotus purpuratus.

Coelomocytes represent the immune cells of echinoderms, but detailed knowledge about their roles during immune responses is very limited. One major challenge for studying coelomocyte biology is the lack of reagents to identify and purify distinct populations defined by objective molecular markers rather than by morphology-based classifications that are subjective at times. Glycosylation patterns are known to differ significantly between cell types in vertebrates, and furthermore they can vary depending on the developmental stage and activation states within a given lineage. Thus fluorescently labeled lectins that recognize distinct glycan structures on cell surface proteins are routinely used to identify discrete cell populations in the vertebrate immune system. Here we now employed a panel of fifteen fluorescently-labeled lectins to determine differences in the glycosylation features on the surface of Strongylocentrotus purpuratus coelomocytes by fluorescence microscopy and flow cytometry. Eight of the lectins (succinylated wheat germ agglutinin, Len culinaris lectin, Pisum sativum agglutinin, Saphora japonica agglutinin, Solanum tuberosum lectin, Lycopersicon esculentum lectin, Datura stramonium lectin, Vicia villosa lectin) showed distinct binding patterns to fixed and live cells of three major coelomocyte classes: phagocytic cells, red spherule cells, and vibratile cells. Importantly, almost all lectins bound only to a subgroup of cells within each cell type. Lastly, we established fluorescently-labeled lectin-based fluorescence activated cell sorting as a strategy to purify distinct S. purpuratus coelomocyte (sub-)populations based on molecular markers. We anticipate that this will become a routine approach in future studies focused on dissecting the roles of different coelomocytes in echinoderm immunity.

Evidence for parallel evolution of a gene involved in the regulation of spermatogenesis.

PHD finger protein 7 (Phf7) is a male germline specific gene in Drosophila melanogaster that can trigger the male germline sexual fate and regulate spermatogenesis, and its human homologue can rescue fecundity defects in male flies lacking this gene. These findings prompted us to investigate conservation of reproductive strategies through studying the evolutionary origin of this gene. We find that Phf7 is present only in select species including mammals and some insects, whereas the closely related G2/M-phase specific E3 ubiquitin protein ligase (G2e3) is in the genome of most metazoans. Interestingly, phylogenetic analyses showed that vertebrate and insect Phf7 genes did not evolve from a common Phf7 ancestor but rather through independent duplication events from an ancestral G2e3 This is an example of parallel evolution in which a male germline factor evolved at least twice from a pre-existing template to develop new regulatory mechanisms of spermatogenesis.

Collaboration of RAG2 with RAG1-like proteins during the evolution of V(D)J recombination.

The recombination-activating gene 1 (RAG1) and RAG2 proteins initiate V(D)J recombination, the process that assembles the B- and T-lymphocyte antigen receptor genes of jawed vertebrates. RAG1 and RAG2 are thought to have arisen from a transposable element, but the origins of this element are not understood. We show that two ancestral RAG1 proteins, Transib transposase and purple sea urchin RAG1-like, have a latent ability to initiate V(D)J recombination when coexpressed with RAG2 and that in vitro transposition by Transib transposase is stimulated by RAG2. Conversely, we report low levels of V(D)J recombination by RAG1 in the absence of RAG2. Recombination by RAG1 alone differs from canonical V(D)J recombination in having lost the requirement for asymmetric DNA substrates, implicating RAG2 in the origins of the "12/23 rule," a fundamental regulatory feature of the reaction. We propose that evolution of RAG1/RAG2 began with a Transib transposon whose intrinsic recombination activity was enhanced by capture of an ancestral RAG2, allowing for the development of adaptive immunity.

Form follows function - the three-dimensional structure of antigen receptor gene loci.

Antigen receptor genes are assembled during lymphocyte development from individual gene segments by a somatic gene rearrangement process named V(D)J recombination. This process is tightly regulated to ensure the generation of an unbiased broad primary repertoire of immunoglobulins and T cell receptors, and to prevent aberrant recombination products that could initiate lymphomagenesis. One important mode of regulation that has recently been discovered for the immunoglobulin heavy chain (IGH) gene locus is the adoption of distinct three-dimensional structures of the locus. Changes in the spatial conformation are thought to ensure the appropriate access of the V(D)J recombinase machinery at each developmental stage, and the formation of extensive chromosome loops has been implicated in allowing equal access to widely dispersed gene elements.

Synthesis and characterization of a SIRT6 open tubular column: predicting deacetylation activity using frontal chromatography.

SIRT6 is a histone deacetylase that has been proposed as a potential therapeutic target for metabolic disorders and the prevention of age-associated diseases. Thus the identification of compounds that modulate SIRT6 activity could be of great therapeutic importance. We have previously reported on the identification of quercetin and vitexin as SIRT6 inhibitors, using SIRT6-coated magnetic beads. In this study, we have immobilized SIRT6 onto the surface of an open tubular capillary and characterized the quercetin binding site using frontal displacement chromatography. Structurally related flavonoids were tested for their activity on SIRT6, including apigenin, naringenin, luteolin, and kaempferol. In addition to obtaining their binding activity using frontal affinity chromatographic techniques, we also ranked the compounds based on their ability to displace quercetin. The data suggest that a single displacement curve is representative of the enzymatic activity of the tested ligand. In addition, using the inhibition data obtained in this study, we developed a preliminary pharmacophore model that confirmed the experimental data.

Transcription, splicing, and release: are we there yet?

A systematic analysis of LPS-induced gene expression in macrophages by Bhatt et al. demonstrates that inflammatory responses are governed primarily at the level of transcription initiation. Unexpectedly, full-length nascent RNAs that contain introns appear to accumulate on chromatin, presumably to complete processing, prior to release of functional mRNA for export to the cytoplasm.

Separation of mutational and transcriptional enhancers in Ig genes.

Secondary Ig gene diversification relies on activation-induced cytidine deaminase (AID) to create U:G mismatches that are subsequently fixed by mutagenic repair pathways. AID activity is focused to Ig loci by cis-regulatory DNA sequences named targeting elements. In this study, we show that in contrast to prevailing thought in the field, the targeting elements in the chicken IGL locus are distinct from classical transcriptional enhancers. These mutational enhancer elements (MEEs) are required over and above transcription to recruit AID-mediated mutagenesis to Ig loci. We identified a small 222-bp fragment in the chicken IGL locus that enhances mutagenesis without boosting transcription, and this sequence represents a key component of an MEE. Lastly, MEEs are evolutionarily conserved among birds, both in sequence and function, and contain several highly conserved sequence modules that are likely involved in recruiting trans-acting targeting factors. We propose that MEEs represent a novel class of cis-regulatory elements for which the function is to control genomic integrity.

Characterizing somatic hypermutation and gene conversion in the chicken DT40 cell system.

The secondary immunoglobulin gene diversification processes, somatic hypermutation (SHM), immunoglobulin gene conversion (GCV), and class switch recombination, are important for efficient humoral immune responses. They require the action of activation-induced cytidine deaminase, an enzyme that deaminates cytosine in the context of single-stranded DNA. The chicken DT40 B-cell line is an important model system for exploring the mechanisms of SHM and GCV, as both processes occur constitutively without the need for stimulation. In addition, standard gene targeting strategies can be used for defined manipulations of the DT40 genome. Thus, these cells represent an excellent model of choice for genetic studies of SHM and GCV. Problems arising from defects in early B-cell development that are of concern when using genetically engineered mice are avoided in this system. Here, we describe how to perform gene targeting in DT40 cells and how to determine the effects of such modifications on SHM and GCV.

Classical Mus musculus Igκ enhancers support transcription but not high level somatic hypermutation from a V-lambda promoter in chicken DT40 cells.

Somatic hypermutation (SHM) of immunoglobulin genes is initiated by activation-induced cytidine deaminase (AID) in activated B cells. This process is strictly dependent on transcription. Hence, cis-acting transcriptional control elements have been proposed to target SHM to immunoglobulin loci. The Mus musculus Igκ locus is regulated by the intronic enhancer (iE/MAR) and the 3' enhancer (3'E), and multiple studies using transgenic and knock-out approaches in mice and cell lines have reported somewhat contradictory results about the function of these enhancers in AID-mediated sequence diversification. Here we show that the M. musculus iE/MAR and 3'E elements are active solely as transcriptional enhancer when placed in the context of the IGL locus in Gallus gallus DT40 cells, but they are very inefficient in targeting AID-mediated mutation events to this locus. This suggests that either key components of the cis-regulatory targeting elements reside outside the murine Igκ transcriptional enhancer sequences, or that the targeting of AID activity to Ig loci occurs by largely species-specific mechanisms.

Targeting of AID-mediated sequence diversification to immunoglobulin genes.

Activation-induced cytidine deaminase (AID) is a key enzyme for antibody-mediated immune responses. Antibodies are encoded by the immunoglobulin genes and AID acts as a transcription-dependent DNA mutator on these genes to improve antibody affinity and effector functions. An emerging theme in field is that many transcribed genes are potential targets of AID, presenting an obvious danger to genomic integrity. Thus there are mechanisms in place to ensure that mutagenic outcomes of AID activity are specifically restricted to the immunoglobulin loci. Cis-regulatory targeting elements mediate this effect and their mode of action is probably a combination of immunoglobulin gene specific activation of AID and a perversion of faithful DNA repair towards error-prone outcomes.

RAG-2 unleashed: lymphocytes beware.

The programmed degradation of the RAG-2 enzyme upon entry to S phase restricts V(D)J recombination to the G0-G1 phase of the cell cycle. In this issue of Immunity, Zhang et al. (2011) show that this is critical to prevent lymphoma formation.

The origins of vertebrate adaptive immunity.

Adaptive immunity is mediated through numerous genetic and cellular processes that generate favourable somatic variants of antigen-binding receptors under evolutionary selection pressure by pathogens and other factors. Advances in our understanding of immunity in mammals and other model organisms are revealing the underlying basis and complexity of this remarkable system. Although the evolution of adaptive immunity has been thought to occur by the acquisition of novel molecular capabilities, an increasing amount of information from new model systems suggest that co-option and redirection of pre-existing systems are the main source of innovation. We combine evidence from a wide range of organisms to obtain an integrated view of the origins and patterns of divergence in adaptive immunity.

The origins of the Rag genes--from transposition to V(D)J recombination.

The recombination activating genes 1 and 2 (Rag1 and Rag2) encode the key enzyme that is required for the generation of the highly diversified antigen receptor repertoire central to adaptive immunity. The longstanding model proposed that this gene pair was acquired by horizontal gene transfer to explain its abrupt appearance in the vertebrate lineage. The analyses of the enormous amount of sequence data created by many genome sequencing projects now provide the basis for a more refined model as to how this unique gene pair evolved from a selfish DNA transposon into a sophisticated DNA recombinase essential for immunity.

A coming-of-age story: activation-induced cytidine deaminase turns 10.

The discovery and characterization of activation-induced cytidine deaminase (AID) 10 years ago provided the basis for a mechanistic understanding of secondary antibody diversification and the subsequent generation and maintenance of cellular memory in B lymphocytes, which signified a major advance in the field of B cell immunology. Here we celebrate and review the triumphs in the mission to understand the mechanisms through which AID influences antibody diversification, as well as the implications of AID function on human physiology. We also take time to point out important ongoing controversies and outstanding questions in the field and highlight key experiments and techniques that hold the potential to elucidate the remaining mysteries surrounding this vital protein.