New Perspectives on Genomic Imprinting, an Essential and Multifaceted Mode of Epigenetic Control in the Developing and Adult Brain.

Mammalian evolution entailed multiple innovations in gene regulation, including the emergence of genomic imprinting, an epigenetic regulation leading to the preferential expression of a gene from its maternal or paternal allele. Genomic imprinting is highly prevalent in the brain, yet, until recently, its central roles in neural processes have not been fully appreciated. Here, we provide a comprehensive survey of adult and developmental brain functions influenced by imprinted genes, from neural development and wiring to synaptic function and plasticity, energy balance, social behaviors, emotions, and cognition. We further review the widespread identification of parental biases alongside monoallelic expression in brain tissues, discuss their potential roles in dosage regulation of key neural pathways, and suggest possible mechanisms underlying the dynamic regulation of imprinting in the brain. This review should help provide a better understanding of the significance of genomic imprinting in the normal and pathological brain of mammals including humans.

Functional circuit architecture underlying parental behaviour.

Parenting is essential for the survival and wellbeing of mammalian offspring. However, we lack a circuit-level understanding of how distinct components of this behaviour are coordinated. Here we investigate how galanin-expressing neurons in the medial preoptic area (MPOAGal) of the hypothalamus coordinate motor, motivational, hormonal and social aspects of parenting in mice. These neurons integrate inputs from a large number of brain areas and the activation of these inputs depends on the animal's sex and reproductive state. Subsets of MPOAGal neurons form discrete pools that are defined by their projection sites. While the MPOAGal population is active during all episodes of parental behaviour, individual pools are tuned to characteristic aspects of parenting. Optogenetic manipulation of MPOAGal projections mirrors this specificity, affecting discrete parenting components. This functional organization, reminiscent of the control of motor sequences by pools of spinal cord neurons, provides a new model for how discrete elements of a social behaviour are generated at the circuit level.

Murine single-cell RNA-seq reveals cell-identity- and tissue-specific trajectories of aging.

Aging is a pleiotropic process affecting many aspects of mammalian physiology. Mammals are composed of distinct cell type identities and tissue environments, but the influence of these cell identities and environments on the trajectory of aging in individual cells remains unclear. Here, we performed single-cell RNA-seq on >50,000 individual cells across three tissues in young and old mice to allow for direct comparison of aging phenotypes across cell types. We found transcriptional features of aging common across many cell types, as well as features of aging unique to each type. Leveraging matrix factorization and optimal transport methods, we found that both cell identities and tissue environments exert influence on the trajectory and magnitude of aging, with cell identity influence predominating. These results suggest that aging manifests with unique directionality and magnitude across the diverse cell identities in mammals.

Single-cell transcriptomics of the naked mole-rat reveals unexpected features of mammalian immunity.

The immune system comprises a complex network of specialized cells that protects against infection, eliminates cancerous cells, and regulates tissue repair, thus serving a critical role in homeostasis, health span, and life span. The subterranean-dwelling naked mole-rat (NM-R; Heterocephalus glaber) exhibits prolonged life span relative to its body size, is unusually cancer resistant, and manifests few physiological or molecular changes with advancing age. We therefore hypothesized that the immune system of NM-Rs evolved unique features that confer enhanced cancer immunosurveillance and prevent the age-associated decline in homeostasis. Using single-cell RNA-sequencing (scRNA-seq) we mapped the immune system of the NM-R and compared it to that of the short-lived, cancer-prone mouse. In contrast to the mouse, we find that the NM-R immune system is characterized by a high myeloid-to-lymphoid cell ratio that includes a novel, lipopolysaccharide (LPS)-responsive, granulocyte cell subset. Surprisingly, we also find that NM-Rs lack canonical natural killer (NK) cells. Our comparative genomics analyses support this finding, showing that the NM-R genome lacks an expanded gene family that controls NK cell function in several other species. Furthermore, we reconstructed the evolutionary history that likely led to this genomic state. The NM-R thus challenges our current understanding of mammalian immunity, favoring an atypical, myeloid-biased mode of innate immunosurveillance, which may contribute to its remarkable health span.

Transitions in paternal social status predict patterns of offspring growth and metabolic transcription.

One type of parental effect occurs when changes in parental phenotype or environment trigger changes to offspring phenotype. Such nongenetic parental effects can be precisely triggered in response to an environmental cue in time-locked fashion, or in other cases, persist for multiple generations after the cue has been removed, suggesting multiple timescales of action. For parental effects to serve as reliable signals of current environmental conditions, they should be reversible, such that when cues change, offspring phenotypes change in accordance. Social hierarchy is a prevalent feature of the environment, and current parental social status could signal the environment in which offspring will be born. Here, we sought to address parental effects of social status and their timescale of action in mice. We show that territorial competition in seminatural environments affects offspring growth. Although dominant males are not heavier than nondominant or control males, they produce faster growing offspring, particularly sons. The timing, effect-size, and sex-specificity of this association are modulated by maternal social experience. We show that a change in paternal social status is sufficient to modulate offspring weight: from one breeding cycle to the next, status-ascending males produce heavier sons than before, and status-descending males produce lighter sons than before. Current paternal status is also highly predictive of liver transcription in sons, including molecular pathways controlling oxidative phosphorylation and iron metabolism. These results are consistent with a parental effect of social experience, although alternative explanations are considered. In summary, changes in paternal social status are associated with changes in offspring growth and metabolism.

The Multipartite Mitochondrial Genome of Enteromyxum leei (Myxozoa): Eight Fast-Evolving Megacircles.

Myxozoans are a large group of poorly characterized cnidarian parasites. To gain further insight into their evolution, we sequenced the mitochondrial (mt) genome of Enteromyxum leei and reevaluate the mt genome structure of Kudoa iwatai. Although the typical animal mt genome is a compact, 13-25 kb, circular chromosome, the mt genome of E. leei was found to be fragmented into eight circular chromosomes of ∼23 kb, making it the largest described animal mt genome. Each chromosome was found to harbor a large noncoding region (∼15 kb), nearly identical between chromosomes. The protein coding genes show an unusually high rate of sequence evolution and possess little similarity to their cnidarian homologs. Only five protein coding genes could be identified and no tRNA genes. Surprisingly, the mt genome of K. iwatai was also found to be composed of two chromosomes. These observations confirm the remarkable plasticity of myxozoan mt genomes.

Genomic insights into the evolutionary origin of Myxozoa within Cnidaria.

The Myxozoa comprise over 2,000 species of microscopic obligate parasites that use both invertebrate and vertebrate hosts as part of their life cycle. Although the evolutionary origin of myxozoans has been elusive, a close relationship with cnidarians, a group that includes corals, sea anemones, jellyfish, and hydroids, is supported by some phylogenetic studies and the observation that the distinctive myxozoan structure, the polar capsule, is remarkably similar to the stinging structures (nematocysts) in cnidarians. To gain insight into the extreme evolutionary transition from a free-living cnidarian to a microscopic endoparasite, we analyzed genomic and transcriptomic assemblies from two distantly related myxozoan species, Kudoa iwatai and Myxobolus cerebralis, and compared these to the transcriptome and genome of the less reduced cnidarian parasite, Polypodium hydriforme. A phylogenomic analysis, using for the first time to our knowledge, a taxonomic sampling that represents the breadth of myxozoan diversity, including four newly generated myxozoan assemblies, confirms that myxozoans are cnidarians and are a sister taxon to P. hydriforme. Estimations of genome size reveal that myxozoans have one of the smallest reported animal genomes. Gene enrichment analyses show depletion of expressed genes in categories related to development, cell differentiation, and cell-cell communication. In addition, a search for candidate genes indicates that myxozoans lack key elements of signaling pathways and transcriptional factors important for multicellular development. Our results suggest that the degeneration of the myxozoan body plan from a free-living cnidarian to a microscopic parasitic cnidarian was accompanied by extreme reduction in genome size and gene content.

Regulation of alternative splicing at the single-cell level.

Alternative splicing is a key cellular mechanism for generating distinct isoforms, whose relative abundances regulate critical cellular processes. It is therefore essential that inclusion levels of alternative exons be tightly regulated. However, how the precision of inclusion levels among individual cells is governed is poorly understood. Using single-cell gene expression, we show that the precision of inclusion levels of alternative exons is determined by the degree of evolutionary conservation at their flanking intronic regions. Moreover, the inclusion levels of alternative exons, as well as the expression levels of the transcripts harboring them, also contribute to this precision. We further show that alternative exons whose inclusion levels are considerably changed during stem cell differentiation are also subject to this regulation. Our results imply that alternative splicing is coordinately regulated to achieve accuracy in relative isoform abundances and that such accuracy may be important in determining cell fate.

Evolution of T cells in the cancer-resistant naked mole-rat.

Naked mole-rats (NMRs) are best known for their extreme longevity and cancer resistance, suggesting that their immune system might have evolved to facilitate these phenotypes. Natural killer (NK) and T cells have evolved to detect and destroy cells infected with pathogens and to provide an early response to malignancies. While it is known that NMRs lack NK cells, likely lost during evolution, little is known about their T-cell subsets in terms of the evolution of the genes that regulate their function, their clonotypic diversity, and the thymus where they mature. Here we find, using single-cell transcriptomics, that NMRs have a large circulating population of γδT cells, which in mice and humans mostly reside in peripheral tissues and induce anti-cancer cytotoxicity. Using single-cell-T-cell-receptor sequencing, we find that a cytotoxic γδT-cell subset of NMRs harbors a dominant clonotype, and that their conventional CD8 αßT cells exhibit modest clonotypic diversity. Consistently, perinatal NMR thymuses are considerably smaller than those of mice yet follow similar involution progression. Our findings suggest that NMRs have evolved under a relaxed intracellular pathogenic selective pressure that may have allowed cancer resistance and longevity to become stronger targets of selection to which the immune system has responded by utilizing γδT cells.

The operonic location of auto-transcriptional repressors is highly conserved in bacteria.

Bacterial genes are commonly encoded in clusters, known as operons, which share transcriptional regulatory control and often encode functionally related proteins that take part in certain biological pathways. Operons that are coregulated are known to colocalize in the genome, suggesting that their spatial organization is under selection for efficient expression regulation. However, the internal order of genes within operons is believed to be poorly conserved, and hence expression requirements are claimed to be too weak to oppose gene rearrangements. In light of these opposing views, we set out to investigate whether the internal location of the regulatory genes within operons is under selection. Our analysis shows that transcription factors (TFs) are preferentially encoded as either first or last in their operons, in the two diverged model bacteria Escherichia coli and Bacillus subtilis. In a higher resolution, we find that TFs that repress transcription of the operon in which they are encoded (autorepressors), contribute most of this signal by specific preference of the first operon position. We show that this trend is strikingly conserved throughout highly diverged bacterial phyla. Moreover, these autorepressors regulate operons that carry out highly diverse biological functions. We propose a model according to which autorepressors are selected to be located first in their operons in order to optimize transcription regulation. Specifically, the first operon position helps autorepressors to minimize leaky transcription of the operon structural genes, thus minimizing energy waste. Our analysis provides statistically robust evidence for a paradigm of bacterial autorepressor preferential operonic location. Corroborated with our suggested model, an additional layer of operon expression control that is common throughout the bacterial domain is revealed.

Evolutionary models accounting for layers of selection in protein-coding genes and their impact on the inference of positive selection.

The selective forces acting on a protein-coding gene are commonly inferred using evolutionary codon models by contrasting the rate of nonsynonymous substitutions to the rate of synonymous substitutions. These models usually assume that the synonymous substitution rate, Ks, is homogenous across all sites, which is justified if synonymous sites are free from selection. However, a growing body of evidence indicates that the DNA and RNA levels of protein-coding genes are subject to varying degrees of selective constraints due to various biological functions encoded at these levels. In this paper, we develop evolutionary models that account for these layers of selection by allowing for both among-site variability of substitution rates at the DNA/RNA level (which leads to Ks variability among protein-coding sites) and among-site variability of substitution rates at the protein level (Ka variability). These models are constructed so that positive selection is either allowed or not. This enables statistical testing of positive selection when variability at the DNA/RNA substitution rate is accounted for. Using this methodology, we show that variability of the baseline DNA/RNA substitution rate is a widespread phenomenon in coding sequence data of mammalian genomes, most likely reflecting varying degrees of selection at the DNA and RNA levels. Additionally, we use simulations to examine the impact that accounting for the variability of the baseline DNA/RNA substitution rate has on the inference of positive selection. Our results show that ignoring this variability results in a high rate of erroneous positive-selection inference. Our newly developed model, which accounts for this variability, does not suffer from this problem and hence provides a likelihood framework for the inference of positive selection on a background of variability in the baseline DNA/RNA substitution rate.

Urocortin-3 neurons in the mouse perifornical area promote infant-directed neglect and aggression.

While recent studies have uncovered dedicated neural pathways mediating the positive control of parenting, the regulation of infant-directed aggression and how it relates to adult-adult aggression is poorly understood. Here we show that urocortin-3 (Ucn3)-expressing neurons in the hypothalamic perifornical area (PeFAUcn3) are activated during infant-directed attacks in males and females, but not other behaviors. Functional manipulations of PeFAUcn3 neurons demonstrate the role of this population in the negative control of parenting in both sexes. PeFAUcn3 neurons receive input from areas associated with vomeronasal sensing, stress, and parenting, and send projections to hypothalamic and limbic areas. Optogenetic activation of PeFAUcn3 axon terminals in these regions triggers various aspects of infant-directed agonistic responses, such as neglect, repulsion, and aggression. Thus, PeFAUcn3 neurons emerge as a dedicated circuit component controlling infant-directed neglect and aggression, providing a new framework to understand the positive and negative regulation of parenting in health and disease.

A machine-learning approach for predicting B-cell epitopes.

The immune activity of an antibody is directed against a specific region on its target antigen known as the epitope. Numerous immunodetection and immunotheraputics applications are based on the ability of antibodies to recognize epitopes. The detection of immunogenic regions is often an essential step in these applications. The experimental approaches used for detecting immunogenic regions are often laborious and resource-intensive. Thus, computational methods for the prediction of immunogenic regions alleviate this drawback by guiding the experimental procedures. In this work we developed a computational method for the prediction of immunogenic regions from either the protein three-dimensional structure or sequence when the structure is unavailable. The method implements a machine-learning algorithm that was trained to recognize immunogenic patterns based on a large benchmark dataset of validated epitopes derived from antigen structures and sequences. We compare our method to other available tools that perform the same task and show that it outperforms them.

The Tug1 lncRNA locus is essential for male fertility.

BACKGROUND: Several long noncoding RNAs (lncRNAs) have been shown to function as components of molecular machines that play fundamental roles in biology. While the number of annotated lncRNAs in mammalian genomes has greatly expanded, studying lncRNA function has been a challenge due to their diverse biological roles and because lncRNA loci can contain multiple molecular modes that may exert function. RESULTS: We previously generated and characterized a cohort of 20 lncRNA loci knockout mice. Here, we extend this initial study and provide a more detailed analysis of the highly conserved lncRNA locus, taurine-upregulated gene 1 (Tug1). We report that Tug1-knockout male mice are sterile with underlying defects including a low number of sperm and abnormal sperm morphology. Because lncRNA loci can contain multiple modes of action, we wanted to determine which, if any, potential elements contained in the Tug1 genomic region have any activity. Using engineered mouse models and cell-based assays, we provide evidence that the Tug1 locus harbors two distinct noncoding regulatory activities, as a cis-DNA repressor that regulates neighboring genes and as a lncRNA that can regulate genes by a trans-based function. We also show that Tug1 contains an evolutionary conserved open reading frame that when overexpressed produces a stable protein which impacts mitochondrial membrane potential, suggesting a potential third coding function. CONCLUSIONS: Our results reveal an essential role for the Tug1 locus in male fertility and uncover evidence for distinct molecular modes in the Tug1 locus, thus highlighting the complexity present at lncRNA loci.

Epitopia: a web-server for predicting B-cell epitopes.

BACKGROUND: Detecting candidate B-cell epitopes in a protein is a basic and fundamental step in many immunological applications. Due to the impracticality of experimental approaches to systematically scan the entire protein, a computational tool that predicts the most probable epitope regions is desirable. RESULTS: The Epitopia server is a web-based tool that aims to predict immunogenic regions in either a protein three-dimensional structure or a linear sequence. Epitopia implements a machine-learning algorithm that was trained to discern antigenic features within a given protein. The Epitopia algorithm has been compared to other available epitope prediction tools and was found to have higher predictive power. A special emphasis was put on the development of a user-friendly graphical interface for displaying the results. CONCLUSION: Epitopia is a user-friendly web-server that predicts immunogenic regions for both a protein structure and a protein sequence. Its accuracy and functionality make it a highly useful tool. Epitopia is available at http://epitopia.tau.ac.il and includes extensive explanations and example predictions.

Evolutionary modeling of rate shifts reveals specificity determinants in HIV-1 subtypes.

A hallmark of the human immunodeficiency virus 1 (HIV-1) is its rapid rate of evolution within and among its various subtypes. Two complementary hypotheses are suggested to explain the sequence variability among HIV-1 subtypes. The first suggests that the functional constraints at each site remain the same across all subtypes, and the differences among subtypes are a direct reflection of random substitutions, which have occurred during the time elapsed since their divergence. The alternative hypothesis suggests that the functional constraints themselves have evolved, and thus sequence differences among subtypes in some sites reflect shifts in function. To determine the contribution of each of these two alternatives to HIV-1 subtype evolution, we have developed a novel Bayesian method for testing and detecting site-specific rate shifts. The RAte Shift EstimatoR (RASER) method determines whether or not site-specific functional shifts characterize the evolution of a protein and, if so, points to the specific sites and lineages in which these shifts have most likely occurred. Applying RASER to a dataset composed of large samples of HIV-1 sequences from different group M subtypes, we reveal rampant evolutionary shifts throughout the HIV-1 proteome. Most of these rate shifts have occurred during the divergence of the major subtypes, establishing that subtype divergence occurred together with functional diversification. We report further evidence for the emergence of a new sub-subtype, characterized by abundant rate-shifting sites. When focusing on the rate-shifting sites detected, we find that many are associated with known function relating to viral life cycle and drug resistance. Finally, we discuss mechanisms of covariation of rate-shifting sites.

Epitope mapping using combinatorial phage-display libraries: a graph-based algorithm.

A phage-display library of random peptides is a combinatorial experimental technique that can be harnessed for studying antibody-antigen interactions. In this technique, a phage peptide library is scanned against an antibody molecule to obtain a set of peptides that are bound by the antibody with high affinity. This set of peptides is regarded as mimicking the genuine epitope of the antibody's interacting antigen and can be used to define it. Here we present PepSurf, an algorithm for mapping a set of affinity-selected peptides onto the solved structure of the antigen. The problem of epitope mapping is converted into the task of aligning a set of query peptides to a graph representing the surface of the antigen. The best match of each peptide is found by aligning it against virtually all possible paths in the graph. Following a clustering step, which combines the most significant matches, a predicted epitope is inferred. We show that PepSurf accurately predicts the epitope in four cases for which the epitope is known from a solved antibody-antigen co-crystal complex. We further examine the capabilities of PepSurf for predicting other types of protein-protein interfaces. The performance of PepSurf is compared to other available epitope mapping programs.

Computational characterization of B-cell epitopes.

Characterizing B-cell epitopes is a fundamental step for understanding the immunological basis of bio-recognition. To date, epitope analyses have either been based on limited structural data, or sequence data alone. In this study, our null hypothesis was that the surface of the antigen is homogeneously antigenic. To test this hypothesis, a large dataset of antibody-antigen complex structures, together with crystal structures of the native antigens, has been compiled. Computational methods were developed and applied to detect and extract physico-chemical, structural, and geometrical properties that may distinguish an epitope from the remaining antigen surface. Rigorous statistical inference was able to clearly reject the null hypothesis showing that epitopes are distinguished from the remaining antigen surface in properties such as amino acid preference, secondary structure composition, geometrical shape, and evolutionary conservation. Specifically, epitopes were found to be significantly enriched with tyrosine and tryptophan, and to show a general preference for charged and polar amino acids. Additionally, epitopes were found to show clear preference for residing on planar parts of the antigen that protrude from the surface, yet with a rugged surface shape at the atom level. The effects of complex formation on the structural properties of the antigen were also computationally characterized and it is shown that epitopes undergo compression upon antibody binding. This correlates with the finding that epitopes are enriched with unorganized secondary structure elements that render them flexible. Thus, this study extends the understanding of the underlying processes required for antibody binding, and reveals new aspects of the antibody-antigen interaction.

eIF2B activator prevents neurological defects caused by a chronic integrated stress response.

The integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a small molecule eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of disease contexts.

Pepitope: epitope mapping from affinity-selected peptides.

UNLABELLED: Identifying the epitope to which an antibody binds is central for many immunological applications such as drug design and vaccine development. The Pepitope server is a web-based tool that aims at predicting discontinuous epitopes based on a set of peptides that were affinity-selected against a monoclonal antibody of interest. The server implements three different algorithms for epitope mapping: PepSurf, Mapitope, and a combination of the two. The rationale behind these algorithms is that the set of peptides mimics the genuine epitope in terms of physicochemical properties and spatial organization. When the three-dimensional (3D) structure of the antigen is known, the information in these peptides can be used to computationally infer the corresponding epitope. A user-friendly web interface and a graphical tool that allows viewing the predicted epitopes were developed. Pepitope can also be applied for inferring other types of protein-protein interactions beyond the immunological context, and as a general tool for aligning linear sequences to a 3D structure. AVAILABILITY: http://pepitope.tau.ac.il/